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Recurrent Pregnancy Loss
Causes, Controversies, and Treatment
Series in Maternal-Fetal Medicine
About the Series

Published in association with the Journal of Maternal Fetal and Neonatal Medicine, the series in
Maternal Fetal Medicine keeps readers up to date with the latest clinical therapies to improve the health
of pregnant patients and ensure a successful birth. Each volume in the series is prepared separately and
typically focuses on a topical theme. Volumes are published on an occasional basis, depending on the
emergence of new developments.
Obstetric Evidence Based Guidelines, Third Edition
Vincenzo Berghella
Maternal-Fetal Evidence Based Guidelines, Third Edition
Vincenzo Berghella
Maternal-Fetal and Obstetric Evidence Based Guidelines, Two Volume Set, Third Edition
Vincenzo Berghella
The Long-Term Impact of Medical Complications in Pregnancy: A Window into Maternal and
Fetal Future Health
Eyal Sheiner
Operative Obstetrics, Fourth Edition
Joseph J. Apuzzio, Anthony M. Vintzileos, Vincenzo Berghella, Jesus R. Alvarez-Perez
Placenta Accreta Syndrome
Robert M. Silver
Neurology and Pregnancy: Clinical Management
Michael S. Marsh, Lina Nashef, Peter Brex
Fetal Cardiology: Embryology, Genetics, Physiology, Echocardiographic Evaluation, Diagnosis,
and Perinatal Management of Cardiac Diseases, Third Edition
Simcha Yagel, Norman H. Silverman, Ulrich Gembruch
New Technologies and Perinatal Medicine: Prediction and Prevention of Pregnancy Complications
Moshe Hod, Vincenzo Berghella, Mary D’Alton, Gian Carlo Di Renzo, Eduard Gratacos, Vassilios Fanos
Problem-Based Obstetric Ultrasound, Second Edition
Amar Bhide, Asma Khalil, Aris T. Papageorghiou, Susana Pereira, Shanthi Sairam, Basky Thilaganathan
Recurrent Pregnancy Loss: Causes, Controversies, and Treatment, Third Edition
Howard J.A. Carp
For more information about this series please visit: https://www.crcpress.com/Series-in
-Maternal-Fetal-Medicine/book-series/CRCSERMATFET
Causes, Controversies, and Treatment
Third Edition
Edited by
Howard J.A. Carp, MB BS, FRCOG
Clinical Professor
Obstetrics and Gynecology
Sheba Medical Center, Tel Hashomer
and
Sackler School of Medicine
Tel Aviv University
Tel Aviv, Israel
Front cover: Disorganized embryo as seen on embryoscopy. Picture courtesy of Thomas Philipp, MD, Vienna, Austria.
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Contents
Preface.....................................................................................................................................................viii
Contributors............................................................................................................................................... ix
Part I Basic Principles

1. The Epidemiology of Recurrent Pregnancy Loss.......................................................................... 1
Ole B. Christiansen
2. The Signaling between Embryo and Mother as a Basis for the Development of Tolerance.... 13
Eytan R. Barnea
3. Recurrent Pregnancy Lossfrom Evidence-Based to Personalized Medicine........................... 22

Howard J.A. Carp
Part II Etiology

4. The Genetics of Spontaneous Abortions....................................................................................... 30
Joe Leigh Simpson
5. The Endometrial Factor in Recurrent Pregnancy Loss.............................................................. 43

Luiza Borges Manna and Ying Cheong
6. Fetal Structural Malformations and Recurrent Pregnancy Loss.............................................. 48

Howard J.A. Carp, Thomas Philipp, Micha Baum, and Michal Berkenstadt
7. The Endocrinology of Recurrent Pregnancy Loss....................................................................... 59

Nicola Pluchino, Serena Bellaminutti, Panagiotis Drakapoulos, Antonis Makrigiannakis,
and Andrea R. Genazzani
8. The Etiology of the Antiphospholipid Syndrome......................................................................... 70

Sara De Carolis, Giuseppina Monteleone, Cristina Garufi, Rotem Inbar, Miri Blank,
and Yehuda Shoenfeld
9. Defects in Coagulation Factors Leading to Recurrent Pregnancy Loss................................... 79

Aida Inbal and Howard J.A. Carp
10. The Immunobiology of Recurrent Miscarriage........................................................................... 89

Marighoula Varla-Leftherioti, Theodora Keramitsoglou, and Christina Tsekoura
11. Immune Testing in Recurrent Pregnancy Loss..........................................................................101

Jeffrey Braverman, Darren Ritsick, and Nadera Mansouri-Attia
12. Uterine Anomalies and Recurrent Pregnancy Loss...................................................................110

Daniel S. Seidman and Mordechai Goldenberg
v
vi Contents
13. The Male Factor in Recurrent Pregnancy Loss......................................................................... 126

Catherine F. Ingram, Nannan Thirumavalavan, Marc Goldstein, and Dolores J. Lamb
Part III The Developing Pregnancy

14. Ultrasound Follow-Up in Early Pregnancy................................................................................ 134
Akhila Vasudeva and Pratap Kumar
15. Threatened Miscarriage and Recurrent Pregnancy Loss.........................................................145

Howard J.A. Carp
16. The Role of Cerclage and Pessaries..............................................................................................153

Israel Hendler and Howard J.A. Carp
17. What Genetic Screening Is Appropriate in Recurrent Pregnancy Loss?............................... 164

Howard Cuckle
18. Obstetric Outcomes after Recurrent Pregnancy Loss...............................................................172

Rakefet Yoeli-Ullman, Howard J.A. Carp, and Shali Mazaki-Tovi
Part IV Management

19. Investigation Protocol for Recurrent Pregnancy Loss.............................................................. 184
Howard J.A. Carp
20. Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? Yes............................ 197
Ashok Kumar and Simar Kaur
21. Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? No............................. 202
Roy Mashiach
22. Human Chorionic Gonadotropin Supplementation in Recurrent Pregnancy Loss............... 206

Carlo Ticconi, Adalgisa Pietropolli, and C.V. Rao
23. Antiphospholipid Syndrome: Management of the Obstetric Patient........................................215

Ashley E. Benson and D. Ware Branch
24. Can Recurrent Pregnancy Loss Be Prevented by Antithrombotic Agents?........................... 223

Audrey A. Merriam and Michael J. Paidas
25. Empirical In Vitro Fertilization for Recurrent Pregnancy Loss: Is It a Valid Concept?.......231
Michal Kirshenbaum and Raoul Orvieto
26. Debate: Should PGT-A Still Be Performed in Recurrent Pregnancy Loss? Yes.................... 239
Carmen M. García-Pascual, Pilar López, Nasser Al-Asmar, Pere Mir, Lorena Rodrigo,
Carlos Simon, and Carmen Rubio
27. Debate: Should PGT-A Still Be Performed in Recurrent Pregnancy Loss? No..................... 243
Raoul Orvieto and Norbert Gleicher
Contents vii
28. Third Party Reproduction in Recurrent Pregnancy Loss........................................................ 249

Gautam Nand Allahbadia, Rubina Merchant, Akanksha Allahbadia Gupta, and A.H. Maham
Part V Immunotherapy
29. Leucocyte Immunotherapy for Recurrent Miscarriage............................................................ 257
Salim Daya
30. IVIg Treatment for Recurrent Pregnancy Loss........................................................................ 268

Carolyn B. Coulam
31. The Role of Filgrastim.................................................................................................................. 275

Fabio Scarpellini and Marco Sbracia
32. Opinion: Immunotherapy Has No Place in the Treatment of Recurrent

Pregnancy Loss.............................................................................................................................. 280
Micha Baum
Index....................................................................................................................................................... 287
Preface
Six years have passed since the second edition of this book, and
thirteen since the first edition. Major advances have necessitated a
new edition. Genetics, in particular, has evolved out of all recognition
with the introduction of higher and higher-resolution analyses which
are being employed to make more accurate diagnoses. These changes
are summarized in Chapters 4 and 6. However, the prevention of
genetic aberrations by PGT-A is a hotly debated issue; the two
sides are summarized in Chapters 26 and 27. The first two editions
contained numerous debates on controversial subjects in recurrent
pregnancy loss (RPL). Many of these contentious issues have now
reached a consensus and can be summarized in chapters rather than
debates. The guidelines of the various professional organizations
have narrowed their differences somewhat. However, none relates
to the resistant patient who continues to miscarry despite the various
treatment modalities recommended in the guidelines. The resistant
patient is addressed in Chapters 19 and 28. New chapters have been
added regarding structural anomalies, empirical in vitro fertilization,
and personalized medicine as opposed to evidence-based medicine and which immune assessment should
be used.
RPL remains a distressing problem. Patients understandably expect answers and solutions. The
physician often does not have these answers. Recommendations vary from inactivity and follow-up
to intensive investigation and treatment. Recommendations are confounded by the lack of a universal
definition of RPL and often failure to distinguish between good and poor prognosis patients. This edition,
like the previous editions of this book, tries to summarize the controversies and discuss the scientific
basis for various causes of RPL in depth and to clarify the various treatment modalities. It is hoped that
we have succeeded in this endeavor.
The book is planned for general gynecologists, and specialists working in the field. Each contributing
author is an authority on a specific area of recurrent pregnancy loss. All chapters have undergone major
revision to include the changes that have occurred since the second edition.
I would like to thank each author for the time and effort taken in preparing the manuscripts to make
the publication of this book possible. I would also like to thank those responsible in a more indirect way
for the publication of this book: my teachers over the years, and my collaborators. However, special
recognition goes to the greatest teachers and collaborators of all, the patients.
Howard J.A. Carp, MB BS, FRCOG
viii
Contributors
Nasser Al-Asmar D. Ware Branch

Igenomix Valencia University of Utah Health
Valencia, Spain Salt Lake City, Utah
Gautam Nand Allahbadia Jeffrey Braverman

Reproductive Endocrinology and IVF, MMC IVF Reproductive Immunology
and New York City, New York
Bourn Hall IVF
Jumeirah, Dubai, United Arab Emirates Sara De Carolis
and Department of Obstetrics, Gynaecology
Orchid Fertility and Andrology Services, DHCC and Pediatrics
and F. Policlinico Gemelli IRCCS
Dr. Amal Elias Fertility Center Università Cattolica del Sacro Cuore
and Rome, Italy
Millennium Medical Center MMC IVF
Dubai, United Arab Emirates Howard J.A. Carp
Department of Obstetrics and Gynecology
Eytan R. Barnea Sheba Medical Center
Society for the Investigation of Early Tel Hashomer, Israel
Pregnancy and
New York City, New York Sackler School of Medicine
Tel Aviv University
Micha Baum Tel Aviv, Israel
Sheba Medical Center
Tel Hashomer, Israel Ying Cheong
University of Southampton
Serena Bellaminutti and
Division of Obstetrics and Gynaecology Complete Fertility Southampton
University of Geneva Southampton, United Kingdom
Geneva, Switzerland
Ole B. Christiansen
Ashley E. Benson Department of Obstetrics and Gynaecology
University of Utah Health Center for Recurrent Pregnancy Loss of Western
Salt Lake City, Utah Denmark
Aalborg University Hospital
Michal Berkenstadt Aalborg, Denmark
Danek Gertner Institute of Human
Genetics Carolyn B. Coulam
Sheba Medical Center Clinical Immunology Laboratory
Tel Hashomer, Israel Rosalind Franklin University of Medicine and
Science
Miri Blank North Chicago, Illinois
Zabludowicz Center for Autoimmune Diseases
Sheba Medical Center Howard Cuckle
Sackler Faculty of Medicine Department of Obstetrics and Gynecology
Tel Aviv University Columbia University Medical Center
Tel Aviv, Israel New York City, New York
ix
x Contributors
Salim Daya Akanksha Allahbadia Gupta

McMaster University Indira IVF
Hamilton, Ontario, Canada New Delhi, India
and
Newlife Fertility Centre Israel Hendler
Mississauga, Ontario, Canada Obstetrics and Gynecology
Sackler Medical School
Panagiotis Drakapoulos Tel Aviv University
Center for Reproductive Medicine Tel Aviv, Israel
Universitair Ziekenhuis Brussel and
Brussels, Belgium Department of Obstetrics and Gynecology
Sheba Medical Center
Carmen M. García-Pascual Tel Hashomer, Israel
Igenomix Valencia
Valencia, Spain Aida Inbal
Thrombosis and Hemostasis Unit
Cristina Garufi Beilinson Hospital
Lupus Clinic, Rheumatology, Dipartimento di Rabin Medical Center
Medicina Interna e Specialità Mediche Petah Tikva, Israel
Sapienza Università di Roma and
Rome, Italy Sackler Faculty of Medicine
Tel Aviv University
Andrea R. Genazzani Tel Aviv, Israel
Division of Obstetrics and Gynaecology
University of Pisa Rotem Inbar
Pisa, Italy Zabludowicz Center for Autoimmune
Diseases and Department of Obstetrics and
Norbert Gleicher Gynecology
The Center for Human Reproduction Sheba Medical Center
and Tel Hashomer, Israel
Foundation for Reproductive Medicine
and Catherine F. Ingram
Stem Cell Biology and Molecular Embryology Baylor College of Medicine
Laboratory Houston, Texas
The Rockefeller University
Simar Kaur
New York City, New York
Department of Obstetrics and Gynaecology
and
Maulana Azad Medical College and Associated
Lok Nayak Hospital
Vienna University of Medicine
New Delhi, India
Vienna, Austria
Theodora Keramitsoglou
Mordechai Goldenberg
Department of Immunology and
Histocompatibility
Chaim Sheba Medical Center
Helena Venizelou Maternity Hospital
Tel Hashomer, Israel
Athens, Greece
and
Sackler School of Medicine Michal Kirshenbaum
Tel Aviv University Department of Obstetrics and Gynecology
Tel Aviv, Israel Chaim Sheba Medical Center
Marc Goldstein and
Department of Urology Sackler Faculty of Medicine
Weill Cornell School of Medicine Tel Aviv University
New York City, New York Tel Aviv, Israel
Contributors xi
Ashok Kumar Rubina Merchant

Department of Obstetrics and Gynaecology Rotunda—The Center for Human
Maulana Azad Medical College and Associated Reproduction
Lok Nayak Hospital Mumbai, India
New Delhi, India
Audrey A. Merriam
Pratap Kumar Division of Maternal-Fetal Medicine
Department of Obstetrics and Gynecology Department of Obstetrics, Gynecology and
Kasturba Medical College Reproductive Sciences
Manipal Academy of Higher Education Yale University
Manipal, Karnataka, India New Haven, Connecticut
Dolores J. Lamb Pere Mir

Department of Urology and Center for Igenomix Valencia
Reproductive Genomics Valencia, Spain
Weill Cornell School of Medicine
New York City, New York Giuseppina Monteleone
Department of Obstetrics, Gynaecology and
Pilar López Pediatrics
Igenomix Argentina F. Policlinico Gemelli IRCCS
Caba, Argentina Università Cattolica del Sacro Cuore
Rome, Italy
A.H. Maham
MMC IVF Raoul Orvieto
Dubai, United Arab Emirates Department of Obstetrics and Gynecology
Chaim Sheba Medical Center
Antonis Makrigiannakis
and
University of Crete
Family Planning and Fertility Regulation
Heraklion, Greece
Sackler Faculty of Medicine
Luiza Borges Manna Tel Aviv University
North Middlesex Hospital Tel Aviv, Israel
London, United Kingdom
and Michael J. Paidas
Obstetrics and Gynaecology Department of Obstetrics, Gynecology and
Wessex Deanery, United Kingdom Reproductive Sciences
Miller School of Medicine
Nadera Mansouri-Attia University of Miami
Braverman Reproductive Immunology Miami, Florida
New York City, New York
Thomas Philipp
Roy Mashiach Gynecology and Obstetrics
Department of Obstetrics and Gynecology Danube Hospital
Sheba Medical Center Vienna, Austria
Adalgisa Pietropolli
Shali Mazaki-Tovi Academic Department of Systems Medicine
Department of Obstetrics and Gynecology Tor Vergata University
Sheba Medical Center Rome, Italy
and Nicola Pluchino
Sackler School of Medicine Division of Obstetrics and Gynaecology
Tel Aviv University University of Geneva
Tel Aviv, Israel Geneva, Switzerland
xii Contributors
C.V. Rao Carlos Simon

Department of Cellular Biology and Igenomix Valencia
Pharmacology and
Herbert Wertheim College of Medicine Department of Obstetrics and Gynaecology
Florida International University Valencia University and INCLIVA
Miami, Florida Valencia, Spain
and
Darren Ritsick Department of Obstetrics and Gynaecology
Braverman Reproductive Immunology Stanford University
New York City, New York Stanford, California
Lorena Rodrigo Joe Leigh Simpson

Igenomix Valencia Department of Human and Medical Genetics
and Herbert Wertheim College of Medicine
Ronda Narciso Monturiol Florida International University
Valencia, Spain Miami, Florida
and
Carmen Rubio Reproductive Genetic Innovation (RGI)
Igenomix Valencia Northbrook, Illinois
Valencia, Spain
Nannan Thirumavalavan
Scott Department of Urology
Marco Sbracia
Baylor College of Medicine
Hungaria Center for Endocrinology and
Houston, Texas
Reproductive Medicine (CERM)
Rome, Italy
Carlo Ticconi
Academic Department of Surgical Sciences
Fabio Scarpellini
Tor Vergata University
Hungaria Center for Endocrinology and
Rome, Italy
Reproductive Medicine (CERM)
Rome, Italy Christina Tsekoura
Department of Immunology and
Daniel S. Seidman Histocompatibility
Department of Obstetrics and Gynecology Helena Venizelou Maternity Hospital
Chaim Sheba Medical Center Athens, Greece
and Marighoula Varla-Leftherioti
Sackler School of Medicine Department of Immunology and Histocompatibility
Tel Aviv University Helena Venizelou Maternity Hospital
Tel Aviv, Israel Athens, Greece
Yehuda Shoenfeld Akhila Vasudeva

Internal Medicine Department of Obstetrics and Gynecology
Zabludowicz Center for Autoimmune Diseases Kasturba Medical College
Sheba Medical Center Manipal Academy of Higher Education
Tel Hashomer, Israel Manipal, Karnataka, India
and
Research of Autoimmune Diseases Rakefet Yoeli-Ullman
Sackler Faculty of Medicine Department of Obstetrics and Gynecology
Tel Aviv University Sheba Medical Center
Tel Aviv, Israel Tel Hashomer, Israel
1
The Epidemiology of Recurrent Pregnancy Loss
Ole B. Christiansen
Substantial disagreement exists about spontaneous prognosis after recurrent pregnancy loss (RPL),
probably due to differences in monitoring intensity between studies. In future studies of prognosis in
RPL it is suggested that the live birth rate per time unit is introduced as the main outcome measure.
Introduction
The term miscarriage (or abortion) is used to describe a pregnancy that fails to progress, resulting
in death and expulsion of the embryo or fetus. The World Health Organization (WHO) definition [1]
stipulates that the fetus or embryo should weigh 500 g or less, a stage that corresponds to a gestational
age of 20 weeks. The European Society for Human Reproduction and Embryology (ESHRE) defines a
miscarriage as an intrauterine pregnancy demise prior to viability confirmed by ultrasound or histology,
whereas miscarriages, biochemical pregnancy losses, and pregnancies of unknown location (PULs)
are jointly termed pregnancy losses [2]. Recurrent miscarriage (RM) has traditionally been defined as
≥3 consecutive miscarriages, and recurrent pregnancy loss (RPL) as ≥3 pregnancy losses. However,
the American Society for Reproductive Medicine (ASRM) RPL defines RPL as ≥2 not necessarily
consecutive clinical miscarriages [3], and recently ESHRE’s RPL guideline group also defined RPL as
≥2 not necessarily consecutive pregnancy losses [4].
Including women with two previous pregnancy losses in studies of RPL is epidemiologically very
problematic. If the ASRM/ESHRE definition of >2 losses is used, the vast majority of patients will
have a good prognosis for live birth. The live birth rate after two consecutive pregnancy losses is
75%–80% in the next pregnancy [5,6] or within 3 years [7]. The ≥2 definition of RM/RPL assumes that
the prognosis for pregnancy losses is similar in women with the same number of previous consecutive
or nonconsecutive pregnancy losses, e.g., a woman with four pregnancy losses after a birth has the
same prognosis in the next pregnancy as a woman with three pregnancy losses followed by a live birth
followed by one miscarriage. Only one study [8] has addressed whether pregnancy losses prior to a live
birth have similar prognosis as those subsequent to a live birth. In a multivariate analysis of 127 patients
with unexplained secondary RPL, each pregnancy loss after the birth, and in particular the presence
of a second trimester miscarriage after the birth, increased the risk for subsequent pregnancy loss with
incidence rate ratio (IRR) = 1.14 (95% confidence interval [CI] 1.04–1.24, p = 0.002) and IRR = 2.15
(95% CI 1.57–2.94, p < 0.0001), respectively, whereas early and late pregnancy losses prior to the birth
did not exhibit any prognostic impact. According to this study [8], a patient with four pregnancy losses
after a birth will have a 50% chance of a live birth compared to a 90% chance in a patient with three
losses prior to but only one loss after the live birth. Knowledge about the prognosis is important for
designing valid trials.
1
2 Recurrent Pregnancy Loss
Epidemiologic Parameters Relevant for RPL

Occurrence
The prevalence of RM/RPL is the number of women in a population who, at a specific time point, meet
the definition, and the incidence is the number of new women per time unit suffering a new pregnancy
loss, and the prevalence or incidence is often expressed as the proportion of individuals at risk for the
disorder. The denominator could be all women in the population, women of fertile age, or women who
had attempted pregnancy at least two or three times. The estimate of the prevalence or incidence of RM/
RPL is uncertain since in most countries there is no nationwide registration of miscarriages or RM/RPL.
In addition, many early pregnancy losses are not treated in hospitals and are thus not registered. There
are a few older estimates of the prevalence of RM based on the definition of ≥3 consecutive miscarriages.
One of the most informative studies was the retrospective study by Alberman [9]. Nine out of 1097 female
doctors (0.8%) who had had three or four previous pregnancies reported ≥3 consecutive miscarriages.
However, since the study was summarized before 1990, many early miscarriages may not have been
registered due to the lack of highly sensitive hCG tests and ultrasound examinations at that time.
Other estimates of the prevalence of RM roughly concord with that of Alberman. In a group of 5901
women with ≥2 pregnancies screened for toxoplasma antibodies, 1.4% had experienced RM [10]. Data
from a questionnaire-based study [11] found in a sample of 493 women with ≥2 intrauterine pregnancies
that 0.6% had had ≥3 consecutive miscarriages and 1.8% had had ≥3, not necessarily consecutive, losses.
Overall, the prevalence of RM according to the old definitions seems to be between 0.6% and 1.4%.
A problem in adapting the new RPL definitions is that the number of women who meet the criteria
will expand substantially. In Alberman’s [9] study among 2062 women who had had two to four previous
pregnancies, 42 reported ≥2 not necessarily consecutive miscarriages (3.25%) which is significantly
higher than the 0.6%–1.4% prevalence of RM using the traditional definition. This suggests that adapting
the new RM/RPL definition will triple the prevalence of the diagnosis. The implications of this are
discussed later.
The observation that the RPL prevalence is >1% indicates that RPL is not a random event but a disorder
affecting women who have an increased risk of pregnancy loss. If RPL (according to the old definition)
were caused by a random accumulation of “sporadic” miscarriages mainly caused by fetal aneuploidy,
the prevalence of RPL would be 0.153 = 0.34% (based on a frequency of sporadic miscarriage of 15%
in the population [9]) rather than 1%. The 1% prevalence indicates that most RPL cases are caused by
nonrandom factors which increase the risk of pregnancy loss in each pregnancy.
Knowledge of changes in the incidence of RPL are important and can inform us about changes in
environmental or genetic risk factors of importance for pregnancy loss. Roepke et al. [12] in a nationwide
register-based study found that the incidence of women with three or more consecutive pregnancy losses
had increased significantly in Sweden from 2003 to 2012. If the denominator was all women in Sweden
aged 18–42 years in the period, the incidence increased from 0.042% to 0.069%, relative increase 74%
(p < 0.0001) and if the denominator was women with a least one pregnancy in the period, the incidence
increased from 0.55% to 0.82%, relative increase 58% (p < 0.0001). Changes in maternal age, body mass
index (BMI), and coding practice during the period could not explain the change.
Number of Previous Miscarriages

Prospective studies of RPL patients show remarkable consistency in finding an increasing risk of
pregnancy loss as the number of previous pregnancy losses increases. The chance of subsequent live birth
in untreated RPL patients with 3, 4, and ≥5 pregnancy losses has been reported to be 42%–86%, 41%–
72%, and 23%–51%, respectively [13–16] (Figure 1.1). The significant variability in the estimate of the
subsequent risk of pregnancy loss in RPL patients can probably be attributed to the time of ascertainment
of the pregnancies (Figure 1.2) since the average age of the patients and the duration of follow-up in the
various studies were not different. The information in Figure 1.2 is based on data directly supplied or
can unequivocally be deduced from the literature [14,16,17]. In studies where the patients were urged
to contact the department for inclusion in a treatment trial as soon as menstruation was overdue and the
The Epidemiology of Recurrent Pregnancy Loss 3
3 misc. 4 misc. 5+ misc.

100
80
Birth rate (%)
60
40
20
0
Ref. 13 Ref. 14 Ref. 15 Ref. 16
FIGURE 1.1 Subsequent birth rate according to the number of previous miscarriages in patients with RPL reported in
four studies (col. 1, ref. 15; col. 2, ref. 14; col. 3, ref. 13; col. 4, ref. 16).
pregnancy test was positive [16], almost all biochemical pregnancies were identified and the patients
would be registered as having a high pregnancy loss rate (47.1%) but a low nonpregnancy rate (14.7%)
during the observation period. In studies where the patients were told to call the department in gestational
week 6–7 and were included in treatment trials [17] or cohorts receiving standard care [14] only after
ultrasonographic demonstration of fetal heart action, most biochemical pregnancies would not be
ascertained and therefore significantly higher nonpregnancy rates (38.3%–55.6%) and significantly lower
pregnancy loss rates (11.1%–14.4%) would be registered compared with the former study (Figure 1.2).
The subsequent probability of live birth in RPL can best be estimated using data from the placebo arm
of placebo-controlled trials [16,17] (Figure 1.2) because in such trials the ascertainment of pregnancies
is generally better than in nonrandomized studies, as patients are included according to strict protocols
and are closely monitored in early pregnancy. Hence, more very early pregnancy losses are included in
placebo-controlled than in nonrandomized studies [18].
The negative prognostic effect of the number of previous pregnancy losses could be due to maternal
age being positively correlated to gravidity. However, in multivariate analyses of clinical parameters of
prognostic impact in RPL, the number of previous pregnancy losses has without exception remained the
strongest prognostic parameter even after adjustment for other risk factors [8,13,19,20].
Not pregnant Miscarriages Births
60
N = 45 N = 153 N = 34
50
*
**
40
Clinical
*
30
%
20
Preclinical
10 ** *
**
0
Ref. 17 Ref. 14 Ref. 16
FIGURE 1.2 Frequency of women registered as not being pregnant, miscarrying, or giving birth in three prospective
cohorts of untreated patients with RPL (col. 1, ref. 13; col. 2, ref. 14; col. 3, ref. 15; col. 4., ref. 16). Ref. 16 indicates the
proportion of both preclinical and clinical miscarriage; all miscarriages in ref. 17 (except one) and in ref. 14 were clinical.
*p = 0.001; **p < 0.0001, χ2 test.
Three miscarriages
Four miscarriages
Five miscarriages
Six or more miscarriages
100
Women with recurrent miscarriage and one or
more live births after first consultation (%)
80
60
40
20
0
0 5 10 15 20 25
Years elapsed after date of first consultation
FIGURE 1.3 Cumulative live birth rates per time unit in women with RPL according to the number of previous pregnancy
losses. (Reproduced with permission from Lund M et al. Obstet Gynecol. 2013;119. 37–43.)
Assessing the outcome of the first pregnancy after referral in order to assess prognosis is problematic.
A 100% follow-up is necessary and if very early biochemical pregnancies are included in the outcome
data (which they should), very close monitoring of the patients must be undertaken. In addition, the
outcome of the first pregnancy after referral is not clinically relevant since most patients have no problems
conceiving and will have further pregnancy attempts. For the patients, the only relevant outcome is a live
birth. We have proposed that the most relevant method of assessing prognosis is to calculate the chance
of a subsequent live birth per time unit after the date of first consultation. In countries with valid national
birth registers and the possibility of identifying all individuals in the registers through unique personal
identification numbers, an almost 100% follow-up of RPL women with regard to live births is possible.
In a study of 987 women with RPL [21], the chance for live birth after 5 years’ follow-up was 71.9%
after three, declining to 50.2% after ≥6 previous pregnancy losses (Figure 1.3). There was only a minor
additional improvement of the live birth chance after 5 years had elapsed.
Maternal Age
In a register-based study of 634,272 Danish women achieving pregnancy between 1978 and 1992 who
attended a hospital during pregnancy [22], the miscarriage rates in women with RPL were almost identical
in women of age 30–34 years and 35–39 years (38%–40%) but it increased to 70% in women of age 40–44
(Figure 1.4). It seems that the impact of age on the miscarriage rate is quite modest in RPL until age 40,
but beyond this age it is the strongest prognostic factor. In concordance with this, several multivariate
analyses [8,13,19] in RPL patients (almost all of whom were younger than 40), found that maternal age was
not a significant predictor of pregnancy loss after adjustment for other relevant independent variables. In
one study [8], the adjusted IRR for new pregnancy loss was 0.99 (95% CI 0.96–1.03) for each additional
year of age in patients younger than 40 years, indicating no impact at all.
Subgroups of RPL
Three different groups of women should be assessed separately: (a) the primary RPL group consists of
women with ≥3 consecutive pregnancy losses with no pregnancy progressing beyond 20 weeks’ gestation,
(b) the secondary RPL group consists of women who have had ≥3 pregnancy losses following a pregnancy,
80
70
60
50
Birth rate (%)
< 30 years
40 31–35 years
36–39 years
30
40–44 years
20
10
0
Ref. 22 Ref. 15
FIGURE 1.4 Subsequent birth rate according to maternal age in patients with RPL reported in two studies (col. 1, ref.
22; col. 2, ref. 15).
that progressed beyond 20 weeks’ gestation, which may have ended in live birth, stillbirth, or neonatal
death, and (c) the tertiary RPL group, which consists of women who have had several pregnancy losses
before a pregnancy that progressed beyond 20 weeks’ gestation followed by ≥3 pregnancy losses [18].
In some studies, secondary RPL is defined as RPL after a live birth [23] or a pregnancy that progressed
beyond gestational week 28; however, in this survey the 20-week cutoff will be used. Unfortunately,
many studies fail to distinguish patients with primary and secondary RPL. It is indeed possible that
secondary RPL is not a particular entity but just the clinical appearance of the RPL syndrome among
patients who, by chance, instead of delivering their child after three or four miscarriages deliver in the
first pregnancy and subsequently experience a series of miscarriages. However, there is support from
immunogenetic studies [24,25], NK cells [23,26], and immunotherapy [27,28] that secondary RPL is a
separate entity with characteristics different from primary RPL. If primary and secondary RPL have
different pathophysiological mechanisms, different prognoses would be expected. Summarizing the
placebo-treated patients included in the author’s placebo-controlled trials of immunotherapy [16,28],
the live birth rate in the first pregnancy was 17/35 = 48.6% in women with primary RPL compared with
11/34 = 32.4% in women with secondary RPL (not significant) when matched for the number of previous
miscarriages and age. Other studies have reported success rates [14,15] in the two subsets that are not
different, which is the commonly accepted view.
RPL patients with second trimester losses constitute a different subset. Drakeley et al. [29] found that
25% of their RPL patients had had at least one second trimester loss. Among 228 RPL patients admitted
to the RPL clinic in Copenhagen 2000–2004, 39 (17.1%) had experienced a mixture of first and second
trimester miscarriages but only three had suffered exclusively second trimester losses. Since almost all
patients with second trimester miscarriages had experienced first trimester miscarriages, early and late
RPL must have pathogenic factors that partially overlap. Several prospective studies indicate that a history
of second trimester pregnancy losses has a strong negative prognostic impact [8,30,31].
Familial Aggregation
Few studies have investigated the occurrence of RPL in families of RPL couples with normal chromosomes.
Results from published family studies are shown in Table 1.1. Johnson et al. [32], Alexander et al. [33],
and Ho et al. [34] compared the prevalence of RPL among relatives of women with RPL with the
corresponding prevalence in relatives of fertile controls. Christiansen et al. [35] obtained information
concerning relatives’ pregnancy outcomes from questionnaires completed by the relatives themselves,
and the stated pregnancy loses were confirmed from hospitals’ and practitioners’ records. The rate of
≥3 pregnancy losses in relatives was compared with an external control group [11]. Table 1.1 shows
that the risk of RPL in first-degree relatives of RPL patients is 2–7 times higher than in the background
TABLE 1.1
Proportion of Recurrent Pregnancy Loss (RPL) in Relatives of
Women with RPL
Reference and Kind RPL Rate in RPL Rate in
of Relatives Studied Relatives (%) Controls (%) P-value
Johnson et al. [32]
Blood relatives 12.2 7.3
Alexander et al. [33]
Mothers and sisters 7.0 0.0 0.02
Ho et al. [34]
First-degree relatives 1.4 0.2 0.0001
Christiansen et al. [35]
Sisters 10.6 1.8 0.00005
Brothers’ wives 6.3 1.8 NS
Abbreviation: NS, Not significant.
population. The relative frequency λ (= the frequency of RPL in relatives divided by the frequency in
the general population) is a measure of the degree of heritability of a disorder [36]. In the Danish study
[35], λ was 5.9 for sisters and 3.5 for brothers’ wives when comparisons are made with the population
prevalence [11], pointing toward a moderate degree of heritability of RPL.
Partner Specificity
It is commonly assumed that unexplained RPL is a partner-specific condition, and a criterion that all
pregnancies should be with the same partner has been included in the definition of primary and secondary
RPL by some authors [37]. However, no study has really addressed the question of partner specificity.
In a multivariate analysis [38], the authors’ group found that after adjustment for all relevant prognostic
factors, the chance of a subsequent live birth was not different in patients with secondary RPL who have
had all pregnancies with the same partner compared with those who have had two different partners,
casting doubt on the concept of partner specificity.
Clinical Associations
An association between RPL and perinatal complications has been reported in many studies. These
complications are fully described in Chapter 18 of this book. It is debatable whether the risk of intrauterine
growth restriction (IUGR) is associated with the previous consecutive miscarriages. However, Christiansen
et al. [39] found that the mean birth weight of women with RPL themselves was 3265 g compared with
3414 g in matched female controls (p < 0.025) and the mean birth weight of women with ≥5 miscarriages
at the time of admission was 2991 g (p < 0.001 compared with controls). The birth weights of the male
partners did not differ from the birth weight of matched male controls. These data strongly suggest that
the association between low birth weight and RPL is an inherent part of the RPL syndrome.
Lifestyle Factors
Lifestyle factors are rarely, if ever, major causes of RPL; however, studies have shown that many lifestyle
factors increase the risk of miscarriage. There is good evidence that obesity [40,41], high daily caffeine
intake [42–44], alcohol consumption [45], and use of nonsteroidal anti-inflammatory drugs [46,47]
increase the risk of miscarriage or RPL significantly. Social class and occupation also impact the rate
of miscarriage, with the greatest risk among women exposed to high physical or psychic stress during
work [48,49]. Several studies also indicate that a previous subfertility/infertility diagnosis or infertility
treatment may increase the risk of miscarriage [20,50].
Integration of Epidemiologic Knowledge in Research and Management of RPL

Occurrence
Knowing the incidence of RPL has several applications: it can be used for comparing risks of RPL
between different populations, and it can be used for comparing change in risk over time, which is
necessary for identifying risk factors. The alarming increase in the incidence of RPL in Sweden [12]
in the last 10 years should be sufficient to allocate funds in order to clarify which risk factors may have
increased in frequency. The focus could be on endocrine disruptors [51,52] or factors responsible for
the concomitant increase in the incidence of most autoimmune diseases. It has been suggested that less
exposure to helminthic infections during childhood in modern societies may have left the immune system
“uneducated” (resulting in a lack of regulatory T cells) thereby increasing the risk of autoimmune disease
and harmful immunity to the fetus and trophoblast.
Number of Miscarriages
The number of previous pregnancy losses is the most important prognostic factor in RPL and should
therefore be taken into account when planning therapeutic trials. The ideal trial should stratify for the
number of previous pregnancy losses, with randomization between control and experimental treatments
within each stratum. Stratifying the sample by the number of previous pregnancy losses may make it
easier to demonstrate the effect of the experimental intervention. It may then be easier to demonstrate
an effect in women with higher number of losses, as the spontaneous success rate is so much lower in
women with fewer losses [18,53].
Due to the new definitions of RPL as two or more losses, an increasing number of studies include women
with only two nonconsecutive pregnancy losses. Two pregnancy losses may in many cases be a chance
phenomenon. Sporadic miscarriages are due to chromosomal abnormalities in 43% of the cases [54]. Thus,
in theory, in 0.43 × 0.43 = 18.5% of women with two miscarriages the cause is due purely to embryonic
aneuploidy. Including women with only two early pregnancy losses will “dilute” the estimate of the risk factor
(in both case-control and cohort studies) or the treatment effect in controlled clinical trials. The proportion
of RPL patients in whom the disorder can be explained by an accumulation of “sporadic” pregnancy losses
declines with the number of previous losses [55]. Conversely, the proportion of euploid embryos increase
with the number of previous losses. This is supported by the fact that the frequency of many immunological
risk factors increases [24,56,57], the possible effect of immunotherapy increases [18,53], and the frequency
of aneuploid miscarriages declines [58] with the number of previous pregnancy losses.
Maternal Age
Because increased maternal age increases the subsequent pregnancy loss rate, therapeutic trials should
stratify for maternal age. However in RPL, age seems to impact on pregnancy outcome after age 40 [8,22]
(Figure 1.4) so it may be sufficient to undertake stratification according to age below and above 40 years.
Advanced maternal age is associated with several other disorders such as uterine fibroids and endocrine
and autoimmune abnormalities; therefore, maternal age should be accounted for in any trial.
Subgroups of RPL
If primary and secondary RPL and RPL with first and second trimester losses have different pathogenetic
backgrounds, the frequency of recognized risk factors for RPL and the efficacy of treatments may differ
between the groups. A series of studies have provided data suggesting that such differences exist (Table 1.2).
The factor V Leiden (FVL) genetic polymorphism is the most common cause of activated protein C
resistance (APCR), which is a risk factor for thrombosis and possibly associated with RPL [59]. Wramsby
et al. [60] found a significant association with primary but not secondary RPL, and Rai et al. [61] found
that APCR was significantly associated with the absence of a previous live birth. In a study of three
congenital thrombophilic factors (including FVL), 25.5% of women with primary RPL were positive
TABLE 1.2
Prevalence of Risk Factors or Effect of Treatments according to Subgroups of Patients
Prevalence/Effect in Prevalence/Effect in Late
Secondary vs. Primary RPL vs. Early Primary RPL
Parental chromosome abnormality Equal N/A
Antipaternal antibodies Higher Higher
Antiphospholipid antibodies Lower or equal Higher
Heriditary thrombophilia factors Lower Higher
NK cell activity Lower N/A
HLA-DRB1*03 Higher N/A
MBL deficiency N/A Higher
Allogeneic lymphocyte immunization Lower N/A
Treatment with i.v. immunoglobulin Higher N/A
Abbreviation: N/A, Cannot be estimated.
for at least one thrombophilic factor compared with 15.1% of women with secondary RPL [62]. Most
studies also claim a higher prevalence of thrombophilias in patients with second trimester miscarriages
compared to early losses [59,63].
In contrast, the prevalence of parental chromosome abnormalities is similar between primary and
secondary RPL. In a review [64] of 79 relevant studies, chromosome abnormalities were found in 3.7%
of secondary and 2.9% of primary RPL couples. Franssen et al. [65] also found that the prevalence of
parental chromosome abnormalities was similar in primary and secondary RPL. Consequently, parental
chromosome testing should be performed in both types of RPL.
A series of immunological parameters may be relevant in RPL and may have a different distribution
between the subgroups of RPL patients.
Antibodies
Alloantibodies directed against paternal/fetal human leukocyte antigens (HLAs) are produced with
increased gestational age [66,67]. Anti-HLA antibodies often persist for years and can therefore be found
more often in women with secondary compared with primary RPL [6]; however, they seem not to be
pathogenic [28,68].
Most autoantibodies can be found with increased prevalence in RPL and are associated with a poor
pregnancy prognosis [13]; however, few studies have differentiated between primary and secondary
RPL. In patients with primary RPL, the prevalence of positive anticardiolipin or antinuclear antibody
concentrations may be higher than in secondary RPL [13,69,70]. None of the differences were statistically
significant but future studies of autoantibodies in RPL should clearly distinguish between primary and
secondary RPL. There is, however, a consensus that antiphospholipid antibodies (aPL) display a stronger
association with late than early RPL [59,71].
NK Cells
NK cell numbers and cytotoxicity have been reported to predict a poor prognosis in RPL [72]. NK cell
activity has been reported to be increased in peripheral blood NK cells in primary but not secondary RPL
when compared with controls [25,26].
Class II HLA Alleles

Class II HLA alleles are associated with most autoimmune disorders. In the largest study of HLA-DRB1
alleles in patients with RPL [24], the immunological high-responder allele HLA-DRB1*03 was found
significantly more often in secondary RPL than in controls (32.4% and 21.0%, respectively; p < 0.006),
but not in primary RPL. In a later retrospective cohort study, Nielsen et al. [25] found that maternal
carriage of HLA class II alleles predisposing to immunity against male-specific HY antigens displayed a
strong prognostic impact in women with RPL after the birth of a boy [25]. Genetic polymorphism in the
MBL-2 gene are associated with low plasma levels of MBL and are more strongly associated with late
than first trimester RPL [73].
Immunotherapy also elicits different effects in primary and secondary RPL. The efficacy of paternal or
third-party leucocytes has been evaluated in a meta-analysis of placebo-controlled trials [74] showing that
immunotherapy did not improve the live birth rate in secondary RPL, whereas it significantly improved
the live birth rate in primary RPL [53]. In a meta-analysis [27], intravenous immune globulin (IVIg)
reduced the pregnancy loss rate significantly in women with secondary RPL (OR = 0.77 (95% CI 0.58–
1.02, p < 0.05) but not in patients with primary RPL. Unfortunately, these subgroup differences were not
taken into account in a recent Cochrane meta-analysis on immunotherapy in RPL [75], which concluded
that neither allogeneic lymphocyte immunization nor IVIg were efficient when all published studies were
analyzed as a single group.
Familial Aggregation
As discussed above, family studies (Table 1.1) support a multifactorial model for inheritance of RPL. The
development of many common diseases (e.g., arterial hypertension, diabetes mellitus, and schizophrenia)
is thought to be determined by a multifactorial model. One risk factor is not sufficient to cause disease
but when several intrinsic and extrinsic factors accumulate in an individual (or couple), the risk exceeds
a threshold level and disease develops. Both thrombophilic [76] and immunogenetic risk factors seem to
aggregate significantly more frequently than expected in RPL patients. Traditionally, the causes of RPL
have been assumed to be single causative factors, e.g., uterine malformations 10%, endocrine factors
10%, aPL 15%, etc. However, this model is probably inadequate, and the threshold of multiple factors
may be more appropriate [77]. In principle, RPL patients should be screened for all potential risk factors
and screening not stopped as soon as the first risk factor has been identified. The recognition that RPL
exhibits a high degree of heritability implies that susceptibility genes for RPL may be inherited by genetic
linkage analyses in families with several siblings experiencing RPL [7,78,79].
Partner Specificity
Early studies on HLA antigens in RPL assumed that increased HLA similarity between partners led to
inadequate maternal protective immune responses and fetal loss. However, after many studies on HLA
sharing in couples with RPL, the role of HLA sharing could not be confirmed [80,81]. If good quality
epidemiological studies showing little evidence of partner specificity in RPL had been performed [38]
prior to the HLA sharing studies, the theories of increased HLA sharing between RPL spouses may not
have developed.
Clinical Associations
A series of factors associated with RPL—aPL, hereditary thrombophilias, and MBL deficiency—have
also been associated with late miscarriage, low birthweight, and perinatal complications [57,59]. Since
RPL per se seems to be associated with perinatal complications and low birthweight, prospective studies
of the effect of the mentioned factors on perinatal complications should be adjusted for the confounding
effect of the number and type of previous miscarriages.
Lifestyle Factors
RPL is a complex disorder where lifestyle factors are expected to modify the effect of non-lifestyle
(intrinsic) factors previously discussed. The prevalence of the most important lifestyle factors among
patients and controls should be given in publications in order to document that the groups studied
for the occurrence of non-lifestyle risk factors or pregnancy outcome are comparable. Since it is
likely that smoking aggravates the effect of thrombophilic risk factors on the risk of pregnancy
loss, details of smoking habits should be reported in all studies of RPL and thrombophilia. It is
generally recognized that women with polycystic ovary syndrome (PCOS) exhibit an increased rate
of miscarriage and RPL. However, when adjustment for obesity is undertaken, the miscarriage rate in
PCOS does not seem to be dependent on polycystic ovarian pathology or PCOS-associated endocrine
abnormalities [41].
Conclusions
Epidemiologic studies can provide essential information for basic laboratory research, case-control
studies, or treatment trials. However, it seems that epidemiologic knowledge is rarely taken into account
in current clinical research and management of RPL.
The incidence of RPL has rarely been assessed but is a much more clinically important parameter than
the prevalence. It should be recognized that applying the new definitions of RPL suggested by ASRM
and ESHRE, the prevalence/incidence of RPL will probably triple, whereas the overall spontaneous
prognosis for live birth in the patients will increase to 75%–80%. However, the subset of patients with
the poor prognosis will not diminish, it will just be hidden in the mass of patients with a good prognosis.
Estimates of the future miscarriage risk in RPL vary significantly. Some studies have estimated the
prognosis too optimistically because preclinical pregnancy losses have been considered to be non-
pregnancy. Therefore, in future treatment trials the baby take-home rate per time unit may be a better
outcome measure than the pregnancy loss rate per pregnancy. The number of previous miscarriages is
not only the strongest prognostic factor but with an increased number of previous miscarriages fetal
aneuploidy seems to become less prevalent and maternal factors more prevalent. Therefore stratification
by the number of previous miscarriages is important in RPL studies.
In conclusion, at least three features should be included in future RPL research: recognition of the
multifactorial/polygenic background of RPL, recognition of the different pathogenetic features of
primary/secondary RPL, and recognition of the importance of the number of previous miscarriages.
Awareness of these features should eliminate the practice of combining data from too heterogeneous
RPL studies for meta-analysis.
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2
The Signaling between Embryo and Mother as
a Basis for the Development of Tolerance
Eytan R. Barnea
Introduction
In mammalian gestation, immunologic acceptance and tolerance are paramount for the successful
interaction between the embryo/graft and its maternal host. Initial immunologic awareness must take
place prior to implantation. The semipermeable zona pellucida forms rapidly post fertilization and
protects the embryo until it reaches the endometrium. The zona is surrounded by maternal immune cells,
and this unit transmits the message that fertilization has occurred. However, in in vitro fertilization (IVF)
and embryo transfer this mechanism is not operative. The main question is when and how the embryo-
maternal communication initiates and creates maternal recognition of pregnancy. Advances in assisted
reproduction suggest that the embryo is the dominant element in the reproductive process; its viability
and ability to signal are critical for embryo-maternal recognition. Furthermore, genetics does not appear
to play a predominant role; donor embryos or xeno-transfer embryos fare very well following transfer
with no discernable difference to semi-allogenic embryos.
Herein we focus on a peptide secreted from the earliest stages of gestation, preimplantation factor (PIF),
which fulfills three fundamental requirements. First, it is only secreted by viable embryos and is only
detected in the maternal circulation in viable pregnancies. Second, it has an essential role in pregnancy,
through autocrine effects on the embryo and as a promoter of implantation and trophoblast invasion.
Third, it regulates global maternal immune responses while preserving the antipathogen action. Evidence
is emerging that PIF can also be effective in the treatment of pregnancy pathologies and preclinical and
clinical nonpregnant immune disorders and transplantation.
Why Is There a Delay between Fertilization and Implantation?

The tube picks up the egg after fertilization when estradiol is at a peak. For implantation to take place,
progesterone must peak. Consequently, the embryo post fertilization needs to be delayed in order to
mature and develop until implantation can occur. Physiologically, maturation takes place in the tube.
However, as IVF has shown, if a four-cell stage embryo is replaced, maturation can take place in the
uterus for a few days until implantation occurs [1]. The delay in implantation suggests that embryo-derived
signaling must be present for the endometrium to become receptive. However, embryo signaling is not
specific for the uterus since implantation can take place in the tube, ovary, and abdominal cavity [2]. The
signal seems to be embryo-derived and the maternal system responds, and not vice-versa.
There are very few conditions that prevent conception and implantation. Even in severe diseases,
although the incidence of pregnancy is reduced significantly, the pregnancy can still develop [3–5], also
indicating that the maternal system is mostly receptive and does not guide the reproductive process.
In addition, there is no need for genetic compatibility, since cross-species fertilization can occur (e.g.,
horse and donkey, resulting in a mule). Embryo signaling overcomes the problem that only half the
genome is maternal and the other half is paternal, to the extreme example of cross-species pregnancy.
As earlier data have shown, the mother recognizes that conception has taken place upon the appearance
13
of a zygote, but not before. In organ transplantation, if the donor’s cells and organs are foreign, rejection
would immediately occur. In birds and mammals, fertilization is internal, and tolerance develops. The
sperm, although genetically and antigenically foreign, is not attacked by the maternal system. In birds,
later rejection is overcome as growth and development of the embryo takes place in an egg covered by a
shell [2]. In mammals, the zygote is surrounded by the zona pellucida, which prevents maternal immune
cells’ entrance. After hatching from the zona pellucida, protective mechanisms are operative to prevent
rejection. At hatching and implantation, the uterus should be sufficiently primed. Unless the embryo
signal is ineffective or the maternal organism is excessively hostile, the viable embryo will implant. The
uterus is not a privileged but a preferential site for the early embryo; the response would be completely
different in the presence of a foreign tissue. However, in cross-species embryo transfer, where the genetics
are completely different, specific embryo signaling allows the zygote to remain implanted and even
thrive until delivery. The hormones estrogen and progesterone are essential to mature the uterus for
implantation. However, they are insufficient to initiate pregnancy. The embryo must take a significant part
in this process though specific signaling. After implantation, there must be accommodation of the embryo
throughout pregnancy. In ectopic pregnancy, priming of the fallopian tube can also occur, as in the uterus.
Embryo-Specific Maternal Communication—PIF

The culture media in which embryos were grown in vitro have been shown to have immune regulatory
properties. Early pregnancy factor (EPF) (Chaperone 10, which facilitates protein folding) is probably
secreted by the ovum (bovine model) and can be detected in pregnant women’s sera 48 hours after
fertilization. In red deer (Cervus elaphus), EPF has been correlated with the number of viable fetuses [6].
Sera that contain EPF have significantly higher blastocyst development rates [7]. An additional embryo-
derived nonspecific compound is platelet-activating factor (PAF), a unique signaling phospholipid
required for fetal development [8]. PAF acts by binding to its receptor, PAF-R, which is a G-protein-
coupled receptor often found in immune cells. Embryo-derived signaling molecules can be detected in the
maternal circulation. The primary role of human chorionic gonadotropin (hCG) is to support the corpus
luteum, allowing maintenance of pregnancy. hCG also has a role in promoting placental angiogenesis
and syncytiotrophoblast formation. There are multiple forms of hCG which have diverse functions. The
hyperglycosylated form of hCG is involved in creating tolerance to the embryo by modulating endometrial
immunity. In dendritic cells, MHC class II, IL-10, and IDO expression is increased, which impairs T cell
proliferation. In patients undergoing IVF, circulating levels of various cytokines has been determined
following hCG injection. It was shown that hCG decreases the anti-inflammatory cytokines IL27 and
IL17; in contrast IL10 levels increased, as well circulating Treg cells [9]. hCG has also been shown to
increase IDO production particularly in dendritic cells, suggesting another mechanism for pregnancy
resistance to autoimmunity [10]. However, hCG is not embryo-specific; it is unique to humans. In rodents,
placental lactogen has the dominant role.
Our team has discovered an additional compound, preimplantation factor (PIF) [2,3,11,12]. PIF seems
to be embryo specific, secreted only by viable embryos, and can be detected in the maternal circulation.
This molecule provides the evidence that embryo-maternal communication starts at fertilization and lasts
throughout pregnancy until delivery [5,11–13]. PIF is found across many mammalian species: human, pig,
horse, cow, and mouse [5,13–17]. PIF levels have been detected in IVF culture and increase up to the blastocyst
stage [11,12,18]. Furthermore, PIF is detected in the extra-villous trophoblast where intimate contact is
established between embryo and the mother [14]. Hence, PIF first acts at a distance from the endometrium
and later through direct contact at the maternal-fetal interface. Therefore the correct signaling is transmitted
to the mother indicating the need for support until delivery. The data generated with IVF embryos provided
important information indicating that only a viable embryo will implant, while a non-viable embryo will not
[19]. If the pregnancy is healthy, PIF levels will increase until the second trimester and then decline [14]. In
contrast, in pathologic pregnancy—for example, miscarriage—the expression of PIF is low and prematurely
declines in preeclampsia and intrauterine growth restriction (IUGR) placentae [20]. Thus, embryo maternal
communication is altered in pathological pregnancy and may provide a danger signal to the mother that
pathology is present. In murine gestation the placenta and uNK cells release PIF-containing granules on day
The Signaling between Embryo and Mother as a Basis for the Development of Tolerance 15
14 in preparation for labor [17]. However, administration of lipopolysaccharide (LPS) led to re-expression
of PIF in the placenta. PIF administration is associated with a twofold reduction in fetal deaths compared
to controls. In premature labor, low expression is a reflection that embryo maternal communication is
disrupted, and there is a failure of effective fetal/maternal nutrient exchange. In most cases inflammation
is the cause, and innate immunity comes into operation and probably neither the mother nor fetus would
survive. By initiating a “rejection” presenting as labor, both mother and progeny have a chance of surviving
if the fetus is the right gestational age and not severely compromised.
PIF Autocrine, Autotrophic, and Protective Actions

Once formed, the zygote is surrounded by the zona pellucida. The embryo is then protected from the
maternal immune system. However, the mother becomes aware of the embryo a very short time after
fertilization due to signaling. Signaling has been shown with respect to platelet emargination [21]. Certain
proteins present in platelets are targeted by PIF (including platelet-derived growth factor beta [PDGFβ]).
In the presence of PIF, platelet and lymphocyte adhesion is increased [22–24]. From fertilization onward,
the secretion of PIF increases reflecting the health of the embryo, while embryos destined to miscarry
do not increase PIF levels in culture [12]. The main receptors to PIF are protein disulfide isomerase/
thioredoxin (PDI-T) and heat shock proteins (HSPs) [25]. In a recent study, the effect of an inhibitor of
PDI-T was assessed which increased oxidative stress of the embryos [26]. Bovine data has shown that
embryos cultured in large groups in the presence of this inhibitor led to growth arrest. When PIF was
added and cultures monitored for 8 days, the number of embryos reaching the blastocyst stage more than
doubled. PIF prevents the PDI-T protein transition from an oxidative to a redox form, thereby protecting
embryos from demise. As an adverse maternal environment can lead to recurrent pregnancy loss (RPL),
the protective effect of PIF was tested in the presence of embryo toxic serum derived from women with a
history of RPL. Early data indicated that if embryos were cultured in an optimized environment, progress
to the blastocyst stage was high (80%) [18]. If PIF was added to those cultures, even high concentrations
did not further improve embryo development. However, when PIF was added to the embryo culture media
that was exposed to RPL serum, PIF protected against embryo demise and also increased the number
of embryos that reached the blastocyst stage. Recognizing that oxygen radicals and toxins are of low
molecular weight (MW), the RPL serum was divided into two fractions, <3 kDa and >3 kDa [26]. The
low MW serum delayed embryo development until the blastocyst stage, whereas in the presence of the
>3 kDa serum fraction there was a significant increase in embryo demise. Embryo demise may have been
due to the presence of antibodies or other proteins in the RPL serum. Notably, in both circumstances
PIF effectively blocked the adverse effects of RPL serum. RPL serum has been tested for the presence of
anti-PIF antibodies, but no anti-PIF antibodies were found in RPL serum. Consequently, RPL is not due
to presence of circulating anti-PIF antibodies [26].
PIF has also been assessed for an autocrine effect. Anti-PIF antibody in the culture decreased
development to the blastocyst stage by 80% [12]. However, in singly cultured cow embryos, which rarely
develop to the blastocyst stage, the presence of PIF for 3 days followed by PIF-free media up to day 7
promoted progress to the blastocyst stage [18]. In analyzing embryo development to determine the exact
site of FITC-PIF uptake as the embryo advanced from the morula to the blastocyst stage, the major uptake
was observed in the leading edge of the blastocyst bulge, where the contact with the endometrium would
be expected (blastocyst extrusion) [25]. Hence, PIF has both an embryotropic and protective effect. This
effect is amplified following implantation when the zona is removed and the interaction between the
embryo and the mother is direct and therefore the most critical stage in reproduction takes place, leading
embryo implantation to succeed or to fail.
Receptive Uterine Environment Is Critical for Reproduction

In addition to the role of the embryo, a receptive uterine environment is also a clear requisite. There is
also an embryo-derived regulatory effect on the uterus [27–29]. Overall, PIF has a significant effect at
pre-, during, and post-implantation periods the requirement shifts from apposition to attachment and
invasion. Disturbance of any of these steps will lead to pregnancy loss: implantation failure, chemical
pregnancy, early or later miscarriage. Therefore, endometrial adaptation must be clearly coordinated.
PIF appears to have an important role in all these steps. The effect of PIF starts prior to implantation
since it increases endometrial integrin expression in human epithelial cells, a major receptivity marker
[28]. This pro-receptivity action is shown in human stromal cells that are activated by estrogen and
progesterone (human embryonic stem cell [hESC]). Detailed studies on gene, protein, and pathway
analysis indicate that PIF has a major effect on local immunity, adhesion, and apoptosis control, which
favor implantation. The increase in IRAKBP1 that interacts with TRL5 has antimicrobial activity. The
decrease in IL12RB2 reduces pro-inflammatory cytokine action. A local mild pro-inflammatory milieu
favors embryo embedding [27]. The effect on embryo adhesion genes was shown by increased Down
syndrome cell adhesion molecules such as SORBS2 and SORBS1 expression. Therefore, the embryo
through PIF creates a favorable endometrial environment. In first trimester decidua, the protective effects
are magnified. PIF leads to protection against an adverse maternal environment which would be highly
detrimental to the embryo during embryogenesis. The earliest embryonic structure is the notochord;
therefore special attention has been paid to determine whether PIF is involved in development of the
notochord. The data indicate that in both the implantation period and in the first trimester, PIF exerts
both neurotrophic and neuroprotective effects [31]. These neuroprotective effects may have implications
for postnatal life. The effect of PIF seemed to protect against childhood diseases including autism [29]
and adult neurodegenerative diseases [30], neonatal neurotrauma as observed in the premature infant,
and reversing advanced neuroinflammation leading to brain remyelination and reversal of paralysis
[5,30–35]. This suggests that PIF could help to reduce pregnancy pathologies by administration during
pregnancy [17].
Implantation Is Insufficient: Effective Trophoblast Invasion Is Also Required

In addition to improving endometrial receptivity, PIF also influences trophoblast invasion [14,20,29,35,36].
Invasion has to be regulated in order to prevent excessive invasion as seen in placenta accreta, or too
shallow an interaction which leads to coagulation disorders or abruption. [15]. The pro-invasive effect
of PIF was investigated on a transformed trophoblastic cell line using primary extravillous human
trophoblasts, which confirmed this effect on invasion. The effects of PIF involve metalloproteinases, tissue
inhibitor of metalloproteinase (TIMP), and integrins [14,20,29,36]. These ligands were also involved in
the effect of PIF on the endometrium [28]. Insights into the effect of PIF on trophoblast invasion were
provided by using specific inhibitors which defined the pathways involved: MAPK, IP3 K, and JAk-Stat
[14]. Furthermore, a detailed genomic analysis identified several associated genes that are affected in the
trophoblast [20]. PIF effectively upregulated Azurocidin-1, which has an important role in chemotactic
and antimicrobial activity. By promoting IL17F, a pro-inflammatory cytokine, trophoblast invasion is
supported, and the effect is also amplified by increased lincRNA MALAT-1 expression. By upregulating
T cell receptor alpha, similarly to that found in the endometrium, PIF regulates the immune response
[27]. PIF increases BCL2, resulting in anti-apoptosis, and affects associated pathways. These mechanisms
contrast with the effect of PIF on the endometrium where hESC cell apoptosis must take place in order to
accommodate the invading trophoblast. The increase in BCL2 involves p53-related pathways where p53
protein inhibition blocks the stimulatory effect of PIF on BCL2 and BAX, thereby establishing a direct
functional relationship. Thus, endogenous PIF that is present in the placenta appears to have a major local
regulatory and protective role that is disrupted in pathological placentae such as IUGR, where decreased
expression impairs fetal growth [20].
PIF Interaction and Synergy with Progesterone and HLA-G

Progesterone is essential for pregnancy development [37]. During implantation, the corpus luteum
increases progesterone secretion, which transforms the endometrium from the proliferative to the
secretory phase [38]. Data show that PIF as monotherapy primes the endometrium independent of
progesterone [28]. Therefore, the role of PIF and progesterone can be questioned as to primordiality and
hierarchy [39]. Progesterone promotes trophoblastic cell proliferation and involvement in differentiation
[40]. PIF is secreted at fertilization, and progesterone’s peak secretion takes place in the mid-luteal phase
[12]. PIF anticipates the secretion of progesterone, which also leads to assessing the possible synergy
between these two essential compounds. From 7 weeks onward, the placenta takes over progesterone
expression, and secretion from the corpus luteum which then undergoes involution [41]. In contrast, PIF
is secreted by the early embryo followed by the trophoblast in direct contact with the maternal milieu [14].
PIF’s regulatory effect of progesterone on the trophoblast was recently examined. PIF has a promoting
effect on progesterone [42]. PIF’s effect was tested on trophoblast JEG-3 cells where PIF was associated
with increased progesterone receptor expression. PIF also increased the secretion of progesterone by the
trophoblast. However, since progesterone enhances tolerance, a side-by-side comparison was performed
with PIF to test the effect on the expression of pro-tolerance HLA antigens [43]. PIF had a greater effect
than progesterone on all the HLA antigens tested [44,45], including HLA-G, E, C, and F. The effect of
PIF was also greater than that of progesterone in regulating the secretion of several cytokines, including
an increase in IL-10, IL-1b, IL8, GM-CSF, and TGF-b1. Progesterone, however, only increased IL-10
secretion, but even here, the effect was half the effect of PIF. In addition, when the effect of PIF was
compared to progesterone on different trophoblast proteins, the effect of PIF was more pronounced in
increasing regulatory T cells (FoxP3+), coagulation factors, and complement regulation. PIF also reduced
PRDX2 and HSPs 70 more than progesterone in negating oxidative stress and protein misfolding, which
could lead to impaired tolerance [42]. Overall, PIF is synergetic with progesterone and therefore might
even be able to reduce the risk of premature labor by amplifying the effect of progesterone.
PIF Interaction and Effect on NK and Dendritic Cells in RPL

Seventy percent of conceptuses fail to implant prior to menses. Subsequently, the loss rate is 10%–15%
in the first trimester and 2%–3% afterward until delivery. Therefore, for the maternal system to be
favorable, the embryo must be viable and able to transmit a specific quiescence signal. Secretion of PIF
can be detected in the maternal circulation 9 days after insemination and 5 days after embryo transfer.
The detection of PIF in the maternal circulation at such an early stage indicates that interaction must
occur with the maternal immune system. The interaction of PIF with human immune cells was examined
in women with RPL. Increased circulating natural killer (NK) cells and killing activity are thought
to be associated with a higher risk of RPL [46]. The effect of PIF was examined on peripheral blood
mononuclear cell (PBMC) using a standard cytotoxicity assay, analyzing the effect on a K562 cell line
in a cohort of RPL patients [47]. The data showed decreased NK cell cytotoxicity through an indirect
action by reducing the CD69 expression-prime inflammatory marker. The direct binding of FITC-PIF to
NK cells is only 10%, and this does not change after activation by a mitogen, PHA [11]. Notably, when
a side-by-side comparison was carried out with the effect of intravenous immune globulin (IVIg) or
intralipid, the PIF inhibitory effect was similar: 40%–50% reduction [47]. However, the concentration
of PIF used to reduce the cytotoxicity was six orders of magnitude ng instead of mg lower, indicating
PIF’s effect specificity. When dose-dependent administration was performed, PIF was effective even in
a very low concentration (2.5 ng/mL as compared to the 25 ng/mL concentration), whereas there was no
significant effect in the control group. These levels are in the range found in the maternal circulation, and
therefore reflect the circulating levels in pregnant women. Furthermore, PIF reduced NK cell cytotoxicity
irrespective of the NK cell/lymphocyte ratio. Since NK cells’ CD69 is a marker of inflammation, the
data showed that in the presence of PIF CD69 expression decreased significantly. Overall, in 86 patients
with RPL, PIF led to reduced NK cell toxicity. As a further step, the effect of PIF was tested in patients
undergoing IVF. A different effect was observed with respect to binding to NK cells as analyzed by flow
cytometry. In healthy controls, PIF binding, as expected, was low, and it was mostly to CD14+ cells,
while in women with RPL the binding was enhanced, reflecting overactive immunity [11]. Chernishov
et al. [47] then tested whether PIF could help in identifying patients at risk for loss following IVF and
embryo transfer (IVF-ET). In 40 unselected patients, the binding of PIF and effect on NK cell cytotoxicity
was determined following exposure to whole blood. The data showed that PIF after 24 hours exerts a
significant effect on both NK activity and cytotoxicity. In addition, PIF mildly activates pre-incubated
naive blood cells. Since the patient population varies (having different infertility etiology), the data show
that the effect of PIF is immune regulatory and protective instead of being immune suppressive. Further, it
suggests that PIF could be used as a screen for IVF patients prior to embryo transfer to determine whether
adverse effects on the embryo may be present. Moreover, it opens the possibility of treating patients with
elevated adverse NK activity prior to undergoing infertility treatment [48]. PIF is taken up by uNK in
vivo in murine pregnancy. uNK cells are important in regulating embryo-maternal interactions toward
tolerance when well controlled [49].
Dendritic cells (DCs) are thought to be important in protecting the embryo against rejection [50].
We compared the proportion of FITC-PIF binding to circulating Th2-promoting plasmacytoid dendritic
cells (pDC), and to Th1/pro inflammatory myeloid DCs in RPL patients. Binding was assessed following
incubation with anti-CD123-antibody lineage cocktail, CD11c, and HLA-DR antibodies. Patients with a
history of RPL (N = 13) were compared with healthy non-pregnant women (NP N = 11). PIF binding to
RPL immune cells was equally reduced in both pDC and the mDC (myeloid dendritic cell) populations
as compared to controls (pDC PIF+: NP 58.2 ± 18.3; RPL 41.2 ± 19.2, p = 0.03) mDC (PIF+: NP
57.9 ± 9.1; RPL 46.1 ± 14.2, p = 0.029). These data suggest that a reduction in PIF binding to DCs can
represent a marker of risk for pregnancy loss.
Embryo-Maternal Communication through the Immune System

PIF could have therapeutic potential in reducing NK cell toxicity and interacting with both pDC and
mDC. Three types of examination were performed in vivo, in vitro, and testing outside pregnancy as a
monotherapy in preclinical and clinical studies [4,5,11,30,31,33,51–57].
First, when FITC-PIF is injected in the murine model it targets the immune system (both spleen and
bone marrow) within 5 minutes and is rapidly cleared from the maternal circulation [52]. Furthermore,
PIF binding to PBMC is significantly different in pregnancy and prior to pregnancy. Prior to pregnancy,
binding is mostly to CD14+ cells, which encompasses both macrophages and neutrophils. However, in
pregnancy the binding to CD3+ cells is significantly increased [11]. The different binding patterns indicate
that the pregnancy is an activated environment. PIF, through interaction with the maternal immune
system, can improve the embryo’s signaling effect, thereby mitigating the maternal immune response to
the presence of the embryo [38]. In addition, such interactions may help the maternal system to decrease
the autoimmune response. Many autoimmune diseases improve in pregnancy, such as multiple sclerosis,
rheumatoid arthritis, hyperthyroidism etc. If pregnancy terminates as miscarriage, autoimmune disease
tends to relapse, e.g., in rheumatoid arthritis [4,32]. Even in normal pregnancy, autoimmune disorders tend
to relapse after delivery, reflecting the conceptus’ role in protection. Thus, PIF, while protecting self (i.e.,
embryo) may also defend against pathogens, as pregnancy is an immune activated and not an immune
suppressive environment. The critical aspect of an immune regulator is whether it can regulate the global
immune system. PIF has been reported to reduce the mixed lymphocyte reaction, which is relevant for
long-term tolerance (as confirmed outside of pregnancy in murine and primate transplant studies for bone
marrow and ovarian tissue, respectively) [55–59]. The second aspect is reducing the activated immune
response when excessive without affecting basal immunity. In this respect, exposure to antigens is highly
diverse, and PIF is effective in regulating exposure to anti-CD3/CD28 antibody, LPS, PHA, TPA, and
other mitogens, reflecting the ability to respond to diverse stimuli rather than being dependent on a single
agent [11,30,51–54,57]. The binding is also to specific immune cell populations beyond CD14+ to CD4+,
CD8+, CD19, and CD4/CD25+/Foxp3+ [60]. This last immune phenotype is particularly important
from the start of pregnancy. The CD4/CD25+/Foxp3+ cells are pro-tolerance cells. Targeting by PIF
could enhance embryo tolerance. The secretory products of the immune cells were also identified, and in
general terms there is a Th2/Th1 cytokine bias as determined by both genetic analysis and assessment of
secreted cytokines [11,34,51]. However, the embryo also needs to protect the mother. PIF increases the
number of Th1 cytokines to maintain antipathogen action. PIF decreases the formation of free oxygen
radicals in LPS-stimulated macrophages [55]. In the presence of a mitogen GMCSF, PIF led to activation
of macrophages, and in co-culture with lymphocytes previously activated by anti-CD3 led to decreased
proliferation. Therefore, PIF’s action is integrative between the innate and adaptive system. PIF targets
the macrophages that act as antigen-presenting cells, which assess the need to respond and activate other
arms of the immune system [51]. Since PIF is taken up in vivo and in immune cells, it is mostly attached
to intracellular receptors [11,52]. As previously shown, specific PIF receptors have been identified using
same-affinity chromatography followed by mass spectrometry in human immune cells. The data showed
high similarity of PIF targets in the immune system as well as the embryo [25]. The most pronounced
targets were as expected in the innate immune system and in CD14+ cells, whereas in both CD4+
and CD8+ cells the binding targets had a 90% homology with that found in CD14+ cells, albeit with a
fourfold lower number of proteins [52]. The data showed that over 60% of the targets identified were also
present in the embryo [25].
In addition to PDI-T and HSPs that are responsible for protection against oxidative stress and protein
misfolding, several other proteins are involved. These include the cytoskeleton, immune response, and
compounds that are involved in coagulation control [52]. With respect to the immune aspect, both local
protection by reduced critical inflammasome-NALP3- and caspase 1 in the placenta have been reported,
and decreased local inflammatory cytokines such as IL18, TNFα, and GRO expression. From an immune
perspective, the reduction in local inflammation induced by LPS was associated with a major decline in
the levels of several circulating cytokines. These include a threefold decrease in INFɣ and lower IL1-β,
IL18, GM-CSF, and GRO, MIP1b, IL12p70, IL22, and IL27. PIF also reduced some anti-inflammatory
cytokines such as IL4 and IL5. Since PIF reduced spontaneous pregnancy loss threefold, it was necessary
to determine the effect on the placenta, where there is only a mild effect on the inflammasome. The results
on circulating cytokines matched those seen in LPS, PIF reduced IL-18, IL-5, IL-12p70, while IL-23,
and MCP1 are also decreased. but not significantly. Hence, some effects are complementary while other
mechanisms are likely to be involved in PIF-induced protection against spontaneous and LPS pregnancy
loss. The result was that PIF optimized fetal weight was not affecting placental weight. In preterm infants,
both low and high placental weight are associated with fetal demise when compared to fetal weight. In
contrast, only the low weight term placenta carries a high risk of fetal death [61]. Therefore, integrated
PIF immune protection also has a trophic effect on the fetus.
The effect of PIF as monotherapy has also been assessed in diverse immune disorders such as
pancreatic, hepatic nervous system, and transplantation disorders [5,32,31,33,34,53–57,59]. In all models,
the protective effect was integrated, involving both local and systemic levels. What PIF does in pregnancy
(protecting self locally at the implantation site while regulating the maternal immunity toward tolerance
without undo immune suppression) mimics how PIF behaves in disease models. The data generated in
those models led to toxicology studies showing PIF safety. A subsequent clinical trial in patients with
autoimmune liver disease documented PIF’s safety after single and multiple subcutaneous injections
[62]. This trial also showed that PIF does not cause deleterious drug-to-drug interactions, irrespective of
whether used for treatment of metabolic diseases or immune suppression. As long-term administration
may be required for treating chronic immune disorders, PIF’s long-term toxicology (90 days daily
subcutaneous PIF administration, clinical grade, at very high doses) is being investigated in partnership
with the NIH/NCATs/BRiDGs program. The study, in coordination with FDA required murine/canine
studies, aims to show that PIF is safe as we progress toward phase II clinical trials.
Conclusions
The embryo has the necessary elements to progress through birth and development to adulthood. The
environment in which the embryo develops has a great impact on future postnatal life. The effective
embryo-maternal communication that starts at conception and is subsequently amplified guides successful
reproduction. PIF that is secreted from the zygote onward follows pregnancy until term but not beyond.
PIF is a specific message for self-preservation through autotrophic/protective actions in a potentially
hostile environment. PIF creates a receptive environment, facilitating effective trophoblast development.
In addition, the maternal immune system must be regulated selectively. Since the embryo-maternal
interaction is finite, a “gentle” form of “rejection” occurs when the fetus is ready for extrauterine life.
However, when there is pregnancy pathology, the role of PIF as a protector is shown in overactive NK
cells, spontaneous loss, and inflammation-induced pregnancy loss. Lessons learned from pregnancy
through the action of PIF are being utilized clinically for the treatment of diverse immune disorders
and transplantation.
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3
Recurrent Pregnancy Loss from
Evidence-Based to Personalized Medicine
Howard J.A. Carp
Introduction
Today there are two trends in selecting the most appropriate treatment for an individual patient: the
evidence-based approach, and the personalized approach, Both have advantages and disadvantages.
At first glance, both seem mutually opposed; however, they do have complementary features that can
assist in selecting the most appropriate treatment in any field of clinical medicine in general, or in
recurrent pregnancy loss (RPL) in particular. Below is an example of the problems that can arise from
both approaches.
In 2003, the United States Food and Drug Administration (FDA) approved the chemotherapeutic agent
gefitinib for the treatment of advanced non-small cell lung cancers. In 2005, Thatcher et al. [1] reported
the results of a randomized placebo-controlled multicenter study in Lancet. The results, using the best
principles of evidence-based medicine (EBM), reported that gefitinib was not associated with a significant
improvement in survival in locally advanced or metastatic non-small cell lung cancer (5.6 vs. 5.1 months,
respectively; HR: 0.89; p = 0.11). Based on this study, the FDA withdrew approval for the use of gefitinib.
However, in 2004, there were two reports that mutations in the epidermal growth factor receptor (EGFR)
predict sensitivity and response to gefitinib [2,3]. Tumors lacking EGFR mutations responded poorly or
not at all. However, only 10% of non-small cell lung cancers harbor the EGFR mutation. This leaves a
problem. Are those patients with an EGFR mutation to be denied effective treatment because they are
only 10% of the population? Should 90% be given a drug that has no effect? In 2015 Burotto et al. [4]
published a meta-analysis of treatment results in patients with non-small cell lung cancers harboring the
EFGR mutation and showed a significant benefit. Burrotto returned to the principles of EBM in order to
have feedback on the results, but used restriction to only include patients with the EGFR mutation. EBM
assesses a clinical problem where there is no clear diagnosis, then audits the results in order to make
decisions about treatment. Personalized medicine seeks an accurate diagnosis of cause, and then offers
targeted therapy. However, there is often little audit of the results.
This chapter assesses both methods of assessment, their strengths and weaknesses, and application
to RPL.
Evidence-Based Approach
EBM developed as a reaction against poorly designed observational treatment research and physicians’
reliance on personal experience with other patients. In EBM, data are collected from valid and
current studies on large cohorts of patients, from which mean values or figures are derived to infer
recommendations. The principles of the evidence-based approach involve searching the literature for
studies and critically appraising them to answer clearly defined and focused questions generated from
encounters with patients presenting with clinical problems. Randomized controlled trials (RCTs) and
meta-analyses are the major tools of EBM. The strength of the evidence-based approach is to audit the
treatment effect and have feedback that treatment improves outcome over natural history, and to avoid
22
Recurrent Pregnancy Loss from Evidence-Based to Personalized Medicine 23
unnecessary treatments which may have side effects. EBM has certainly made an important contribution
to questioning unsubstantiated therapeutic claims.
EBM requires a hierarchy of evidence in order to assess therapy. The best evidence is the RCT or meta-
analysis. Personal experience and expert opinion are the lowest levels of evidence [5]. This classification
has been modified several times with the inclusions of subgroups. The principles of EBM require us to
use the best evidence available. However, the meta-analysis or RCT has become the gold standard [6],
and often all other grades of evidence are excluded.
In the first and second editions of this book, Daya described a stringent set of conditions for inclusion
in an RCT of recurrent miscarriage. Some of these conditions are discussed below.
Reducing Selection Bias

Maternal age and number of previous miscarriages are the two most important prognostic factors. Most
trials include patients of all ages and either two or more or three or more miscarriages in a trial. They
then show demographic details that the trials are matched for age, number of miscarriages, etc. However,
if there is heterogeneity, the results will be affected. Patients with ten miscarriages have a much worse
prognosis than patients with two miscarriages. In any trial there will be many more patients with two
or three miscarriages than ten miscarriages. Hence the mean number of miscarriages in a demographic
table may not reflect the results for patients with a poor prognosis.
In the SPIN study [7], which assessed empirical enoxaparin and aspirin in women with two or more
unexplained pregnancy losses, there was no increased chance of having a successful pregnancy with
pharmacologic intervention (OR = 0.91, CI = 0.52–1.59). However, 57.1% of patients had experienced
only two previous miscarriages. The authors concluded that the current study may not be directly
applicable to women with three or more losses.
Articulating the Research Question

Articulating the research question is all-important, as the question determines which studies are to be
included in a meta-analysis and which are to be excluded. An example is paternal leucocyte immunization.
If all trials are included, a Cochrane database meta-analysis in 2014 [8] of 12 studies (641 participants)
reported that the treatment effect on the live birth rate was not significant, with OR of 1.22 and CI of
0.89–1.69. However, the meta-analysis had heterogeneity as the trial of Ober et al. [9] used cells stored in the
refrigerator overnight, whereas all other trials used fresh cells. Storage may have negated the immunogenic
effect [10]. If the research question was, “Does immunization with fresh cells improve the live birth rate?”
and the Cochrane results recalculated excluding Ober et al.’s trial, there is a statistically significant benefit
(OR 1.63, CI 1.13–2.35) [11]. The most recent meta-analysis of Liu et al. [12] includes 18 (739 treated and 999
control patients). There was a significant improvement in the live birth rate (OR = 4.02, CI of 3.23–5.00).
Drawbacks of EBM
There are many drawbacks of EBM. The main drawback is that the assessment of treatment looks for
a mean effect and assumes a “one size fits all” scenario. In general, outliers are essentially ignored.
However, outliers deserve adequate treatment and should not be ignored merely because they have a
different response to the majority of patients included in the trial.
There is confusion between no evidence of effect and evidence of no effect. No evidence of effect
means that the treatment under question has not been shown to have a statistically significant effect in the
majority of the study population fitting predetermined criteria. No evidence-based trials can show absence
of effect, as there may be an effect in a subgroup of outliers (see gefitinib trials in the Introduction).
However, the conclusions of some trials or meta-analyses state that the treatment under consideration
should not be used. Evidence may also change if more trials are added to the meta-analyses (see paternal
leucocyte immunization, above).
Lack of evidence of effect is seized upon by insurance companies and public health authorities to
restrict treatment that is believed to be ineffective. However, in clinical practice patients do not fall
into tightly controlled groups with strict criteria as found in trials. If treatment is denied by insurance
companies or public health services, the physician is limited in the choices he can make, and the patient
has her autonomy denied by not allowing her a say in her treatment.
EBM trials were originally designed to be objective by formulating a question, and then either doing
the research to test the hypothesis under question or assess all the literature on the subject under question.
However, formulation of the question under review is subjective, depending on the investigators. In
addition, in a meta-analysis the authors decide which trials to include and which to exclude. This choice
may be entirely subjective. (See example above about paternal leucocyte immunization. Should a study
using refrigerated cells be included or not?)
In summary, EBM has created enormous benefits for population health. By separating useful
from useless therapies, EBM has provided the basis for effective population-level control of risk
factors for myocardial infarction and stroke, has played a critical role in the transformation of HIV
from a fatal infection to a chronic disease, and was instrumental in testing drugs that can now cure
hepatitis C virus [13].
Personalized Medicine
Personalized medicine is older than EBM. Personalized medicine relies on an accurate diagnosis and
targeted therapy. Until recently, diagnosis was based on clinical criteria, laboratory results, histology
of biopsied specimens, or imaging. More recently, diagnosis has been made by genomic analysis. A
classic example of genomic diagnosis is blood transfusion. Blood transfusion is never given without
genomic analysis of the red cells (better known as blood typing) and a sensitivity assay (better known as
cross-matching) to determine the most appropriate blood to administer. If blood were to be given in an
intention-to-treat study without grouping or cross-matching, we should reach the conclusion that there is
no evidence of a beneficial effect, and that the risks outweigh the benefit. However, the ability to provide
a precise diagnosis in a routine clinical setting depends on the availability of adequate diagnostic tests,
including molecular profiling tests.
In oncology, the term precision medicine has replaced the term personalized medicine. The approach
currently individualizes treatment mainly on the basis of genomic tests. Sensitivity studies are then used,
which may include spectrometry and computational power and real-time imaging of drug effects in the
body. The ability to practice precision medicine is also dependent on the knowledge bases available to
assist clinicians in taking action based on test results [14].
EBM regarded the physician’s experience as the lowest form of evidence. Professional experience might
have been biased when the physician’s experience was limited to a single doctor. Nowadays, however,
advances in computing and informatics make it possible to access and analyze the collected experience
of tens of thousands of physicians caring for hundreds of thousands of patients—in fact, far more patients
than could ever be enrolled in a single clinical trial. The analysis of aggregate physician experience
may identify wide variations in clinical practice. Heterogeneity of practice patterns is an advantage, as
it enables consideration of patients’ clinical courses under diverse treatment modalities and for patients
with diverse histories. Consequently, choice of treatment can be focused on issues of clinical practice,
where choices for an individual patient should be centered.
Biomarkers
In 1998, the National Institutes of Health Biomarkers Definitions Working Group defined a biomarker
as “a characteristic that is objectively measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” [15]. The
World Health Organization (WHO) has defined a biomarker as “any substance, structure, or process
that can be measured in the body or its products and influence or predict the incidence of outcome or
disease” [16]. The principle of a biomarker is essential to the concept of personalized medicine, as it
influences treatment and can be used as a surrogate marker to assess the effect of treatment. Biomarkers
may include pulse and blood pressure, basic chemistry, or more complex laboratory tests. The key issue is
determining the relationship between any given measurable biomarker and relevant clinical endpoints. To
identify a biomarker as a surrogate endpoint requires determination of relevance and validity. Relevance
refers to a biomarker’s ability to appropriately provide clinically relevant information. Validity refers to
the need to characterize a biomarker’s effectiveness or utility as a surrogate endpoint. However, validity
is not typically black or white but usually consists of various shades of gray. In addition, research into
biomarkers and diagnostics for personalized medicine has fallen short of expectations.
Drawbacks of Personalized Medicine

Personalized medicine has many major drawbacks. The development of new drugs and testing for
genomic biomarkers is expensive. For example, cetuximab is used for treating late-stage colorectal
cancer. However, it is only effective in patients in whom the the KRAS gene is not mutated [20]. KRAS
mutational analysis is commercially available from a number of laboratories. Cetuximab is approved
by the FDA in the United States after KRAS testing in order to identify those individuals who have a
high chance of responding. However, the average extension of life is 5 months. Consequently, in 2006,
the UK’s National Institute for Health and Clinical Excellence did not approve this drug for use on the
National Health Service (NHS), because at a cost of over £11,000 this was “not compatible with the best
use of NHS resources.”
Personalized techniques are not always superior to basic clinical approaches. One case in point
is warfarin, a drug that has a different effect on different patients and requires different doses
to produce an acceptable international normalized ratio (INR). Genetic variants in VKORC1, the
gene that codes for warfarin’s target (vitamin K epoxide reductase), and variants in CYP2C9, the
gene coding for the enzyme that is principally responsible for warfarin metabolism, are associated
with increased sensitivity to warfarin [17]. The European Pharmacogenetics of Anti-Coagulant
Therapy (EU-PACT) trial concluded that patients who initially received pharmacogenetic-based
warfarin dosing were more likely to be in the therapeutic INR range compared to patients who
initially received standard warfarin dosing [18]. In contrast, however, the Clarification of Optimal
Anticoagulation through Genetics (COAG) trial concluded that genotype-guided warfarin dosing did
not improve anticoagulation control compared to a non-genotype-based dosing algorithm containing
other clinical variables, and was associated with less time in the therapeutic INR range among
African-American patients [19].
When drugs are used for certain biomarkers, resistance often emerges, as the drug is targeted to a
genetic mutation and not directed to the different metabolomic pathways.
There is little audit of the results of treatment in personalized medicine. In order to have adequate
feedback, it is necessary to have a trial that is restricted to patients with the same condition, biomarker,
or genetic mutation. This was shown with gefitinib in non-small cell lung cancers, as described
above. Ordinary meta-analysis for gefitinib in non-small cell lung cancer showed no effect. When
restricted to patients with mutations in the EFGR gene, gefitinib was found to be efficacious. In other
words, personalized and evidence-based approaches were both necessary to define the patients and
show efficacy. It is this author’s opinion that evidence-based and personalized medicine should be
complementary.
Biomarkers in Recurrent Pregnancy Loss

Unfortunately, most trials in RPL have only used the evidence-based approach and have not sought
biomarkers or a personalized approach. Most trials have tried one form of treatment on a heterogeneous
population with either two or more or three or more pregnancy losses, with no restriction for different
diagnostic criteria whatsoever. This “one size fits all” approach has created an illusion of futility, and
that no treatment modality has any effect. Hence patients are advised to try another conception with no
treatment, or empirical treatment given on a compassionate basis. However, there are some hints as to
which biomarkers may be effective in directing treatment.
Uterine Anomalies
Uterine anomalies have been described as a cause of RPL, and Chapter 12 gives a full account of uterine
anomalies and RPL. The treatment of the uterine septum is septotomy, usually performed by hysteroscopy.
In other words, the uterine septum is a biomarker indicating the need for septotomy. Using these criteria,
Ogasawara et al. [21] have reported a 20% benefit after septotomy. If fundal incision had been used in all
patients with RPL there would have been no benefit. The same authors also used the bicornuate uterus as
a biomarker for uterine surgery and found no benefit. Hence, the bicornuate uterus may not be a biomarker
for uterine surgery.
Pregestational Testing for Aneuploidy (PGT-A)

Pregestational testing for aneuploidy (PGT-A), formerly known as PGS, is a controversial procedure, as the
debate in this book shows. At present, there is only one comparative study in the literature, a retrospective
cohort study of 300 RPL patients by Murugappan et al. [22]. In this study, neither the pregnancy rate per
attempt, biochemical pregnancy rate, ectopic pregnancy rate, live birth rate, nor the clinical miscarriage
rate per pregnancy differed significantly between treatment and control groups. However, patients in the
treatment group conceived in a median of 6.5 months while patients in the control group conceived in a
median of 3 months. It is of interest that of a total of 128 transfers after PGT-A, there were 18 miscarriages
of embryos said to be euploid at PGT-A. The problem with Murugappan et al.’s study is that it included all
patients with at least two prior miscarriages between 6 and 20 weeks’ gestational age.
The author [23] has previously published clinical criteria for selecting patients for PGT-A. These criteria
include repeat fetal aneuploidy, fetal aneuploidy in the presence of parental karyotypic aberrations, and the
older age groups (if there are enough embryos to biopsy). We believe that these criteria, although published
in 2004, are still valid. In other words, fetal aneuploidy is the biomarker for using PGT-A. Today, when
complete chromosome analysis is performed on the products of conception by molecular techniques, it
is much easier to determine fetal aneuploidy than the previously used karyotyping techniques. However,
there is no trial of PGT-A that is restricted to previous or suspected embryonic aneuploidy.
Murugappan et al.’s [22] study does provide a hint, because when women in the 25–35 age group were
considered, the control group tended to better than the PGT-A group in terms of subsequent live births, but
in the 35–39 and 40–45 age groups, where aneuploidy is suspected to be greater than in the younger age
groups, the patients with PGT-A had more live births. The figures are not statistically significant for the
older age groups, but non-significance may be due to the small number of patients between ages 35–45.
Anticoagulants
Heparins have been used in a number of trials in order to assess their effects in RPL. Clark et al. [7]
compared heparin and aspirin to surveillance alone. Two studies have compared anticoagulants to
placebo [24,25] and two have compared enoxaparin to aspirin [26,27]. Not one has found a beneficial
effect. However, when a specific biomarker is used to restrict treatment to specific patients, a different
picture emerges. In patients with antiphospholipid (aPL) syndrome, meta-analyses [28,29] have shown
the beneficial effect of heparin in APS. In other terms, aPLs are the biomarkers of APS and indicate the
need for treatment by heparins. However, even aPLs might not be specific enough as biomarkers. The
control arm of several trials has shown that many patients with aPL have successful pregnancies without
treatment. In APS, an additional trigger is necessary in order to develop the syndrome. This second trigger
is as yet unknown, but may be a better marker than aPL.
Hereditary thrombophilia has also been used as a biomarker for testing the use of heparins. However,
no beneficial effect has been reported [30]. Hence, either hereditary thrombophilias have no effect on
RPL or a different treatment may be indicated. It is more likely that hereditary thrombophilias are only
related to late pregnancy losses [31] and that treatment with anticoagulants may only be appropriate for
women with late pregnancy losses and hereditary thrombophilias.
Aspirin is often used to prevent pregnancy loss. Indeed, aspirin has numerous effects which may
prevent pregnancy loss. Aspirin exerts its pharmacological effects by irreversibly acetylating a serine
Aspirin Control Weight OR

Study Births/Total Births/Total (%) with 95% CI
Tulppala et al. 1997 1/6 1/6 11.70% |||| 1 (0.048 to 20.8294)

Pattison et al. 2000 16/20 17/20 47.75% |||||||||||||||| 0.7059 (0.1362 to 3.6582)
Cowchock & Reece 1997 10/11 8/8 11.83% |||| 0.4118 (0.0148 to 11.4571)
Kahwa et al. 2006 * 28/28 20/20 * * * (*)
Del Ross et al. 2013 83/86 45/46 28.72% |||||||| 0.6148 (0.0621 to 6.0838)
META-ANALYSIS: 138/151 91/100 100% ||||||||||||||||||||||||||||| 0.6307 (0.1922 to 2.0697)
0.01 0.1 1 10 100

OR (log scale)
FIGURE 3.1 Meta-analysis of aspirin and live birth rate in APS. (Adapted from Empson M et al. Obstet Gynecol.
2002;99:135–44 [37]; Amengual O et al. Lupus. 2015;24:1135–42 [38].)
residue in the cyclooxygenase site of prostaglandin-H2-synthetases. It thus reduces the number of TH-17
cells [32], may inhibit Th-1 cytokines such as TNF-α [33], and increase Treg cells [34]. However, aspirin
has not been found to have an effect in unexplained RPL [35,36]. Even in APS, aspirin has no beneficial
effect in reducing pregnancy loss, as reported by two meta-analyses [37,38] summarizing five papers
(Figure 3.1). Although aspirin is widely used in RPL, there is no supporting evidence for its use, and no
biomarker has been identified.
Immunotherapy
The various types of immunotherapy are described in Chapters 29–31 and the arguments against using
immunotherapy in Chapter 32. The argument against immunotherapy is that there is insufficient evidence
of effect. This argument has been used in the leading guidelines, including the ASRM, RCOG, and ESHRE
guidelines [39–41]. However, all trials of immunotherapy have tested treatment on unselected patients
with two or more or three or more miscarriages. None have excluded patients with fetal aneuploidy, which
could have confounded all the results. However, even after excluding fetal aneuploidy, it is still not clear
how immunologically mediated pregnancy losses should be assessed. Chapter 13 describes some of the
markers that may indicate the need for immunotherapy. However, most of the biomarkers previously
suggested, including HLA antigen sharing, mixed lymphocyte reactivity, anti-paternal complement
dependent antibody, and natural killer (NK) cell levels and activity, have not proved entirely satisfactory.
The biomarkers used today include Treg cells and TH-1/TH-2 ratios. Whether these biomarkers will be
effective remains to be seen. The most widely used biomarker for immunotherapy is NK cell levels or
activity. However, to date, no trial has been performed that is restricted to NK levels or activity.
Progestogens
Progestogens are probably the most widely used agents that attempt to prevent pregnancy loss. Opinion is
divided on their efficacy. There is a debate in this book as to the efficacy of progestogens (Chapter 22). As
with the agents described above, progestogens have been used in trials in all patients with RPL without
selection. No trial excluded patients with embryonic aneuploidy. Therefore all are heavily confounded.
Meta-analyses have reported progestogens to significantly lower the number of pregnancies ending in
miscarriage [42,43]. However, the need for progestogens is controversial, and there is little information
on selection of patients. The serum progesterone level has been used as a biomarker for the need for
progestogen supplementation [44,45], but using serum progesterone levels is problematic. Progesterone
secretion is pulsatile. Blood may be drawn at a pulse peak or nadir. Hormone levels may be normal but
there may be deficiency of progesterone receptors. In addition, an aneuploidy embryo may produce low
hCG levels subsequently leading to low progesterone levels. Low progesterone may be the mechanism
rather than the cause of miscarriage.
The pregnancy-induced blocking factor (PIBF) was long thought to be a possible biomarker. PIBF
is a Th-2 cytokine produced by T-lymphocytes when treated with progesterone. Production rises with
trophoblast invasion [46]. PIBF blocks NK cell cytotoxic activity [47]. PIBF increases the production
of IL10, IL3, and IL4 [48] and mediates progesterone-induced suppression of decidual lymphocyte
cytotoxicity [49]. PIBF has been shown to be lower in women with subsequent miscarriage [44], and
leukocyte immunization has been shown to cause an increase in PIBF [50]. However, despite PIBF
being described almost 30 years ago, it has not become a standard test in clinical practice and remains
confined to research in university laboratories. Hence, there is no clinical study assessing progesterone
supplementation using PIBF as a biomarker. Such a study is sorely needed.
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4
The Genetics of Spontaneous Abortions
Joe Leigh Simpson
Introduction
Genetic factors are the most common causes of spontaneous miscarriage. Half of first trimester clinical
miscarriages show numerical chromosomal abnormalities. Pregnancy loss is also associated with single-
gene mutations, but at present far less is known concerning the role single genes play in spontaneous
miscarriages. In this chapter, we therefore focus on frequency and the most common genetic causes
of sporadic and recurrent miscarriages—chromosomal abnormalities. We shall not consider the role
Mendelian genes play indirectly, often categorized incorrectly into “non-genetic” causes, examples being
heritable thrombophilias. Not considered also for lack of data are chromosomal microdeletions and
microduplications. Overall, perturbations of chromosomes are the dominant causes of miscarriages.
Laboratory Methods in Evaluating Miscarriages

Cytogenomic Testing for Increased Sensitivity in Distinguishing
Aneuploid from Euploid Embryos
By the 1970s, the gold standard for cytogenetic testing became the G-banded karyotype, which could
diagnose structural abnormalities greater than 5–10 Mb (million base pairs). The standard G-banded
karyotype remains invaluable in identifying a balanced translocation in a couple with recurrent pregnancy
loss. However, newer methods have evolved. Today, the American College of Obstetrics and Gynecology
(ACOG) and other organizations recommend that women who desire an invasive prenatal diagnostic
test be offered chromosomal microarrays (CMAs) because this provides more comprehensive analysis
of the entire genome at a finer resolution than a routine karyotype. A National Institute of Child Health
and Human Development (NICHD) prospective prenatal cytogenetic array study was conducted on 4401
women having varying indications for prenatal genetic diagnosis. All fetal trisomies, sex chromosome
abnormalities, and unbalanced translocations were identified by both routine karyotype and CMA [1].
Compared with results from a karyotype alone, clinically significant findings (copy number variants
[CNVs]) were detected in an additional 6% of the 755 fetuses having structural anomalies or growth
abnormalities by ultrasound. The frequency of clinically significant CNVs was even higher (13%) when
two or more organ systems were involved, compared to an isolated anomaly (5.1%). Even if no structural
anomalies were evident, CMA revealed 1.7% more chromosomal abnormalities than karyotype alone. It
is for these reasons that ACOG and the Society for Maternal Fetal Medicine (SMFM) recommend that
CMA replace conventional cytogenetic analysis (karyotypes) in patients undergoing invasive diagnostic
testing for the evaluation of a fetus with one or more structural anomalies [2,3]. The Royal College
of Obstetricians and Gynaecologists (RCOG) and the European Society of Human Reproduction and
Embryology (ESHRE) likewise recommend the status of the miscarriage to be pivotal [4,5].
Of special value in assessing CMAs is that, unlike karyotypes, no cell culture is required. Culture
methods succeed in no more than 60%–70% of attempts to obtain karyotype. Evaluating 532 stillbirths,
the NICHD Stillbirth Collaborative Research Network demonstrated that a much higher success rate
for informative cases was shown for CMAs than with conventional karyotypes (87.4% vs. 70.5%) [6].
30
The Genetics of Spontaneous Abortions 31
This increased informative success of CMA compared to culture for chromosome abnormalities was
accompanied by detection of more chromosomal anomalies (8.3% vs. 5.8%).
A disadvantage of CMA compared with traditional karyotypes is that in some methods CMAs cannot
distinguish between balanced translocations and normal karyotypes.
Cell-Free DNA Analysis to Detect Trisomies in Miscarriages

Noninvasive prenatal testing screening (NIPS) detecting cell free DNA in the maternal circulation is
widely applied in ongoing pregnancies for detecting fetuses with trisomies 13, 18, and 21. This method
can be envisioned as equally applicable in the management of miscarriages. Thus, a maternal blood
sample is capable of providing information concerning whether a miscarriage was aneuploid or euploid.
This molecular approach uses massive parallel sequencing analysis of the fragments of DNA (circulating
free DNA [cfDNA]) present in blood of all individuals—pregnant or not. During pregnancy, maternal
blood contains cell free DNA from multiple maternal organs and fetal tissue mostly generated from the
placenta. NIPS has proved capable of 99.9% detection for trisomy 21, 98% for trisomy 18, and at least
80% for trisomy 13 [7,8]. NIPS, usually performed at 10–12 weeks’ gestation, can be used to screen for
sex chromosome abnormalities in addition to the common trisomies (13, 18, and 21) both in cohorts of
maternal age ≥35 years and in cohorts <35 years.
The circulating cell free DNA alluded to during pregnancy is based on small fragments of cfDNA
(50−200 base pairs). Millions of these fragments can be sequenced simultaneously and then assigned to
a given chromosome region. The number of chromosome 21 transcripts in a sample can be compared to
number expected in a reference DNA sample known to be from or representative of a normal individual.
(Or, if from a pregnant woman, from mother plus fetus.) Pregnancies carrying a trisomy 21 fetus will have
relatively more chromosome 21 counts (transcripts) than a pregnant woman carrying a normal diploid
fetus. The additional chromosome results in a small additional amount of DNA specific for the trisomic
chromosome in maternal plasma; however, this is enough to differentiate a euploid form an aneuploid
pregnancy.
Commercially available NIPS assays currently test only for chromosomes 13, 18, and 21; only these
trisomies are capable of surviving to become live borns. When screening, however, DNA sequences are
unavoidably obtained on all chromosomes. Results from chromosomes other than 13, 18, and 21 are
“filtered” out, i.e., not disclosed. Unmasking would provide information on all chromosomes in products
of conception (miscarriages). In order for NIPS to provide reliable information on miscarriages, 4% or
more of cell free DNA must be of fetal origin. The average fetal fraction at 10–22 weeks’ gestation is 10%,
independent of gestational age and maternal age, race/ethnicity; however, fetal fraction is lower below 10
weeks. Fortunately, analytical validity should be robust in assessing miscarriages because greater than
4% fetal DNA should exist in maternal blood when a woman is experiencing a miscarriage. Placentas
undergoing apoptosis send considerable cell free DNA into the maternal circulation; thus, chromosomal
status of a miscarriage should be able to be determined readily [9]. This is also possible weeks after fetal
or embryonic demise because placental apoptosis continues.
In conclusion, cfDNA analysis to determine chromosomal status of a miscarriage is under investigation
and yielding good results. Cell-free DNA for this purpose will soon become standard to determine
chromosomal status of every miscarriage.
Chromosomal Abnormalities in Preimplantation Embryos

A high frequency of preimplantation losses in early pregnancy has long been known [10]. Of
morphologically normal embryos, 50%–80% show numerical chromosomal abnormalities (aneuploidy
or polyploidy). Aneuploidy rates increase with advancing maternal age [11,12], reaching 85%–100% in
embryos of women aged 45 years and above. These data are consistent with 6% aneuploidy rates in sperm
from ostensibly normal males [13] and with 20% aneuploidy rates in oocytes [14]. Aneuploidy rates of
this magnitude constituted the original rationale for preimplantation genetic testing, to identify euploid
embryos suitable to transfer.
Not surprisingly, chromosomal abnormalities are more frequent in morphologically abnormal embryos.
Using the only technology previously available (FISH) and testing with 5–7 chromosome-specific
fluorescent probes, abnormality rates of 75% were observed in morphologically abnormal cleavage stage
3-day embryos. Contemporary studies of a morphologically abnormal cohort of embryos subjected to
24-chromosome array comparative genomic hybridization (CGH) or next-generation sequencing (NGS)
have not been reported, but it would be a surprise if more than a small percentage of these embryos were
euploid.
Some 8%–10% of morphologically normal blastocysts assessed by NGS show aneuploid mosaicism
[15,16]. Another 10% show segmental (duplication/deficiency) mosaicism. Mosaic aneuploid embryos
can yield viable pregnancies if transferred, but at much lower rates than euploid embryos. The predictive
value of an array CGH showing an aneuploid embryo is 4% versus 42% for a euploid embryo [17]. In the
study cited, embryo transfer occurred without knowledge of chromosomal status. Of relevance is that the
technology used was array CGH, not the more sensitive NGS; thus, some of the 4% “aneuploidy” could
actually have had aneuploid mosaicism with an unappreciated normal cell line. A “diagnosis” of non-
mosaic embryology is best explained by site of biopsy. The biopsy involves trophectoderm, whereas it is
the inner cell mass (ICM) that differentiates into the embryo.
Irrespective of complexities in diagnosis, transferring only “euploid” embryos increases the pregnancy
rate by 15%–20% [18–20]. The greatest benefit occurs at maternal age 35–40 years [20,21], and probably
in recurrent aneuploid miscarriages. Likewise, younger patients undergoing preimplantation genetic
testing (PGT) for monogenic disorders (PGT-M) who also undergo aneuploidy testing show a 15%–20%
benefit.
Chromosomal Abnormalities in Clinically Recognized Spontaneous Abortion

Frequency at Different Gestational Ages
Beyond the preclinical (preimplantation) stage, 10%–15% of surviving embryos undergo clinical
pregnancy loss later in gestation. At least 50% show chromosomal abnormalities and in maternal age
>35–40 years over 70% [22,23]. Because it is problematic to recover and analyze expelled miscarriages,
reliable quantification first benefited from chorionic villus sampling (CVS), which could be performed
soon after ultrasound diagnosis of fetal demise; 75%–90% of villi (representing products of conception)
were aneuploid [24,25]. Contemporary studies utilizing chromosomal microarrays on products of
conception are consistent or show higher rates. In 2004, Schaeffer et al. [26] reported 41 abortuses
previously analyzed by karyotype and diagnosed as normal; array CGH revealed heretofore unrecognized
abnormalities in 4 of 41 cases. Coupled with data on abortuses recognized as deceased at the time of
a scheduled CVS, the frequency of chromosomal abnormalities in women age 35 or above is at least
60%–75%. Similar findings were found by Sahoo et al. [27]
In the second trimester, chromosomal abnormalities are less frequent. A pitfall in determining
prevalence is that fetal demise precedes spontaneous expulsion of the products of conception by several
weeks. This can be deduced because the loss rate after 8 weeks [28] is only 3%, whereas the frequency
of first trimester miscarriage (1–13 weeks) is 10%–12%. By analogy, some miscarriages “diagnosed”
in the second trimester probably underwent demise in the late first trimester but were retained in utero
for several weeks. Second trimester losses can be accepted as such only if they occur following proven
viability until at least 10–12 gestational weeks, or preferably at 13 weeks or more. In second trimester
losses, the incidence of chromosomal aberrations is similar to that in live-born infants having applicable
trisomies (13, 18, and 21), monosomy X, or sex chromosome polysomies. The frequency of anomalies in
second trimester losses is estimated to be approximately 15%.
In the third trimester (stillborn infants), the frequency of chromosomal abnormalities is less, traditionally
considered to approximate 5% based on karyotypes. Using array CGH, the rate is higher [2]. Irrespective,
the frequency of chromosomal abnormalities in stillborns is obviously higher than the 0.6% incidence in
live borns in the general population.
Spectrum of Chromosomal Abnormalities

Autosomal Trisomy
Autosomal trisomies comprise approximately 50% of cytogenetically abnormal spontaneous abortions,
or 25% of all abortuses. Trisomy for every chromosome has been observed. Among those of most clinical
relevance, the frequencies are numbers 16, 22, 21, 15, 13, and 14 in that descending order. Trisomy 16
is the most common in miscarriages, but rarely, if ever, is observed in live borns in non-mosaic form.
A morphological correlation between placenta and embryo for any given trisomy is imprecise.
Confounders include nonspecific villous changes occurring following fetal demise but before expulsion.
Although low predictive value exists when placental histology is used to differentiate aneuploidy from
euploidy, a few correlations are valid [29,30]. Fetuses with trisomies incompatible with life grow more
slowly than those with trisomies compatible with life (e.g., trisomies 13, 18, 21). In one series, the
crown−rump length for the latter was 20.65 mm, compared with only 10.66 mm for the former [30]. The
explanation could be that fetuses with nonlethal trisomies survive longer than those with lethal trisomies,
or fetuses with lethal trisomies exhibit greater intrauterine growth retardation, or both. Abortuses from
nonlethal trisomies (13, 18, and 21) tend to manifest anomalies similar to with those found in full-term
live-born trisomic infants. These are more recognizable in the second trimester and beyond.
A maternal age effect is evident in most trisomes, but the relative effect varies among chromosomes.
Maternal age correlates positively with errors at meiosis I, the assumed cytological explanation for most
autosomal trisomies. The relative proportion of trisomies arising at meiosis I versus those arising at
meiosis II varies among aneuploidies. Virtually all trisomy 16 cases are maternal in origin, originating in
meiosis I [31]. In trisomies 13 and 21, 90% are maternal, again usually arising at meiosis I. The exception
is trisomy 18; two-thirds of the 90% of maternal origin cases originate at meiosis II [32,33]. These data
would benefit from contemporary studies because not taken into account is the recent recognition by
Kuliev et al. [34] that premature chromatid separation is common during polar body meiosis. Chromatid
correction of meiosis I errors may occur in meiosis II. Thus, the proportion of meiosis I errors was only
marginally higher (41.7% vs. 35.2) in oogenesis than meiosis II errors; errors in both meiosis I and II can
also occur.
Numerical chromosomal abnormalities correlate with advanced maternal age. The explanation is
considered to be decreased or absent meiotic recombination. Recombination obligatorily occurs between
homologous chromosomes [31–33,35–39], assuring physical proximity between homologous chromosomes
if assured until orderly separation (disjunction) that results in two equivalent haploid products. Oocytes
ovulated earlier in a woman’s reproductive lifespan are believed more likely to have undergone sufficiently
robust recombination to render the oocyte/embryo less predisposed to nondisjunction. Again, however,
premature chromatid separation in oocytes is an alternate explanation for aneuploidy not usually
considered [34].
Errors in paternal meiosis account for only 10% of trisomies that involve acrocentric chromosomes
(13, 14, 15, 21, and 22). In non-acrocentric trisomies, paternal meiotic errors are equally likely to arise
at meiosis I or II [39]. Paternal meiotic errors account for 10% of trisomy 21 cases, and some cases of
trisomy 2 [39]. Paternal contribution is uncommon in other trisomies.
Double Trisomy
Double trisomies occur in 1%–2% of all abortuses [40–42]. This frequency is higher than expected by
chance. Among 517 abortuses in one study, double trisomies were found in 2.2% of 321 successfully
karyotyped abortuses [41]. Chromosomes involved in double trisomy most commonly are the X
chromosome and autosomes 21, 18, 16, 22, 13, 2, 15 in descending order. Diego-Alvarez et al. [41]
tabulated the exact combination of 178 reported double trisomies. Advanced parental ages were a
striking feature—39.7 ± 3.4 maternal; 43.4 ± 8.7 paternal. Of the seven cases analyzed, four originated
in maternal meiosis; the origin could not be determined in the other three. In the series by Reddy et al.
[40], gestational age in double trisomy miscarriages was 8.7 ± 2.2 weeks compared to 10.1 ± 2.9 weeks
for a single trisomy. The mean maternal age was 35.9 ± 5.3 years. The sex ratio was approximately equal.
Morphological examination of double trisomy in miscarriages usually shows only an empty sac;
an embryo of normal morphology is uncommon. In one study, five of seven double trisomies showed
no morphological details [42]; one other was an embryonic and the remaining embryo 48,XXX + 18
showing only hydropic changes.
Autosomal Monosomy
Autosomal monosomy is usually lethal prior to or just beyond implantation. However, 56 cases of live-
born autosomal mosaic monosomies (e.g., 46,XX, 45,XX, 21) have been reported [43]. Most involve
smaller chromosomes (e.g., 21 or 22). This once arcane observation has taken on recent importance given
NGS not infrequently revealing mosaic aneuploidy. As discussed earlier, mosaicism occurs in 5%–10% of
embryos. If nonetheless transferred, pregnancies can result in normal euploid (non-mosaic in lymphocytes)
offspring. Possible explanations include the uncommon monosomic cell having been fortuitously removed
during biopsy, selection against aneuploid cells, or monosomy never having been present in the inner
cell mass. DNA levels connoting monosomy or trisomy of less than 20% are considered within normal
(background-noise) range, whereas greater than 80% are aneuploidy. DNA aneuploidies in the 20%–80%
range are topics of ongoing discussion, with some data indicating that up to 40% non-model DNA could
be an equally acceptable threshold for a satisfactory outcome if a mosaic embryo is chosen for transfer.
Triploidy
In triploidy, three haploid chromosomal complements exist. An association exists between diandric
triploidy (two paternal haploid complements) and a diploid hydatidiform mole [44–47]. Triploidy is often
characterized as a “partial mole,” molar tissue and fetal parts coexisting. Partial (triploid) moles are
distinguishable from the more common “complete” hydatidiform moles. The latter are always 46,XX,
exclusively androgenetic, and exclusively consisting of villous tissue.
Placental findings in diandric triploid placentas include a disproportionately large gestational sac, focal
hydropic degeneration of placental villi, and trophoblast hyperplasia. Placental hydropic changes are
progressive and hence difficult to identify early in pregnancy. Irrespective of chromosomal status, placental
villi undergo nonspecific hydropic degeneration following fetal demise. This has made correlations between
histological and cytogenetic findings difficult. There appears to be no apparent correlation between
embryonic morphology and parental origin (diandry or digyny) [46]. Malformations recognized commonly
in triploid abortuses include neural tube defects and omphaloceles, both anomalies also occurring in
triploid conceptuses surviving to term. Facial dysmorphia and limb abnormalities have been reported [48].
Triploid abortuses are usually 69,XXY or 69,XXX. The origin has been presumed to be due to dispermy
[49], following either fertilization by two haploid sperm or fertilization by a single diploid sperm.
Tetraploidy
Tetraploidy (4n = 92) is less common than triploidy and rarely progresses beyond 2−3 weeks of embryonic
life. Tetraploidy in embryonic tissue should be distinguished from the not uncommon, and clinically
insignificant, tetraploid cells found in amniotic fluid. Their basis is multinucleated syncytiotrophoblasts.
Live-born tetraploidy exists but is rare [50] and probably always actually reflects diploid/tetraploid
mosaicism. Origin is probably failure of cytokinesis, as shown in molecular studies and in sync with
origin of 92,XXXX and 92,XXYY [51,52].
X Chromosome Monosomy
Monosomy X accounts for 15%–20% of chromosomally abnormal miscarriages. Early monosomy X
abortuses usually consist of only an umbilical cord stump. If a 45,X embryo survives until later in gestation,
anomalies characteristic of Turner syndrome may be manifested [53]. These include cystic hygromas,
generalized edema, and cardiac defects. Unlike most live-born 45,X infants, abortuses show ovarian germ
cells. Approximately 80% of live-born monosomy X is the result of paternal sex chromosome loss [54].
Sex Chromosomal Polysomy (X or Y)

47,XXY and 47,XYY each occur in about 1 per 800 live-born male births. 47,XXX occurs in 1 per 800
female births. X or Y polysomies are only slightly (10%) more common in abortuses than in live borns
[55]. Thus, there is little additional lethality, and that occurring probably is due to increased anomalies
(e.g., cardiac).
Recurrent Aneuploidy and Its Clinical Consequence

The Concept of Recurrent Aneuploidy
In recurrent miscarriages, aneuploidy in successive pregnancies occurs more often than expected by
chance [56,57]. Chromosomal complements in a given kindred are more likely to be either recurrently
euploid or recurrently aneuploid (Table 4.1). If the complement of the first abortus is abnormal, the
likelihood is increased that the complement of the second abortus will also likely be abnormal [57].
Similarly, frequency of aneuploid embryos in a PGT-A cohort is higher if a prior cycle subjected to
trophectoderm biopsy showed a greater proportion of aneuploid embryos, compared to a cohort that
showed a greater proportion of euploid embryos [58]. Rubio observed the same in a sample of women
undergoing cleavage stage PGT-A for recurrent miscarriages [59].
Recurrence usually involves trisomy, an observation having major ramifications for clinical management
in subsequent pregnancies. The first in a sequence of miscarriages may have involved a lethal chromosome
(e.g., No. 12), whereas a subsequent miscarriage may involve a trisomy that is potentially live-born
trisomy (e.g., No. 21). Thus, prenatal genetic diagnosis should be offered. If no information exists on
chromosome status of miscarriages in a couple having multiple clinical losses, Bianco et al. [60] provide
useful risks of an aneuploid miscarriage based on number of prior miscarriages and maternal age.
The biological basis of recurrent aneuploidy is not known, but multiple plausible explanations exist. The
possibility that the non-random distributions of aneuploid miscarriages in sequential pregnancies merely
reflects increasing maternal age was once considered a potential explanation by Warburton et al. [56], but
a later publication by the same group did not support this [57]. However, autosomal recessive genes can
perturb meiosis and result in many monogenic disorders. Heterozygotes for some disorders may show
increased rates of cancer (e.g., ataxia telangiectasia). Defects in DNA repair, chromosomal synapsis, and
homologous recombination are among processes that disrupt chromosomal disjunction. Their perturbation
would be expected to result in chromosomal abnormalities that manifest as miscarriages, often recurrent
but not obligatorily consecutive.
Recurrent Aneuploidy with Higher-Order Losses

Miscarriages in higher-order losses ostensibly may be more likely to be cytogenetically normal
[61]. So-called maternal factors (sometimes erroneously termed “nongenetic”) could be more likely
TABLE 4.1
Recurrent Aneuploidy Relationship between Karyotypes of Successive Abortuses
Complement of Second Abortus
Complement of First De novo
Abortus Normal Trisomy Monosomy Triploidy Tetraploidy Rearrangement
Normal 142 18 5 7 3 2
Trisomy 33 30 1 4 3 1
Monosomy X 7 5 3 3 0 0
Triploidy 7 4 1 4 0 0
Tetraploidy 3 1 0 2 0 0
De novo rearrangement 3 0 0 0 0 1
Source: Data from Warburton D. et al. Am J Hum Genet. 1987;41(3):465–83 [56].
explanations, especially when numbers of losses exceed four. Consecutive higher-order recurrent
miscarriages would also be expected to be associated with a deleterious maternal environment; genetic
segregation in a couple at risk for an autosomal recessive disorder would not often be expected to result
in consecutively affected pregnancies. The likelihood is 1/4 × 1/4 × 1/4 = 1/64. One caveat to this
conclusion is that gestational ages in a higher-order group are often higher, reflecting age increasing
with increasing number of losses. Second trimester losses are more likely to have uterine abnormalities;
thus, fewer aneuploid losses would be expected. Carp et al. [62] found that among women having three
or more abortuses, the overall likelihood that the abortus would have an abnormal karyotype was 29%.
If the abortus was aneuploid, the likelihood of a subsequent live birth was 68% (13 of 19). If the abortus
was euploid, the subsequent live birth rate was 41% (16 of 39). However, inclusion criteria in the study
of Carp et al. [62] extended to 20 weeks’ gestation greatly decreases the likelihood that aneuploidy is
observed. In conclusion, low aneuploidy rates reflect not strictly high-order losses but higher gestational
age of such a cohort.
Clinical Management of Recurrent Aneuploidy

Clinical management of recurrent miscarriage should logically be stratified on the basis of prior aneuploidy.
An algorithm based on euploid versus aneuploid status is evolving to become standard in management of
recurrent miscarriage. If the abortus proves aneuploid, prenatal genetic diagnosis in future pregnancies
is indicated because the risk for aneuploid offspring is increased over background maternal age.
There is concern that couples experiencing recurrent aneuploidy are at increased risk for an aneuploid
live born. The chromosome involved is likely to be different from that in a previous pregnancy. The
(different) trisomy may not be lethal as that occurring in the first trimester but compatible with life.
Live-born trisomy 21 following an aneuploid miscarriage carries a risk of about 1% [63]. Based on first
trimester screening results (maternal serum analytes, ultrasound), Snijders and Nicolaides [64] reported
a recurrence rate of 0.7% following trisomy 21 and coincidentally also 0.7% following trisomy 18. Both
rates are higher than age-related background.
Bianco et al. [58] have published the outcome of subsequent pregnancies after a prior abortus of
unknown karyotype. If no information is available on the chromosomal status of the abortus, calculations
based on Table 4.2 can be made. For example, if the a priori maternal age of trisomy 21 risk were 1 in
300, one calculates that risk of aneuploidy after 3 miscarriages for a woman older than 35 years would
be 1/300 × 1.68 or 1 in 179.
TABLE 4.2
Risk of Aneuploidy by Number of Prior Miscarriages Stratified by Maternal Age
No. of Prior Spontaneous Abortions Adjusted OR for Trisomy 13, 18, 21a Adjusted OR for All Aneuploidies
A. Maternal Age <35 years

0 1.00 1.00
1 1.27 (0.74–2.08) 1.19 (0.78–1.84)
2 1.31 (0.80–2.13) 1.21 (0.94–1.58)
≥3 1.36 (0.46–2.73) 1.41 (0.56–3.19)
B. Maternal Age >35 years

0 1.00 1.00
1 1.23 (1.04–1.52) 1.23 (1.00–1.52)
2 1.34 (1.01–1.82) 1.30 (0.99–1.74)
≥3 1.56 (1.03–2.31) 1.68 (1.12–2.52)
Source: Data from Bianco K et al. Obstet Gynecol. 2006;107(5):1098–102 [60].

Note: Comparison is with women with no spontaneous abortions, controlling for parity and indications for prenatal
diagnosis.
a OR, odds ratio; 95% confidence intervals.
Structural Chromosomal Rearrangements

The presence of a balanced reciprocal rearrangement in one parent can result in an unbalanced
translocation in offspring. Phenotype depends on the specific duplicated or deficient chromosomal
segments. Likelihood of a successful pregnancy in a given cycle is decreased because many embryos
are unbalanced, not capable of resulting in a viable pregnancy. Table 4.3 shows results of a non-atypical
preimplantation genetic testing sample.
In Robertsonian translocations, two acrocentric chromosomes undergo centric fusion; chromosome
number is reduced to 45. Ribosomal genes present on the miniscule short arm do not express unique
sequences. Acrocentric short arms code for ribosomal DNA and show DNA redundancy with other
acrocentric chromosomes.
Frequency Management of Translocation Heterozygotes

A balanced translocation is found in 1%–5% of couples experiencing repeated losses; the mean is
approximately 2% [61,65–71]. The prevalence of balanced translocations is higher in females than
males, and higher still if there is a family history of a stillborn or abnormal live born. Translocation
heterozygosity is not correlated with maternal age or the number of previous miscarriages. Tabulations
by Simpson et al. [69,70] provided rates in females of a balanced translocation after two, three, four, and
five losses, 0.8%, 1.7%, 2.3%, and 2.9%, respectively. For males, the rates were 1.2%, 1.9%, 2.4%, and
0 (0/39). Goddijn et al. [71] found that the odds ratios after two, three, and four losses to be 1.4 (95% CI
0.4–4.8), 2.2 (12.5), and 2.1 (0.3–15.4), respectively.
Likelihood of Abnormal Live Borns Resulting from a Balanced Translocation

Detection of a balanced translocation traditionally has led to a recommendation for prenatal genetic
diagnosis. Most guidelines recommend parental karyotypes on all couples [3], but guidelines from
RCOG and ESHRE differ [4,5]. RCOG and ESHRE reason that the likelihood of detecting a balanced
translocation is low overall (2%) and the likelihood of abnormal live-born outcome lower yet; thus,
TABLE 4.3
Chromosomal Complements (Next-Generation Sequencing) in a
Cohort of Embryos from a Couple Undergoing PGT-STR Because of
a Balanced Reciprocal Translocation t(9;16) in One Partner
Embryo Results (NGS) Diagnosis
1 46, XY, der(16)t(9;16) Unbalanced
2 45, XX, -7, der(16)t(9;16) Unbalanced/Aneuploid
3 46, XX, der(9)t(9;16) Unbalanced
4 47, XX, +7, der(16)t(9;16) Unbalanced/Aneuploid
5 46, XY,+9, -16 Unbalanced
8 46, XX/ngs(1–22,X)x2 Normal or balanced female
Source: Data from Svetlana Rechitsky, PhD, Reproductive Genetic Innovation
(RGI), Northbrook, Illinois.
Note: Only 1 of the 11 embryos was suitable for transfer. Using next-generation
sequencing, it was not possible to determine if embryo 8 had a balanced
translocation or was a genetically normal embryo without translocation.
TABLE 4.4
Determining Priority for Obtaining Parental Karyotypes in a Couple Experiencing
Recurrent Miscarriages
Prior Miscarriages
Presence or Absence of Sib
Maternal Age at Second Miscarriage ≥3 2 Having Miscarriage (+ or −)
<23 years 10.2 7.3 +

5.7 4.0 −
23–33 years 10.0 7.2 +
5.7 4.0 −
34–36 years 5.8 4.1 +
3.2 2.2 −
37–38 years 4.0 2.8 +
2.2 1.5 −
≥39 years 1.8 1.2 +
1.0 0.7 −
Source: Modified from Franssen et al. BMJ 2005;331:137–41 [72].
Note: Probability of a balanced translocation being present depends on maternal age, number of
prior miscarriages, and presence (+) or absence (−) of a sib having had miscarriages.
parental karyotypes may not be cost effective. Franssen et al. [72] provide one useful tabulation to
estimate likelihood of a balanced translocation. This is based on maternal age, number of prior losses,
and presence or absence of a sib who had a miscarriage. Likelihood of detecting a translocation was
highest for women under 25 years who experienced multiple losses and who also had a sib experiencing
miscarriages (Table 4.4).
Theoretical versus Empirical Risks for Recurrent Miscarriages or Abnormal Offspring

Rarely do empirical data exist to estimate risks of an adverse outcome for a specific reciprocal
translocation. Even if the same chromosomes are involved, the exact break points are likely to differ.
Generalizations must be made on the basis of pooled data derived from many different translocations.
Importantly clinically, theoretical risks for abnormal offspring are almost always greater than empirical
risks [68–71].
The theoretical risk for a parent carrying a Robertsonian translocation having a live-born child with, for
example, Down syndrome is 33%. For a female carrier, the empirical risk is 10% for t(14q;21q), the most
common Robertsonian translocation. Risk is only 2% for a male carrier with t(14q;21q). Centric fusion
(Robertsonian) translocations involving chromosomes other than chromosome 21 show lower empirical
risks. In t(13q;14q) for example, the risk for live-born trisomy 13 is 1% or less. This lower risk presumably
reflects the lethality of many segregant products (trisomies and monosomies).
Empiric risks are based on type of transfer sex and mode of ascertainment. Recurrence risks based
on sex differences are apparent in Robertsonian translocation but not reciprocal translocation. Empirical
risks are 12% for offspring of either female or male heterozygotes [69]. In reciprocal translocation, pooled
data encompassing all chromosomes show empiric risks of 12% for abnormal offspring, whether carrier
is either female or male [69].
A chromosomal rearrangement also confers deletion effects beyond the two or more chromosomes
involved. Even when normal transmission occurs between chromosomes involved in the translocation,
a separate numerical chromosomal abnormality could arise due to interchromosomal effect perturbing
chromosomal disjunction. Additionally, the mode of ascertainment is important in counseling couples
with a balanced translocation. The frequency of unbalanced offspring is lower if a parental balanced
translocation was ascertained through repetitive miscarriages (3%) rather than through anomalous live
borns (20%). There is a greater likelihood for severely unbalanced products in the latter.
Cumulative Likelihood of Live Births in Translocation Heterozygotes

It is clinically useful to stratify the likelihood of achieving pregnancy lifelong into a 1- or 5-year likelihood.
The cumulative likelihood of pregnancy in a couple having a translocation is 65%–70%, not different from
the live birth rate observed in the general population [71,73,74]. In the cohort of 1324 couples of Goddijn
et al. [68], 41 couples had a structural rearrangement. Among 43 pregnancies of 25 couples desiring
subsequent offspring, 70% had a healthy child and 26% a miscarriage. In the reciprocal translocation
cohort of Stephenson and Sierra [71] (N = 28), 35 pregnancies occurring after ascertainment resulted in
22 (63%) live births. In the Robertsonian translocations group (N = 12), 9 of 13 pregnancies resulted in
live births (69%). The outcomes cited are comparable to those of couples not having a rearrangement.
A less favorable cumulative prognosis was reported by Sugiura-Ogasawara et al. [73]. Of 1284 couples
with two or more miscarriages, 4.5% had a balanced translocation. Loss rates following ascertainment
were 61% (11/18) for couples in which the male partner had a translocation and 72.4% (21/29) when the
female partner had the translocation. In couples with normal karyotype, the miscarriage rate was 28.3%
(335/1184). Carp et al. [74] reported a 45.2% (33/73) cumulative live birth rate in couples in which a
partner was a translocation heterozygote, compared to 55.3% (325/588) live birth rate in couples without
a translocation. The same group later reported a similar percentage of normal and balanced karyotypes
(74%) in embryos conceived by translocation heterozygotes compared to embryos of couples not having
a translocation (77%) [75].
Preimplantation Genetic Testing for Structural Rearrangement (PGT-SR)

Although couples having a balanced translocation cumulatively achieve pregnancy in percentages
equivalent to that in the general population (60%–70%), perturbation in meiotic segregation unavoidably
results in a delay because of the frequency of embryos being imbalanced; typically 40%–60% (see Table
4.3). The clinical consequence is increased time to achieve pregnancy. In older women, the 5- to 6-year
mean delay until pregnancy could preclude live borns. Consequently, PGT-SR can be recommended to
identify and transfer the (few) balanced or genetically normal embryos [76].
A special circumstance occurs when a translocation involves homologous acrocentric chromosomes
(e.g., t(13q;13q) or t(21q;21q)). A normal live-born infant is theoretically precluded. If the father carries
a homologous structural rearrangement, donor sperm may be appropriate. If the mother carries the
rearrangement, donor oocytes or donor embryos should be discussed.
Chromosomal Inversions
There are two types of inversions. In pericentric inversions, breaks occur in both arms. In paracentric
inversions, the two breaks occur in the same arm. The frequency of inversions in couples having repetitive
miscarriages is less than 1% but would be detected by karyotype, as would balanced translocation.
Stephenson and Sierra [71] detected 7 inversions among 1893 couples (0.37%). Goddijn et al. [68] reported
9 inversions among 1324 couples. Typical array CGH or NGS platforms cannot identify an inversion
because DNA content is unchanged, analogous to a balanced translocation.
If heterozygous for an inversion, crossing over within the inverted segment may lead to duplication for
some regions and deficiencies for others [77]. Based on pooled data involving many different chromosomes,
females in one series with a pericentric inversion had a 7% risk of abnormal live borns; males had a 5%
risk [78]. Pericentric inversions ascertained through phenotypically normal probands are less likely to
result in abnormal live infants, presumably reflecting lethality of unbalanced products. The clinical
outcome is somewhat paradoxical in that inversions resulting from a recombinational event involving
only a small portion of the total chromosomal length confers a greater likelihood of lethality [77,78]. The
recombinational products are longer duplications or deficiencies. By contrast, if recombination occurs
in a larger inversion loop (i.e., 30%–60% of the total chromosomal length) embryos are more likely to
survive because deficiencies and duplications involve less DNA. Inversions less than 100 Mb appear
not to exert undue untoward outcomes [78]. Few recombinants are observed when an inversion is less
than 50 Mb (40% of chromosome) in length. Only a few recombinants arise when inversions involved
40%–50% of length chromosome, whereas a much higher proportion occurred when the inversion was
greater than 100 Mb.
Data are limited on recurrence risk involving paracentric inversions. Theoretically, there should be
almost zero risk of unbalanced products of clinical consequence because all paracentric recombinants
should be lethal. However, both abortions and abnormal live borns have surprisingly been reported, even
within the same kindred. A tabulated pooled risk for unbalanced viable offspring was 4% [79].
Single-Gene Causes of Miscarriages

Casual thinking has led some investigators to conclude that those miscarriages that do not show numerical
chromosomal abnormalities must be “nongenetic” in etiology. This deduction is incorrect and in fact
illogical. At birth, Mendelian and polygenic/multifactorial disorders are both more common than
chromosomal abnormalities. Single-gene and polygenic etiologies account for 2% or more of congenital
anomalies, whereas chromosomal abnormalities account for only 0.6%. Mendelian and polygenic/
multifactorial factors are known to play pivotal roles in embryonic mortality, but the topic is difficult to
investigate. Genes required for proper differentiation have only rarely been elucidated.
Determining the role single genes play in miscarriages will require studies in human embryos, not
simply extrapolation based on animal studies. Whole genome or whole exome sequencing will be required.
Two studies illustrate the likelihood of identifying gene perturbation. Studies on miscarriages have been
conducted involving embryoscopy, ultrasound, and cytogenetic testing [80,81]. Structural anomalies are
detected in abortuses having a normal chromosomal complement. This is consistent with genetic etiology,
but etiology different from cytogenetic, normally single gene or polygenic. Philipp and Kalousek [80]
reported that while aneuploid abortuses usually showed one or more external anomalies, so, too, did
many euploid embryos. Feichtinger et al. [81] concluded that only one-fourth of euploid embryos were
morphologically normal.
As the cost of DNA sequencing plummets, whole exome sequencing and even whole genome sequencing
can be expected to be applied to miscarriages. Causative genes will be found. Recall that of the 21,000
human genes, function is known for only one third. Many in the pool of miscarriages of “unknown”
etiology will prove to be due to perturbation of genes pivotal for differentiation and cell lineage.
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5
The Endometrial Factor in Recurrent Pregnancy Loss
Luiza Borges Manna and Ying Cheong
Introduction
The human female reproductive tract undergoes cyclic changes every month aimed at accommodating a
new pregnancy. Whereas most species prepare for pregnancy in response to embryonic signals, humans do
so solely in response to endocrine cues, regardless of the presence of a conceptus [1]. The hallmark of these
cycles is the process of decidualization, during which the endometrial stromal cells undergo a myriad of
morphological and molecular changes that transform them into secretory cells capable of accommodating an
embryo. However, the role of the decidua goes beyond passive receptivity; it not only favors implantation, but
also aids in the selection of embryos that are likely to result in a successful pregnancy. This is of paramount
importance in order to safeguard maternal resources against investment in a pregnancy likely to fail. In
addition, however, the decidua develops a unique immune cell profile in order to protect the semi-allogenic
conceptus from maternal immune responses. The decidua is therefore responsible for finding a balance
between maternal and embryonic competing interests while at the same time ensuring the perpetuation
of human species. This entire process, with its numerous checkpoints that ensure an equilibrium between
endometrial receptivity and selectivity, is carried out under tight spaciotemporal control, with over 3000
genes becoming differentially expressed at different points of the cycle [2]. It is not surprising that disruption
in this intricate process can result in either conception delay or recurrent pregnancy loss (RPL). The latter,
which is the focus of this chapter, can occur when three main functions of endometrium function are
disrupted: its ability to mount a satisfactory decidual response, abnormal receptivity of the decidua to the
conceptus, and inability of the decidua to favor developmentally competent embryos.
The Decidualization Process

The formation of a decidual layer from endometrial stromal cells is imperative to all mammals with
invading embryos [1]. Whereas most mammalian species have an endometrial response triggered by
embryonic signals [3], the endometrium in humans decidualizes in each menstrual cycle in response to
the concerted efforts of two key hormones, estrogen and progesterone.
The human endometrial cycle is classically divided into three distinct phases marked by profound
stromal remodeling of the endometrium [4]. An initial proliferative phase, which follows menstruation
and precedes ovulation, is dominated by estrogen and characterized by endometrial thickening, stromal
edema, and increased size and tortuosity of endometrial glands. The progesterone-predominant secretory
phase starts after ovulation and is responsible for dramatic morphological changes in endometrial stromal
cells: these spindle-shaped cells are transformed into plump epithelioid and secretory decidual cells that
are amenable to implantation, placental formation, and early nutrition of the embryo [4,5]. In the event of a
failing pregnancy or lack of a conceptus, declining progesterone levels trigger necrosis and fragmentation
of these endometrial cells with subsequent shedding and overt bleeding (menstruation) [5].
The differentiated decidual cells are capable of undergoing complex adaptations during the process
of implantation, and it has been postulated this is circadian clock-regulated through modulations of the
secretomic milieu [6,7]. In simple terms, during the first stage of the secretory phase, the decidualized
cells create a pro-inflammatory microenvironment that favors implantation [3]. This is followed by an
43
anti-inflammatory response that is supportive of embryo development [8]. When orchestrated well, this
sequence of events enables the implantation of healthy embryos and the formation of a well-developed
placenta capable of supporting the fetus throughout pregnancy. Hence, some researchers propose that
disruptions in the pro- and anti-inflammatory balance of the decidua can result in implantation failure,
miscarriage, or poor outcomes at later gestation. Interestingly, several later pregnancy complications such
as preeclampsia, intrauterine growth restriction, and placental abruption have been linked to abnormalities
in this crucial step [2], highlighting the immense influence of the early developmental environment on
obstetric outcomes and perhaps beyond.
The regular physiological investment in an inexistent pregnancy might seem counterproductive and
reproductively “inefficient.” However, the development of a menstrual cycle can be seen as an evolutionary
strategy, in which the endometrium’s extraordinary plasticity and regenerative capacity preconditions
the endometrium to the highly invasive human placenta and the significant oxidative stress, vascular
remodeling, and angiogenesis associated with early pregnancy in our species [4].
Inadequate Decidual Responses and Recurrent Pregnancy Loss

The absence of a decidual response is incompatible with pregnancy. However, suboptimal endometrial
decidualization can allow the implantation of embryos and establishment of a clinical pregnancy
without the means to carry it safely to term. Therefore, RPL can occur in the event of poor stromal cell
differentiation.
Multiple attempts have been made to identify biomarkers of adequate decidualization, although their
clinical applicability is still controversial [9]. Prolactin (PRL) and insulin-like growth factor binding
protein-1 (IGFBP-1) have been historically used as markers of an appropriate decidua [5]. Salker et al.
[10] have shown that PRL levels are approximately 100-fold lower in the endometrium of women suffering
from RPL when compared to controls, indicating that this population might be unable to mount an
adequate decidual response. In addition, expression of PROK1, a key regulator of endometrial receptivity,
was significantly increased in patients affected by RPL and this continued to rise aberrantly throughout
the cycle, pointing toward an impairment of temporal regulation of decidual changes. In a different study,
similar disruption of decidual response was caused by untimely expression of serum- and glucocorticoid-
inducible kinase 1 (SGK1). Whereas continuous expression of this kinase was associated with complete
infertility, a blunted increase was found in RPL cases, likely due to persistently high reactive oxygen
species that prevented the maintenance of pregnancy [11]. An abnormal decidual response to embryonic
signals has also been demonstrated. Human chorionic gonadotrophin (hCG), secreted early by the
trophoblast, is known to inhibit both PRL and IGFBP-1 in a dose- and time-dependent manner [12] in
normal pregnancies. Whereas hCG was successful in reducing PRL and PROK1 in controls, the opposite
occurred in RPL samples [10].
Lack of endometrial plasticity is also accountable for poor decidual responses. New endometrial
mesenchymal stem cells, which possess elevated concentrations of pluripotent factors, are recruited in
the beginning of each endometrial cycle and differentiated into mature stromal cells [8]. Their stem cell
features render them amenable to reprogramming and adaptation following insults or environmental cues.
Endometrial stromal cells from women with RPL have been shown to have an abnormal methylation
profile, with loss of epigenetic signatures related to stem cells, leading to increased senescence and
reduced differentiation potential [8].
The Window of Implantation and the Embryo Selection Hypothesis

The dynamic and temporal changes in the decidualized endometrium by no means occur by chance.
Either a poor decidual response or, alternatively, an adequately decidualized endometrium without the
ability to change its secretome in a timely manner can affect the outcomes of pregnancy.
Timing is a key factor in implantation. The seminal work from Wilcox et al. [13] showed that
pregnancies were more likely to be successful if implantation occurred 6−10 days after ovulation.
The Endometrial Factor in Recurrent Pregnancy Loss 45
Hence this time period is known as the “window of implantation.” This small “window,” during
which the decidua is the most favorable to implantation, also coincides with the pro-inflammatory
phase of decidualization and with the embryo’s developmental stage during which it is most capable
of implanting [8,10]. Inflammation is therefore imperative to successful implantation, but undesirable
to an ongoing pregnancy [14]. Limiting this period of receptivity is the first endometrial mechanism
to select viable embryos: those with any developmental delay will not be in synchrony with the
endometrium [10,15]. A prolonged window of implantation, however, might prevent the endometrium
from engaging in embryo quality control [1]. It can also lead to implantation of viable embryos in an
unsupportive environment, leading to either early pregnancy loss or later pregnancy complications
[14]. It has been reported that the window of implantation is prolonged in women suffering from RPL
[10,11,14]. The overacceptance of developmentally incompetent embryos has led to the paradoxical
hypothesis of “superfertility” in RPL. The superfertility concept suggests that, rather than rejecting
good quality embryos, the endometrium of women with RPL allows the establishment of more clinical
pregnancies, which are destined to fail, and would have otherwise been rejected in fertile women.
Hence, RPL women miscarry more, as a consequence of implanting more. The finding of shorter
time-to-pregnancy intervals in RPL speaks in favor of the “superfertile” hypothesis [10]. Although
the superfertile hypothesis may be overly simplistic, aberrant receptivity influencing selectivity of the
endometrium is an attractive and plausible hypothesis.
The selectivity of the endometrium does not stop at the implantation window and, in the event of failure
of this first checkpoint of quality control, there are other mechanisms to avoid compromised embryos.
Once the luminal epithelium is breached for implantation, the decidua engages in a molecular dialog
with the conceptus and is capable of tailoring its microenvironment accordingly [16,17]. The decidua
is therefore much more than a passive bystander subject to the embryo’s invasive potential; instead,
the decidua has an active role in implantation by acting as a biosensor of developmentally competent
embryos. Molecular cues to this blastocyst-decidual interaction have been studied in in vitro models. A
study that compared the response of decidualized endometrial cells to different quality embryos showed
that those adequately developed trigger a surprisingly modest decidual response, whereas poor-quality
embryos led to the inhibition of several factors important to early pregnancy [18]. Similarly, genome-wide
expression profiling showed that whereas only 15 decidual genes were differentially expressed in response
to high-grade embryos, 449 were altered in response to developmentally incompetent embryos [19]. It
is thought that the latter downregulate the expression of key molecules such as HSPA8, which triggers
an endoplasmic reticulum stress response that compromises the secretion of PRL and IGFBP-1 [4,19].
In addition, these embryos inhibit the secretion of several interleukins known to be key implantation
factors and immunomodulators [18]. The decidual response is therefore stronger in response to abnormal
embryos, which are likely to have more intense metabolic activity [1]. Moreover, endometrial stromal
cells are programmed to undergo directional migration to encapsulate the blastocyst to ensure that, if
developmentally competent, they become embedded in a nurturing environment [16]. While healthy
endometrial stromal cells show reduced migration toward compromised blastocysts and change their
secretome into a less favorable one once the surface epithelium is breached by a poor-quality embryo,
the same has not been observed in the endometrium from RPL subjects [16,17].
Immunological Factors
An additional role of the decidua is to ensure immune tolerance to the conceptus while at the same time
protecting the mother from external insults. A change in the immune cell composition of the endometrium
occurs after decidualization in order to recognize and accept the semi-allogenic embryo. The most
abundant subtype of leucocytes in the decidua are the uterine natural killer (uNK) cells [20], representing
approximately 70% of all endometrial leukocytes after the secretory phase [5]. uNK cells are a unique
subset of natural killer cells, with a different antigen profile to their circulating counterparts—while the
latter stain heavily for CD56 and CD16 antigens and are highly cytotoxic, uNK cells stain only for CD56
and show little evidence of cytotoxic activity [21]. Instead, they synthesize several angiogenic factors
essential for the establishment of early pregnancy [20]. The amount of uNK cells significantly increases
6−7 days after the luteinizing hormone surge, a time that coincides with implantation and continues to
rise in the first trimester of pregnancy [20,22]. These characteristics, taken together with the fact that
uNK cells tend to cluster at the site of trophoblast invasion and around spiral arteries, suggest that they
might play a role in their remodeling and implantation [23].
The concentration of uNK cells has been found to be increased in women with RPL [24,25]. Both
implantation and early placental development occur in a relatively hypoxic environment, and oxygen
tension remains low until 10−12 weeks’ gestation, when the spiral artery plugs are dissolved [26]. It has
been postulated that the abundance of uNK cells could lead to early spiral artery remodeling, increasing
oxygen tension and oxidative stress above the optimal levels for adequate implantation [26]. However,
the role of oxidative stress remains controversial. A meta-analysis carried out by Tang et al. [27] showed
no association between uNK cell concentrations and pregnancy outcomes, although few of the studies
analyzed used consistent methods. There is still a lack of consensus on timing of endometrial sampling,
methods for uNK cell quantification, and reference ranges, which precludes adequate interpretation of the
results of the various studies [20], resulting in significant heterogeneity in the literature which questions
the validity of uNK as a diagnostic marker for RPL.
T lymphocytes make up approximately 10% of the decidual leukocyte pool [22] and have the ability to
differentiate in response to internal and external signals [28]. T helper (Th) cells can further differentiate
in subtypes with diverse cytokine release patterns [29]. It is an interesting question as to whether
inappropriate immune responses may be responsible for or the superfertility described above.
Challenges and Clinical Implications

It is frustrating to patient and clinician alike that only approximately 50% of RPL cases have an identifiable
cause [30]. Half of the unexplained cases are likely related to endometrial factors, either directly or
indirectly. Being a tissue highly responsive to hormonal and environmental stimuli, endometrial function
can become impaired by common causes of reproductive failure such as endocrine pathologies or
endometriosis [30]. Treating primary systemic causes is therefore more straightforward than treating
intrinsic endometrial abnormalities.
For primary endometrial etiologies to be targeted, quick and reliable diagnostic tests must become
available. However, despite major advancements in the understanding of early pregnancy events, as well
as the role of the endometrium in establishing and maintaining pregnancy, reliable diagnostic tests remain
a challenge. Consequently, little has been achieved in terms of clinical interventions for endometrial
factors [9]. The inexhaustible adaptive capacity of the endometrium precludes a formulaic therapeutic
approach—its dynamic nature makes a single test assessment unlikely, and little is known about inter- and
intra-individual variability between cycles [1]. A “multi-omics” approach for endometrial studies seems
promising for the future, but interpretation and clinical application of such high output data remains
problematic [31].
uNK cells have been investigated in RPL since the identification of uNK cells over 20 years ago
[32]. Although the biological plausibility of uNK cell involvement in RPL is unquestionable, there is
much debate on whether uNK cells should be measured routinely—and if abnormal, treated—in affected
women [32,33]. Unfortunately, the lack of consistent data means that uNK quantification has sometimes
been used for the financial exploitation of frustrated couples in assisted reproduction clinics [32]. Several
immunomodulators and corticosteroid treatment have been studied [32,34] and reported in other chapters
of this book, some with the aim of reducing uNK concentrations in in the endometrium of RPL cases,
but lack of adequate clinical data regarding pregnancy outcomes, and inadequate follow-up of potential
side effects, make empirical use questionable. Similarly, the role of immunopotentiation and progesterone
supplementation have not been investigated as modulators of superfertility in RPL.
Finally, a major challenge in advancing treatment options for endometrial factors of RPL is the
fact that research in humans is marred by both ethical and technical challenges. Future research must
creatively utilize innovative computational engineering technologies alongside digital health platforms
to re-examine and interrogate the endometrium, in order to find new answers to this age-old clinical
management conundrum.
The Endometrial Factor in Recurrent Pregnancy Loss 47
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6
Fetal Structural Malformations and
Howard J.A. Carp, Thomas Philipp, Micha Baum, and Michal Berkenstadt
Introduction
Structural malformations were classically described as occurring in 2% of live births, which is still the
case in much of the world [1]. However, in the Western world, the incidence is far lower due to the wide use
of diagnostic techniques (ultrasound, amniocentesis, chorion villus sampling, and noninvasive screening
such as nuchal translucency screening, PAPP-A, free βhCG, and α-feto protein triple or quadruple testing)
to identify at-risk populations. As many patients with fetal structural malformations elect to terminate the
pregnancy, the present incidence of anomalies at birth is probably lower than previously reported. There is
evidence that the prevalence of fetal anomalies is higher in women with recurrent pregnancy loss (RPL).
Sheiner et al.’s [2] study reports 2 anomalies in 29 patients. Although a very small series, the figures are
higher than expected. Analysis of the figures in the RMITG trial [3] showed an anomaly rate of 4%. In
the author’s series, there were 3 anomalies in 99 developing pregnancies in nontreated patients. However,
in the RMITG and author’s series, no control group is available. In Thom et al.’s report [4], women with a
history of RPL were found to have a higher risk of delivering a child with congenital malformations (RR
1.8%, 95% CI 1.1–3.0) than normal controls. However, many embryos with severe anomalies will be lost
as miscarriages, either as unexplained pregnancy losses in the first trimester, or possibly as diagnosed
anomalies in the second trimester.
Ultrasound is the main diagnostic tool used to detect anomalies. However, despite the major advances
in ultrasound resolution, ultrasound is insufficient for precise visualization in the first trimester below
11 weeks (corresponding to a crown-rump length of less than 30 mm), when almost 90% of recurrent
miscarriages occur. In order to diagnose earlier anomalies, advanced techniques such as embryoscopy
are required. In most cases, the usual teratogens such as viruses, infectious organisms, drugs, etc.
cannot be found and no apparent cause can be identified. In previous years, when karyotyping banding
techniques were used, most anomalies were said to be “multifactorial” and no genetic cause could be
found. However, genetic testing has changed significantly in recent years with the introduction of higher-
resolution molecular techniques, such as comparative genomic hybridization, next-generation sequencing
(NGS), and whole exome/genome sequencing. Many anomalies which were previously diagnosed as being
nongenetic are now known to have a genetic cause. There is still much work to be performed on embryonic
anomalies and RPL. This chapter addresses some of the issues.
Embryoscopy as a Diagnostic Modality

Embryoscopy allows visualization of the embryo in utero. With the transcervical approach, before
curettage in cases of missed abortion, subtle morphologic details, undetectable by ultrasound, can
accurately be assessed without any artificial damage [5,6] (Figure 6.1). In this chapter the diagnostic
value of a detailed morphologic and genetic evaluation of the demised embryo are discussed.
48
Fetal Structural Malformations and Recurrent Pregnancy Loss 49
FIGURE 6.1 (a) Ultrasound prior embryoscopy showed an embryo measuring 24 mm crown-rump length (CRL) without
heartbeat. Head (H), umbilical cord (U), and upper (UL) and lower limbs (LL) can be seen. (b) Embryoscopic anterolateral
view of the upper portion revealed a well-preserved embryo. Delicate structures like the nostrils are clearly discernible.
Note the developing eyelids. Distinct fingers can be clearly seen.
Technique
After confirmation of embryonic demise, and prior to curettage, a rigid hysteroscope with both biopsy
and irrigation working channels is inserted into the uterine cavity. Saline is infused continuously at a low
pressure, 40–120 mm Hg. In first trimester missed miscarriages, the decidua capsularis and parietalis
have not yet fused, so the uterine cavity can be assessed at the same sitting. The gestational sac is localized,
and the chorion incised with microscissors due to its opacity. The embryo can then be viewed through the
amnion. At 8 weeks the embryo possesses several thousand named structures. The embryoscope should
be advanced as close as possible to the embryo in order to document the minute developing structures
such as the limbs (Figure 6.1). The hysteroscope can then be inserted into the amniotic cavity. The details
of the embryo can be seen better from inside the amniotic cavity. However, care must be taken not to
increase the pressure of the saline, or the embryo will be flushed out and lost.
Complete examination of the conceptus includes visualization of the head, face, dorsal and ventral
walls, limbs, and umbilical cord. The incidence of developmental defects is particularly high in early
miscarriage specimens [5,6]. However, in early pregnancy, the embryonic anatomy is constantly changing.
Hence, diagnosis of developmental defects requires expertise and knowledge of the anatomy of the
developing embryo. The diagnosis of an embryonic anomaly is dependent on precise aging [7]. The term
gestational age, which is used in clinical and ultrasound terminology, should not be used for studying
missed abortions, as most of these specimens are usually retained in utero after embryonic demise.
In RPL, particularly if a phenotypically abnormal embryo is found, accurate genetic analysis of the
conceptus is essential. Transcervical embryoscopy allows selective and reliable sampling of chorionic
tissues with minimal potential for maternal contamination. Ferro et al. [8] have described the advantages
of biopsying the embryo at the point of insertion of the umbilical cord (Figure 6.2). If genetic analysis
is performed on the curettings rather than an embryoscopic biopsy, the results may be confounded if the
decidua overgrows the trophoblast specimen. If possible, genetic analysis should be performed on the
embryo in order to exclude confined placental mosaicism if the trophoblast alone is assessed. Hence,
genetic assessment of the trophoblast may not reflect the true ploidy of the embryo [9].
In twin pregnancy, both chorionic sacs can be biopsied separately (Figure 6.3). After the embryoscopy,
curettage can be completed.
Morphologic Defects Diagnosed Embryoscopically

Disorganized Embryos
Abnormal embryonic development can be localized to specific organs or the entire embryo may have
growth disorganization as shown in Figure 6.4. Four grades of disorganized growth have been described
FIGURE 6.2 Direct chorionic villus sampling is performed under visual monitoring using a microforceps (M). Note the
chorionic villi (V) at the tip of the microforceps. (A) Marks remnants of the amnion. A microcephalic 45, X0 embryo (E)
with a crown-rump length of 28 mm is visible in the background of the picture.
based on the degree of abnormal embryonic development [10]. An empty or anembryonic sac (known
clinically as a blighted ovum) is the most severe form, known as Grade 1 (GD 1). No embryo can be
visualized. GD 2 conceptuses show embryonic tissue of 3–5 mm. in size, but with no recognizable
external embryonic landmarks and no retinal pigment. It is not possible to differentiate caudal and
cephalic poles (Figure 6.4). GD 3 embryos are up to 10 mm long. They lack limb buds, but retinal pigment
is often present. A cephalic and caudal pole can be differentiated. The GD 4 embryos have a crown-rump
length over 10 mm with a discernible head, trunk, and limb buds. The limb buds show marked retardation
in development, and the development of the facial structures is highly abnormal. Growth disorganized
embryos show a high prevalence (92%) of autosomal trisomies, trisomy 16 being the most common,
accounting for 46% of abnormal karyotypes [5].
FIGURE 6.3 (a) Transvaginal ultrasonogram before embryoscopy examination of a patient’s fourth consecutive
pregnancy loss showed bichorionic twin pregnancy with two embryos (I+II), measuring 14 and 19 mm in crown-rump
length. No abnormalities were identified on sonography. (b) Embryoscopic examination from an anterolateral view of
the upper part of twin I. External developmental defects are severe microcephaly and facial dysplasia. The hand plates
are formed (UL) but finger ray development is missing, indicating retarded upper limb development relative to the CRL.
(c) Anterior view of the upper part of twin II. Distinct grooves are formed between the fingers of the microcephalic
embryo, but the upper limbs are not bent at the elbows, indicating retarded development for an embryo of this size. The
two chorionic sacs were biopsied separately. Chromosome analysis revealed trisomy 15 (47,XX,+15) (twin I) and trisomy
21(47,XX,+21) (twin II).
FIGURE 6.4 The microscissor (M) is pointing to a growth disorganized embryo (GD2) measuring 3 mm crown-rump
length. No recognizable external embryonic landmarks can be seen embryoscopically. An abnormal karyotype (47,XX,+4)
was diagnosed cytogenetically.
Localized Defects
Localized defects may be isolated or involve multiple organs. See Figures 6.3 and 6.5–6.9. Below are
some examples.
Head defects may show microcephaly, anencephaly, exencephaly, encephalocele, facial dysplasia,
cleft lip, cleft palate, fusions of the face to the chest, anophthalmia, unfused eye globes, and proboscis
development. Facial dysplasia shows poorly developed branchial arches and midface structures.
Microcephaly and facial dysplasia are usually observed in combination.
Neural tube defects (anencephaly, encephalocele, spina bifida) can be multifactorial in origin, caused
by one or more lethal gene defects or aneuploidy [10–13], or nongenetic mechanisms such as amniotic bands.
Lateral and median cleft lip can be distinguished embryoscopically, but not until after 7 weeks of
development, as fusion does not occur until that time. Cleft lip may be part of a malformation syndrome.
FIGURE 6.5 Close-up lateral view of the upper part of an embryo measuring 14 mm crown-rump length after the
amniotic membrane (A) had been opened. The microcephalic embryo showed a fusion face to the chest. Upper limbs (UL)
showed hand plate formation, but no digital rays, indicating retarded development of the limbs for an embryo of this size.
Chromosome analysis revealed an abnormal karyotype (69,XXY).
FIGURE 6.6 Embryoscopic lateral view of an embryo measuring 13 mm in length. External developmental defects of the
embryo are severe microcephaly, facial dysplasia, profoundly retarded upper limb (UL) and lower limb (LL) development.
(U) marks the umbilical cord. The missed abortion was the patient’s third consecutive pregnancy loss and resulted from
IVF. An apparently normal karyotype was diagnosed cytogenetically (46,XY).
FIGURE 6.7 Lateral (a) and close-up anterior view of the upper part (b) of an embryo measuring 12 mm crown-rump
length. External developmental defects of the embryo are severe microcephaly, facial dysplasia, profoundly retarded upper
limb (UL) and lower limb (LL) development, and abnormal short cord (U). The dark brown areas in the facial region are
due to maceration. The missed abortion was the patient’s sixth consecutive pregnancy loss. An apparently normal karyotype
was diagnosed cytogenetically (46,XY).
Irregular clefting may be caused by amniotic bands. Clefts often occur with chromosomal aberrations,
especially trisomy 13. Cleft palate can only be diagnosed in the fetal period, since fusion is completed
after the 10th week of development.
Trunk defects include spina bifida, omphalocele, and gastroschisis. The phenotype of spina bifida is
different in the embryo than in the fetus or neonate. In the embryo, spina bifida is frequently observed as
a plaque-like protrusion of neural tissue over the caudal spine [14]. The physiological midgut herniation
is a macroscopically visible process which starts in the 6th week after fertilization. The midgut only fully
returns to the abdominal cavity at the end of 10 weeks of development. As herniation is still physiological
at 8 developmental weeks, omphalocele can only be diagnosed in the fetal period. Gastroschisis differs
from the physiological herniation of the midgut as the umbilical cord is not involved and no sac is present.
Gastroschisis is rarely observed in the embryo. The pathogenesis of gastroschisis is controversial. The
FIGURE 6.8 Close-up of the face of an embryo with a crown-rump length of 27 mm. A median cleft lip (box) is present.
(UL) marks the right upper limb. Trisomy 9 (47,XY,+9) was diagnosed.
FIGURE 6.9 Embryoscopic lateral view of the upper portion of a well-preserved embryo with anencephaly. The exposed
brain tissue (*) is still intact (exencephaly). The digital rays of the hand (H) are notched. Parts of the external ear (E) are
clearly discernable. Remnants of the amnion are labeled (A). A normal karyotype was diagnosed cytogenetically (46,XX).
theory of abdominal wall disruption as a result of an “in utero” vascular accident has gained the most
acceptance. Therefore, gastroschisis is considered to be a sporadic event with a negligible risk of recurrence.
Limb defects such as polydactyly, oligodactyly, syndactyly, split-hand/split-foot malformation, and
transverse limb reduction defects are the most commonly observed malformations. Polydactyly may occur
as isolated malformation or may be part of a malformation syndrome, either of which may be genetic
or of unknown origin. Postaxial polydactyly is common in trisomy 13 [15]. Syndactyly may be part of a
genetic malformation syndrome. Syndactyly, which can be seen from the end of the 8th week when the
fingers become free, is common in triploidy [15]. The split-hand/split-foot malformation can be a part
of numerous syndromes, such as ectodermal dysplasia, ectodactyly, and clefts, and is often found in
chromosome 15 trisomy. In transverse limb reduction defect, the distal structures of the limb are absent,
with proximal parts are being more or less normal. These are due to a disruption sequence presumed to
be due to peripheral ischemia [16]. The recurrence risk in future pregnancies is minimal [15].
Umbilical cord defects, such as knots, torsion, stricture, cysts, and abnormal thin and/or short cords,
are rarely observed embryoscopically. Umbilical cord cysts and abnormally thin and/or short cords are
usually found in chromosomally abnormal embryos.
Genetic Aberrations as the Cause of Abnormal Embryonic Development

The highest incidence of chromosome anomalies (86%) can be found in conceptuses with combined
localized developmental defects. In growth disorganized embryos, 70% are genetically abnormal. The
lowest incidence of chromosomal abnormalities (41%) is found in phenotypically normal specimens
[6] (Table 6.1). Transcervical embryoscopy allows selective reliable sampling of chorionic tissues
with minimal potential for maternal contamination. In addition, abnormal embryonic development, as
documented by embryoscopy in patients with apparently normal chromosomes, adds valuable information.
This information would be completely lost if morphological examination of the demised embryo had not
been carried out, and abnormal embryonic development would have remained undetected. A grossly
abnormal embryo with a normal chromosomal analysis is a particularly valuable finding, as it points to
a possible etiologic factor in the mother or syndrome of unknown origin (Figures 6.6 and 6.7). Table 6.2
shows a summary of embryoscopic and genetic findings in the first 53 patients investigated with recurrent
miscarriage (>2 consecutive early pregnancy losses). In a larger study [17] of 75 women with three or
more losses, embryoscopy showed 81% of miscarriages to have abnormal morphology. Five patients
underwent embryoscopy three times; of those, three patients (60%) had recurrent abnormal morphology
and two patients a mixed pattern of abnormal morphology. Seventy-eight morphologically abnormal
embryos were genetically analyzed. Twenty-nine were euploid and 49 aneuploid. Six patients underwent
TABLE 6.1
Specimen Morphology and Karyotype of 514 Missed Abortions
Total Specimens Specimens with Abnormal
Total Specimens Successfully Karyotyped Karyotype
Morphology No. %a No. %b No. %c
Normal 58 11.3 56 96.2 23 41.1
Growth disorganization 237 46.1 225 95 156 69.3
Combined defects 198 38.5 193 97.3 166 86.0
Isolated defects 21 4.1 21 100 14 66.7
Total 514 100 495 96.3 359 72.5
a Percentage of total number of specimens with that morphology.
b Percentage of each morphologic category successfully karyotyped.
c Percentage of each morphologic category with an abnormal karyotype.
TABLE 6.2
Summary of Specimen Morphology and Karyotypic Outcome in 53 Patients with Recurrent Miscarriages
(Three or More Consecutive Miscarriages)
Total Specimens Successfully Specimens with Abnormal
Total Specimens Karyotyped Karyotype
Morphology N %a N %b N %c
Normal 8 15.1 7 87.5 3 42.9
Growth disorganization 26 49.1 24 92.3 15 62.5
Combined defects 18 34 18 100 13 72.2
Isolated defects 1 1.9 1 100 1 100
Total 53 100 50 94.3 32 64
a Percentage of total number of specimens with that morphology.
b Percentage of each morphologic category successfully karyotyped.
c Percentage of each morphologic category with an abnormal karyotype.
embryonic genetic analysis three times; two patients (33.3%) showed recurrent aneuploidy, three patients
(50%) recurrent euploidy, and one patient a mixed pattern.
It is unlikely that maternal factors such as antiphospholipid antibodies, thrombophilic disorders,
endocrine factors, or uterine anomalies cause the developmental defects observed embryoscopically.
After exclusion of a chromosomal disorder, these developmental defects might be heterogenous in their
origin. Recent studies using molecular techniques, such as NGS or whole exome sequencing (WES), have
shown that imbalances in genes required for embryonic growth and morphogenesis and mutations exist
in karyotypically apparently normal spontaneous miscarriages, malformed fetuses, and embryos with
developmental abnormalities documented by embryoscopy [18].
Ultrasound as a Diagnostic Modality

Ultrasound is the main diagnostic modality used today for diagnosing anomalies and has been so since its
introduction approximately 50 years ago. As the technology improved and resolution became higher, ever
more anomalies have been diagnosed. However, ultrasound has mainly been used to diagnose anomalies
in order to prevent the birth of infants with anomalies rather to diagnose causes of pregnancy loss. The
landmarks of fetal development detectable on ultrasound are described in Chapter 14. In the previous
section, the anomalies that may cause miscarriage are described when using embryoscopy as a diagnostic
tool. However, despite embryoscopic findings having been described over 15 years ago, the procedure
has not been widely adopted and ultrasound has been more often used. In recent years, ultrasound has
undergone major advances that allow anomaly scanning at the end of the first trimester. The so-called
first trimester scan is completed together with nuchal translucency screening at 11–13 weeks.
Nuchal Translucency Screening

First described by Nicholaides et al. in 1992 [19], the scan looks at the diameter of the translucent area
at the back of the neck. An increased thickness, originally described as being above 3 mm, can give a
predictive value for the detection of genetic anomalies, including Down syndrome. A thickened nuchal
translucency has been described as conferring a 35% risk for chromosomal abnormalities [19], whereas
the risk was only 1% if the nuchal translucency was 2 mm or less. However, even minor errors can have
substantial effects on false-positive and false-negative diagnoses, which in turn can lead to unnecessary
invasive tests (chorionic villous sampling or amniocentesis) or missed diagnoses of aneuploidy. Hence,
the nuchal translucency is not used alone but assessed together with maternal age and serum tests:
pregnancy-associated plasma protein A (PAPP-A), hCG, or cell free DNA (cfDNA). The combination of
these three measurements detects 87% of cases of trisomy 21 at 11 weeks, 85% at 12 weeks, and 82% at
13 weeks, at a 5% false-positive rate [20]. A fetus with thickening of the nuchal translucency is also at
elevated risk for abnormalities other than aneuploidy. Even if the chromosomes are normal, a fetus with
a thickened nuchal translucency at 10–14 weeks is at increased risk of intrauterine demise and structural
abnormalities, especially cardiac, gastrointestinal, or musculoskeletal systems [21,22].
Soft Signs
There are other features that can be diagnosed on ultrasound at the end of the first trimester that are
nondiagnostic but suggestive of anomalies. These include visualization of four chambers in the heart and
tricuspid valve examination as a marker for aneuploidy. These views can be obtained in two-thirds of
13-week fetuses [23]. In experienced hands, cardiac defects can be diagnosed as early as 10 weeks. Nasal
bone hypoplasia is another feature associated with anomalies. In a case series of hypoplastic nasal bone
[24], 42% had common aneuploidies and 10% had clinically relevant copy number variants (CNVs). In
addition to the association with trisomy 21, a hypoplastic nasal bone may indicate facial dysmorphism
associated with clinically relevant CNVs. By using the profile view to measure nuchal translucency and
visualize the nasal bone, changes can be observed in the posterior brain enabling the early diagnosis of
neural tube defects.
Consequently, a structured protocol has been introduced [25] that provides a checklist for anatomical
assessment. However, most of the anomalies assessed are those that are compatible with life, and are often
performed later than the stage in which previous miscarriages have occurred.
Early First Trimester Screening

As the vast majority of anomalies occur in the first trimester, it would be desirable to have an anomaly
scan prior to the stage of previous miscarriages. Such a diagnosis requires a thorough knowledge of
embryology in order to know when different organs develop and at which stage pathological effects can
be seen. In addition, equipment needs to be of a higher resolution than currently available. However,
there are some hints, as described by Arslan et al. [26], who reported some anomalies can be relatively
easily detected in the first trimester, such as anencephaly. Anencephaly has been diagnosed as early as 9
weeks. There are anomalies that reveal signs later in gestation and have no possibility of early detection,
such as cerebellar hypoplasia. There is also a third group of anomalies that can be detected in the first
trimester with meticulous examination using high-tech devices. This group of anomalies includes spina
bifida occulta, skeletal dysplasia, and some kinds of cardiac defects. However, ultrasound is still not able
to compete with embryoscopy in detecting the very early anomalies that may lead to miscarriage.
Role of the Counselor

The question the counselor has to face is whether the structural malformation or aneuploidy is sporadic
or recurrent. Recurrent morphological anomalies have been described [17], as has recurrent aneuploidy
(Table 6.3). The patient may only have information from one pregnancy loss, or there may be information
from more than one pregnancy loss.
Information from One Loss

If there is only information from one pregnancy loss, the etiology may be genetic or of no known
genetic mechanism. If there is no known genetic mechanism, there are databases available to assist
clinicians in taking action based on test results [27]. If there is a genetic explanation for the anomaly,
more information can be accessed. It is possible to obtain information from other pregnancy losses even
if genetic analysis has not been previously performed. If previous miscarriages have been evacuated by
curettage, histological specimens of previous miscarriages, either fixed slides or paraffin blocks, can be
used for chromosomal microarray analysis or NGS testing [28,29]. However, the DNA may be of poor
quality, so skill and experience are required to interpret the results.
With this additional information, counselors identify the risk of recurrence. Analysis of family history
also gives important information regarding inheritance patterns. Karyotyping of the parents by banding
techniques is a poor alternative. When anomalies are present, most have negative results on standard
TABLE 6.3
Repeat Aneuploidy in Abortus
Series Repeat Embryonic Aneuploidy
Carp et al. [25] 8/43 (19%)
Sullivan et al. [26] 3/30 (10%)
Sugiura-Ogasawara et al. [27] 32/42 (75%)
Total 43/115 (37.3%)
Note: In each of these reports, which include patients with >3 losses, an
aneuploidy embryo was found. In Carp et al.’s [25] series, 43 had a
subsequent miscarriage. Eight of these were also aneuploid (19%). The
overall figures suggest that repeat aneuploidy may occur in 37.3% of
patients, but in 62.7% of patients aneuploidy was an isolated event.
karyotyping. Higher resolution testing is required in order to determine if one of the parents is a carrier,
thereby increasing the risk of recurrence in subsequent pregnancies. Higher resolution testing requires
techniques such as WES on both parents and fetus. All of the above tests basically bring the couple into
the next group where information is available from more than one loss.
Information from More Than One Loss

If the malformation was sporadic, the couple can be assured of the low risk of recurrence and advised to
conceive again. However, tissue should be stored so that if there is another anomalous embryo, the tissues of
both anomalous embryos can be compared. In the subsequent pregnancy, ultrasound should be performed
for anomaly scans. Embryoscopy would not be advised on a live embryo due to the possibility of miscarriage
in a patient with RPL. However, in the case of another fetal demise, embryoscopy should be repeated.
If a genetic etiology is found to explain the malformation, pregestational testing (PGT) is the only way
to prevent recurrence. Later diagnosis by chorionic villus sampling or amniocentesis will prevent the live
birth of an infant with genetic aberrations but will involve the patient having an artificial termination
of pregnancy. The issue of PGT-A is hotly debated in RPL in Chapters 26 and 27. In rare mutations, the
specific sequence should be sought in order to reach a diagnosis.
If PGT fails to provide the answer (all tested embryos were abnormal, or no pregnancies ensued),
gamete donation may be indicated.
At the end of testing, there is still a group of patients with embryonic structural malformations, in whom
no genetic diagnosis can be made. In addition, a search for known teratogens such as viruses, drugs, etc.
fails to find risk factors. The malformation may be caused by epigenetic mechanisms (acetylation or
methylation of regions on the DNA molecule) that alter the reading of the genetic code without altering
the coded areas themselves. In such cases, it is difficult to know what to advise. At present, we do not
even know if the primary cause is in the embryo or the mother. However, future genomic analyses may
provide additional information and should be periodically updated.
Role of Testing
The question inevitably occurs as to when the above testing is necessary. Genetic testing, particularly
WES, is expensive. Chapter 19 shows the investigation protocol that one of the authors (HC) recommends.
Patients with two miscarriages have an 80% chance of a live birth. According to the American Society
of Reproductive Medicine (ASRM) [30] and the European Society of Human Reproduction and
Embryology (ESHRE) [31] protocols, patients with ≥2 miscarriages should be treated. However, the
costs and time involved, and 80% chance of a subsequent live birth should be taken into account when
discussing management. The patient with ≥3 losses has a 60% chance of a live birth. The Royal College
of Obstetricians (RCOG) [32] recommends testing and treatment after ≥3 miscarriages. Chapter 19
defines a group of patients we characterize as “poor prognosis patients” (≥5 pregnancy losses, etc.). In
these patients, the authors consider that comprehensive testing be performed.
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7
The Endocrinology of Recurrent Pregnancy Loss
Nicola Pluchino, Serena Bellaminutti, Panagiotis Drakapoulos,

Antonis Makrigiannakis, and Andrea R. Genazzani
Introduction
Among all pregnancy losses, it is estimated that approximately 8%–12% are due to endocrine factors. The
maintenance of pregnancy depends on numerous endocrinological events that may lead to the successful
growth and development of the fetus. Although the great majority of pregnant women have no preexisting
endocrine abnormalities, a small number may have endocrine alterations that could potentially lead to
recurrent pregnancy losses.
Progesterone is essential for successful implantation and maintenance of a normal pregnancy.
Therefore, disorders related to inadequate progesterone secretion by the corpus luteum may affect the
outcome of the pregnancy. Luteal phase deficiency, hyperprolactinemia, and polycystic ovarian syndrome
are some examples of endocrine disorders affecting pregnancy outcome. Several other endocrinological
abnormalities such as thyroid disease, hypoparathyroidism, uncontrolled diabetes, and decreased ovarian
reserve have been implicated as etiologic factors for recurrent pregnancy loss. Inhibins and activins are
nonsteroidal glycoproteins thought to have important roles in reproductive physiology and are proposed
as markers of fetal viability.
Luteal Phase Deficiency and Pregnancy Loss

Embryo implantation is a critical step of the reproductive process and needs a receptive endometrium during
the so-called “implantation window” (5–10 days after LH surge), a good quality embryo, and a synchronized
dialog between maternal and embryonic tissues [1]. The ovarian steroids progesterone and estrogen are
the essential hormones involved in uterine receptivity. The preovulatory increase in the secretion of
17b-estradiol (E2) supports the proliferation and differentiation of uterine epithelial cells and is followed
by the production of progesterone, which induces the proliferation and differentiation of stromal cells [2].
Progesterone is a sex steroid produced by the corpus luteum after ovulation and it is fundamental to
maintain the pregnancy until approximately the tenth week of gestation; thereafter it is produced by the
placenta [3].
Progesterone stimulates the endometrial receptivity, the decidualization of the stroma cells, and the
production of a series of cytokines, such as IL-1 and EGF, which are involved in the regulation of
integrin and prostaglandins, while they may also enhance the non-inflammatory T-helper 2 response
due to progesterone-induced blocking factor (PIBF) production [4]. PIBF mediates the NK response
to trophoblast and inhibits Th-1 cytokines (such as TNF alpha, which is responsible of cytotoxic and
inflammatory reactions) [5]. Current evidence shows that Th2 cytokines promote normal development
of the pregnancy, while an excess of Th1 cytokines leads to pregnancy loss [6]. Progesterone increases
nitric oxide production, thus improving the blood flow and oxygen to the endometrium [7] and reduces the
contractility of the myometrium at the time of implantation [8]. All these processes cease if progesterone
production is reduced due to luteal phase deficiency or a failing pregnancy that produces low hCG leading
to inadequate progesterone levels. In the latter case, low progesterone levels seem to participate to the
mechanism leading to expulsion of the embryo rather than causing the miscarriage [6.].
59
Luteal phase aberrations have been reported in the past to account for up to 35% of recurrent pregnancy
losses (RPL) [9]. However, there is no consensus as to the methods to be used to diagnose luteal phase
deficiency. Although serum progesterone levels below ≤12 ng/mL have been associated with increased
risk of miscarriage [10], serum progesterone levels can vary up to ten times between blood sampled
at a pulse peak or nadir. Luteal phase deficiency was originally thought to arise from insufficient
production of progesterone by the corpus luteum and subsequent inadequate endometrial maturation to
allow appropriate placentation. Luteal phase defect may also be due to reduced follicular development,
diminished progesterone production by the corpus luteum, and a dysfunctional endometrial response to
normal progesterone levels.
There are other causes for luteal phase deficiency, including stress, exercise, weight loss,
hyperprolactinemia, and the extremes of reproductive life, at the onset of puberty or perimenopausally [11].
Luteal phase support (LPS) is routinely given as part of in vitro fertilization (IVF) treatment. The
use of agonistic or antagonistic gonadotropin-releasing hormone (GnRH) protocols in stimulated
IVF/intracytoplasmic sperm injection (ICSI) cycles cause disruption of the luteal phase, leading to
inadequate development of the endometrium and asynchrony between endometrial receptiveness and
embryo transfer [12]. The etiology of luteal phase defect (LPD) in IVF has been extensively reviewed,
and different mechanisms have been proposed. Recently, it has been postulated that one of the principal
causes is related to the supraphysiological levels of steroids secreted by a high number of corpora
lutea during the early luteal phase, which directly inhibit LH release via negative feedback actions at
the hypothalamic-pituitary axis level. In addition, higher concentrations of progesterone can also lead
to an accelerated transformation to secretory endometrium at the time of embryo transfer, affecting
implantation rates [6].
In the case of RPL, the last Cochrane review published in 2018 concluded that progesterone
supplementation of patients with luteal phase deficiency could prevent recurrent miscarriage (average
risk ratio 0.69%, 95% CI 0.51–0.92, 11 trials, 2359 women, moderate-quality evidence) and increase the
live birth rate, especially in women with a history of at least three miscarriages [13]. However, it should
be stated the quality of evidence of included RCTs was judged as moderate. A metanalysis of three studies
on the use of progesterone in recurrent miscarriage showed that dydrogesterone administration was
associated with a 29% reduction in the odds of miscarrying (Figure 7.1) [5]. However, the PROMISE trial,
a randomized control trial that evaluated the use of micronized progesterone in women with recurrent
miscarriage, showed comparable results in terms of live birth rates compared to placebo [14]. It seems
that dydrogesterone but not micronized progesterone may be associated with a lower risk of recurrent
miscarriage [15]. As far as timing of progesterone administration is concerned, there is evidence that
supplementation should start after ovulation with or without the use of ovulation induction agents or 2–3
days after the basal body temperature increases (or after a positive urinary LH test) and continued up to
7–11 weeks of gestation [16, 17]. However, additional head-to-head trials of progesterone types, dosing,
and route of administration are required.
Hyperprolactinemia and Pregnancy Loss

Prolactin (PRL) is mainly synthesized and secreted by the lactotroph cells of the pituitary [18], and
in minor part by other sites such as mammary gland, placenta, uterus, and T lymphocytes [19]. Many
studies show that PRL plays an essential role in reproductive functions [20]. Recent research on rodents
has revealed that PRL receptors are involved not only in generating, but also in maintaining pregnancy.
However, the specific cellular mechanism of PRL action in the human ovary remains unclear [6]. Prolactin
can act directly on human granulosa cells, stimulating the expression of Type II 3b-hydroxysteroid
dehydrogenase involved in the last step of progesterone synthesis, and increase IGF-II secretion [21]. PRL
can also suppress progesterone and estrogen secretion [22]: it inhibits estrogen production by antagonizing
the stimulatory effects of FSH on aromatase activity [23] and directly inhibiting aromatase synthesis
itself [16]. In fact, PRL is required at low doses (<20 ng/mL) for progesterone production by granulosa
cell cultures, but at higher concentrations (>100 ng/mL), PRL inhibits progesterone production, resulting
in LPD [24].
Study Progestogen Controls Weight% OR with 95% Cl
Abortions/Total Abortions/Total
Gerhard, 1987 [P in O] 0/17 1/17 1.20% 0.3143 (0.0119 to 8.2735)

Pagliano. 2004 [MP] 4/25 8/25 8.06% 0.4048 (0.1039 to 1.5769)
Yassaee, 2014 [MP] 6/30 10/30 9.59% 0.5 (0.1547 to 1.6162)
OR (Vaginal Progesterone) 10/72 19/72 100% 0.47 (0.2 to 1.1)
Ehrenskjöld, 1967 [DYD] 14/72 23/81 20.91% 0.6087 (0.2854 to 1.2984)

Mistó, 1967 [DYD] 0/7 2/9 2.10% 0.2 (0.0081 to 4.9095)
The Endocrinology of Recurrent Pregnancy Loss
EI-Zibdeh, 2009 [DYD] 15/86 15/60 17.50% 0.6338 (0.2827 to 1.4208)

Omar, 2005 [DVD] 3/74 11/80 12.16% 0.265 (0.0709 to 0.9911)
Pandian, 2009 [DYD] 12/96 27/95 28.48% 0.3598 (0.1697 to 0.7627)
OR (Dydrogesterone) 41/435 78/325 100% 0.47 (0.3 to 0.7)
OR (All Studies) 54/407 97/397 100% 0.464 (0.32 to 0.6728)
0.001 0.01 0.1 1 10

OR (log scale)
FIGURE 7.1 Meta-analysis of progestogens in threatened miscarriage.

61
To restore fertility in hyperprolactinemic women, dopamine agonists are the first-line treatment due
to their efficacy in restoring ovulation [24]. A randomized control trial of 64 hyperprolactinemic women
with RPL treated with bromocriptine showed a higher incidence of live births (85.7% vs. 52.4%), while
PRL levels were significantly higher in women who miscarried. Bromocriptine was administered before
conception and continued until the end of the ninth week of gestation in the group of patients in whom
the serum PRL levels were normalized [25]. However, it should be stated that prolactin testing is not
recommended in women with RPL in the absence of clinical symptoms of hyperprolactinemia (oligo/
amenorrhea) [26].
In conclusion, normal PRL levels seem to be critical for the growth and maintenance of early gestation
but further studies are required to elucidate the exact role of prolactin in the pathogenesis of recurrent
miscarriage and to establish whether, in cases of hyperprolactemia, continuation of treatment during
pregnancy could be advantageous.
Thyroid Abnormalities and Pregnancy Loss

Thyroid hormones are vital for fetal development. A recent review on thyroid function and reproduction
stated that thyroid disorders and increased thyroid peroxidase antibodies (TPOAb) are correlated with
disturbed folliculogenesis, spermatogenesis, fertilization, and embryogenesis, indicating an important
role for thyroid hormone pathology and thyroid autoimmunity in subfertility and pregnancy loss [27].
Hyperthyroidism
Hyperthyroidism is found in approximately 0.1%–0.4% of pregnancies [28]. No studies have reported
hyperthyroidism as an independent cause of RPL, even if pregnant women with untreated overt
hyperthyroidism have been shown to be at increased risk for spontaneous miscarriage, congestive heart
failure, thyroid storm, preterm birth, preeclampsia, fetal growth restriction, and increased perinatal
morbidity and mortality [29,30]. Furthermore, treatment of overt Graves’ hyperthyroidism in pregnancy
may be related with better outcomes [31].
Hypothyroidism
The most common cause of hypothyroidism in pregnant women, affecting nearly 0.5% of patients, is
chronic autoimmune thyroiditis (Hashimoto thyroiditis) [32]. Other causes of hypothyroidism include
endemic iodine deficiency, prior radioactive iodine therapy, and thyroidectomy.
One plausible explanation for the relationship between hypothyroidism and pregnancy loss is LPD
linked to a hypofunctioning thyroid. Thyroid hormones have an impact on oocytes at the level of the
granulosa and luteal cells interfering with normal ovulation [33]. Low thyroxine levels have a positive
feedback on thyroid-releasing hormone (TRH), while elevations in TRH have been related with increased
PRL [34]. High PRL levels alter the pulsatility of gonadotropin-releasing hormone (GnRH) and interfere
with normal ovulation.
Untreated hypothyroidism in pregnancy is associated with a greater risk for adverse pregnancy
complications, such as miscarriage, premature birth, low birth weight, and detrimental effects on
fetal neurocognitive development [35,36]. Severe forms of hypothyroidism lead to anovulation and
infertility. Even if an association exists between reduced thyroid function and pregnancy loss, the latest
guidelines published in 2017 did not identify any high-quality studies evaluating the association between
overt hypothyroidism and RPL [26]. A recent study investigated the association between subclinical
hypothyroidism and RPL and detected similar cumulative live birth rates in women with subclinical
hypothyroidism and euthyroid women, and no difference in the prevalence of miscarriage or obstetrics
outcomes between RPL women and controls [37]. In another case-control study evaluating patients with
a history of RPL, the incidence of subclinical hypothyroidism was found to be significantly higher in the
TPOAb-positive group compared to the TPOAb-negative group (52% vs. 16%), although there was no
The Endocrinology of Recurrent Pregnancy Loss 63
difference in the prevalence of miscarriage or obstetric complications between RPL and controls [38],
suggesting that treated thyroid dysfunction is not associated with RPL. Consequently, patients should
be screened for thyroid disease, and thyroid function should be normalized before conception. When
thyroid function is found to be abnormal, follow-up is required to assess TSH, TPOAb levels, and T4
testing [6,26].
Evidence is controversial regarding the upper limit of normal serum TSH for diagnosing subclinical
hypothyroidism. There is a trend with new TSH assays to decrease the upper limit of normal TSH range
from 4.5–5.0 mU/L to 2.5 mU/L. This upper limit is recommended by the National Academy of Clinical
Biochemistry guideline, based on the fact that 2.5 mU/L represents more than two standard deviations
above screened euthyroid volunteers [39].
In conclusion, screening and treatment of subclinical hypothyroidism is recommended in women with
RPL. Thyroxine administration seems to be effective in reducing the number of miscarriages when given
during the early stages of pregnancy [40].
Thyroid Autoimmunity and Pregnancy Loss

Autoimmune thyroid disease is the most common endocrine disorder in women of reproductive age, with
a whole prevalence of 10%–15% [41], and among pregnant women of 5%–20% [42].
Although TPOAb predispose to hypothyroidism, the majority of women having TPOAb are euthyroid
[26.] However, an association between thyroid autoimmunity and RPL has been found in a meta-analysis of
13 studies. The odds of miscarriage with thyroid autoantibodies (TAI) was increased for RPL women (OR
4.22; 95% CI 0.97–18.44; 3 studies; n = 221) and it was even higher in women with thyroid autoantibodies
and normal thyroid function (OR 1.86; 95% CI 1.18–2.94; 10 studies; n = 2753) [43,44].
Ticconi et al. detected thyroid autoantibodies (anti-thyroglobulin [TGAb], TPOAb, or anti-TSH
receptor [TSHrAb] autoantibodies) in 28.75% of women with RPL and in 13% of the control group, with
no difference between women having two or more miscarriages. Furthermore, in the RPL group, 91.3%
of women positive for thyroid autoantibodies were also positive for other autoantibodies (mostly ANA),
compared to only 53.1% of women without thyroid autoantibodies [45]. The exact process that relates
TAI to RPL is still unclear, and the potential mechanisms are classified as thyroid dependent and thyroid
independent.
Concerning the thyroid-dependent mechanism, women with TAI might have a subtle deficiency
in thyroid hormones or the thyroid might be less able to adapt to the increased requirements of
pregnancy. Thyroid-independent mechanisms include altered humoral and innate immunity, cross-
reactivity of thyroid antibodies with extrathyroid sites, and the presence of concurrent autoimmune
diseases [46]. Pregnancy induces the immune system to maintain tolerance of the fetal semi-allograft
cell-mediated immunity is depressed, immunoglobulin secretion increases, and pregnancy-specific
proteins suppress lymphocyte function, shifting to a largely T-helper-2-(Th2) type cytokine profile
[47,48]. The dominant proinflammatory Th1 immune responses typical of TAI patients with an
increased production of IL-2, INF, and IL-17 are related to recurrent spontaneous miscarriages and
multiple implantation failures [49]. TAI may be associated with infertility, thus delaying conception;
hence when women with thyroid autoantibodies do become pregnant, they are older and have a higher
risk of miscarriage [50,51].
In women with thyroid autoantibodies and serum TSH <2 mU/L, treatment is not warranted;
however, serum TSH and free T4 should be measured later in gestation, preferably at the end of the
second trimester [52]. However, for women with thyroid autoantibodies and TSH between 2–4 mU/L
in early gestation, treatment with thyroxine should be considered [60]. Selenium plays a key role
in thyroid homeostasis, being integrated into the molecular structure of several thyroid enzymes
critically involved in the protection against oxidative damage [53]. Several studies have suggested that
selenium treatment reduces antibody levels, allowing a lower dosage of thyroxine supplementation
with beneficial effects on mood and health-related quality of life in patients with Hashimoto thyroiditis
[54]. Unfortunately, there are no randomized studies evaluating selenium supplementation in women
with RPL [55].
Diabetes Mellitus and Pregnancy Loss

Pregestational diabetes type 1, type 2, and other rare types of diabetes complicate 0.5%−1% of all
pregnancies [56]. Many studies have shown that patients with diabetes have a significantly increased risk
of spontaneous abortion, preterm labor, hypertensive disorders, and operative deliveries [57–59]. The
main underlying cause is lethal embryonic malformations due to glucose teratogenicity at high levels if
diabetes is poorly controlled in the periconceptional period and first trimester [40,60,56]. A large UK
study showed that the odds of a malformation increase by 30% for each percentage (11 mmol/mol) of
HbA1c, suggesting that periconception HbA1c is the most important predictor of malformations with a
linear risk between 6.3% and 11% (45–97 mmol/L) [61].
Current evidence has shown that well-controlled diabetes is not a risk factor for RPL, and the focus
should first be optimal metabolic control of diabetic women during the preconceptional period. Regarding
treatment, metformin is a low-risk and effective oral hypoglycemic agent for type 2 diabetes and is
considered safe and effective for gestational diabetes [26].
Polycystic Ovary Syndrome, Insulin Resistance, and Pregnancy Loss

It has been estimated that 40% of pregnancies in women with polycystic ovary syndrome (PCOS) will
result in spontaneous pregnancy loss. PCOS is a complex disorder involving abnormalities in interactions
between the pancreas, the hypothalamus/pituitary, the ovaries, the liver, and adipose tissues [62].
Polycystic ovarian morphology per se is not predictive of pregnancy loss in women with PRL. Patients
with PCOS may have several underlying contributing and interrelated factors also reported in women
with RPL, such as obesity, hyperinsulinemia, insulin resistance, hyperhomocysteinemia, high levels
of plasminogen activator inhibitor-1 factor, hyperandrogenemia, and poor endometrial receptivity [63].
A retrospective case-control study comparing the characteristics of RPL women with PCOS and
without PCOS described significantly higher body mass index, LH/FSH ratio, postprandial blood sugar,
and homocysteine levels in women with PCOS compared to those without PCOS, with no difference in
prolactin, TSH, or fasting blood glucose [64]. Obesity can act on female reproductive function through
hyperinsulinemia and consequently effect androgen production. Hyperinsulinemia and hyperandrogenemia
are closely associated, but the presence of an independent link between hyperandrogenemia and RPL remains
contentious. Elevated androgens in the local microenvironment of the developing follicles impair follicular
development and cause anovulation in PCOS patients. Elevated androgen levels can also decrease oocyte and
embryo quality, and together with elevated insulin may have detrimental effects on endometrial development.
Several recent studies have highlighted the presence of hypofibrinolysis associated with high levels of
plasminogen activator inhibitor-1 (PAI-1) as a possible cause of RPL in women with PCOS [65,66]. PAI-1
levels are associated with dyslipidemia, hyperinsulinemia, and hypertension, three factors that contribute to
the establishment of hyperhomocysteinemia [67] that can once more elevate PAI-1 level, eventually leading
to thrombosis. A recent case-control study found higher levels of maternal serum fructosamine (a marker of
glycemic control) in women with RPL as compared to controls, which could indicate an association between
subclinical glucose intolerance and RPL; however, further evaluation is required [68,26].
In patients with PCOS, metformin was found to significantly reduce the incidence of miscarriage
[69]. One mechanism could be enhancement of luteal phase uterine vascularity and blood flow that
could reduce the incidence of first trimester spontaneous miscarriage [70,71]. Based on the available
data, it could be suggested that treatment with metformin increases the chance of a live birth in women
with PCOS and a history of recurrent pregnancy loss; however, no properly designed placebo-controlled
studies have been conducted on women with RPL and PCOS.
Elevated FSH and Pregnancy Loss

An increased level of basal follicle-stimulating hormone (FSH), low level of anti-Müllerian hormone (AMH),
and low antral follicle count (AFC) have been associated with increased miscarriage rates [72]. In addition,
a high incidence of diminished ovarian reserve has been observed among women with recurrent pregnancy
loss [73]. Diminished ovarian reserve (DOR), defined as altered ovarian reserve markers with regular
menstrual cycles, can be also seen in the general population of young women conceiving naturally and is not
necessarily considered as a pathological entity [74]. DOR can also be a consequence of a partial destruction
of the primordial follicular pool due to chemotherapy, surgery on the ovaries (oophorectomy, cystectomy),
autoimmune oophoritis, or genetic factors such as permutations in the FMR1 gene. Furthermore, ovarian
aging leads to reduced ovarian reserve associated with an increase in fetal aneuploidy and miscarriage,
which makes the investigation of a direct relationship between DOR and pregnancy loss complicated.
The underlying challenge present in certain women with unexplained RPL may rely on the quality and
quantity of their oocytes. In a retrospective comparative analysis, Trout et al. [75] measured FSH levels on
day 3 of the cycle and estradiol (E2) in patients with unexplained RPL and in control RPL patients with
a known etiology. Women with unexplained RPL were found to be more likely to have abnormal ovarian
reserve, with an elevation of FSH and/or E2 compared to the control group. However, the evidence from
the current medical literature is questionable: (i) most studies were carried out in an infertile population,
(ii) the sample sizes were small, and (iii) no study evaluated DOR of different origins [1]. Therefore, it can
be concluded that assessment of ovarian reserve is not a diagnostic test, but a screening tool; an abnormal
test does not exclude the possibility of a live birth, so complete counseling is recommended.
Inhibins and Pregnancy Loss

Inhibins are nonsteroidal glycoproteins with important roles in reproductive physiology. Inhibin A is
the major circulating bioactive inhibin found in early pregnancy. Inhibin B is not detectable in early
pregnancy in the human [76]. Although the major function of inhibin is in the negative feedback control
of gonadotrophin secretion, inhibins can also promote and modulate placental secretory activity and
placental immune regulation, controlling the feto-maternal communication required to maintain
pregnancy. Circulating levels of inhibin A and pro-α C have been implicated in the process of implantation
and early pregnancy development [77].
Inhibins have also been proposed as markers of fetal viability. In the nonpregnant female, inhibins
are secreted and synthesized by both the developing Graafian follicle and corpus luteum [78,79]. In
pregnancy, however, the human placenta, syncytiotrophoblast, decidua, and fetal membranes are the
major sites of production and secretion of inhibin A and inhibin B found in the maternal serum, amniotic
fluid, and cord blood [80]. The local actions during placental growth and differentiation are mirrored
by changes in the circulating levels of dimeric inhibins and inhibin pro-a C as pregnancy progresses
[81]. Circulatory concentrations of inhibin A increase progressively in early pregnancy [82]. Studies
demonstrating lower levels of inhibin A in failing pregnancies have implicated inhibin A in the processes
of successful implantation and early pregnancy development [83].
Recently, inhibin A concentrations have been measured in the maternal circulation of spontaneously
pregnant women delivering a healthy term singleton infant, in patients with missed abortion (either
fetal demise or anembryonic gestational sac), and with complete miscarriage [81], in order to ascertain
whether inhibin A measurement might provide a rapid and useful marker of early pregnancy viability, in
comparison to hCG levels. Patients with complete miscarriage had the lowest hCG and inhibin A levels,
then missed abortion, and the highest levels were seen in ongoing pregnancies (Figure 7.2). The potential
value of inhibin A as a marker of early pregnancy complications should be examined in conjunction with
other established biochemical markers such as serum β-hCG, progesterone, and glycodelin. Muttukrishna
et al. [71] found a statistically significant correlation between serum concentrations of inhibin A and
β-hCG (the degree of correlation varied according to the population group: normal controls r = 0.55,
sporadic miscarriage r = 0.79, recurrent miscarriage r = 0.66). The study by Muttukrishna et al. [71]
confirms a statistically significant positive correlation between inhibin A and β-hCG in the women who
had live births (r = 0.46, P = 0.4) but not in those who had a miscarriage. Given the small size of this
and previous studies, it is not possible at this stage to establish whether serum inhibin A is a better marker
than β-hCG, or whether combined inhibin A and β-hCG measurement is superior to β-hCG alone. Further
studies are required to address these two questions [84,85].
5
Serum inhibin A (MoM)
0
Healthy controls Ongoing Failing Incomplete Complete
Threatened abortion Miscarriage
FIGURE 7.2 Maternal serum inhibin A levels in healthy pregnant women (control), patients with threatened abortion
with ongoing and failing pregnancy, and incomplete and complete miscarriage. Individual values are plotted (expressed as
mean of mean) and horizontal bars represent the group medians. *P, 0.05, **P, 0.001, ***P, 0.001 versus healthy controls
and threatened abortion with ongoing pregnancy.
Endometriosis, Progesterone Resistance, and Early Pregnancy Loss

Endometriosis is one of the most frequent causes of infertility and chronic pelvic pain affecting 1 in 10
women of reproductive age [85]. Women with endometriosis are twice as likely to have infertility [86] and
miscarriage [87]. While an inflammatory response is essential for both menstruation and implantation
[88], chronic inflammation is disruptive and a significative cause of menstrual bleeding disorders and
infertility [89]. Endometriosis results in systemic and local cytokine expression modifications that
overwhelm normal endometrial function [65–67,90]. Strong evidence exists to suggest that endometrial
changes are related with decreased cycle fecundity as a result of endometriosis [89]. Early studies on
endometrial proteins that participate in embryo attachment and invasion reported an endometriosis-
associated decrease in expression of key proteins [89,92], such as anb3 integrin and L-selectin ligand.
The changes in endometrial gene expression associated with defective endometrial receptivity due to
a significant decrease in the number of progesterone receptor isoforms, particularly the PR-B isoform,
reflect a shift away from normal progesterone action [93]. As progesterone’s action is impaired in
endometriosis, endometriosis may mimic progesterone withdrawal and thereby stimulate a premature
inflammatory (premenstrual) response [94], elevated levels of secretory-phase estrogen receptor (ESR1)
resulting in an excessive estrogen activity. The shift toward estrogen dominance induces factors that
promote inflammation, inadequate differentiation of the stroma, remodeling of the endometrium,
angiogenesis, cell proliferation, and immunosuppression, leading to a non-receptive endometrium for
implantation.
Treatment of endometriosis has been shown to be beneficial for future fertility and improved pregnancy
outcomes. Studies from IVF have documented decreased pregnancy rates that can be improved with
GnRH agonist (GnRHa) suppression, surgery, or aromatase inhibitor therapy. Although previous studies
on donor oocytes have shown that the primary defect associated with endometriosis may reside in the
ovary and oocyte quality, larger and more recent investigations have suggested that defective implantation
is also likely involved [89].
The changes in systemic and local cytokine expression that destroy normal endometrial function, such
as p450 aromatase overexpression and leukemia inhibitory factor (LIF) downregulation, are reversible by
surgical removal of endometriomas [95]. Mild and severe endometriosis have both been associated with a
higher prevalence of miscarriages compared with control women; this relationship was stronger in mild
endometriosis (rASRM I/II) than in severe endometriosis (rASRM III/IV). Early stages of the disease
with more active lesions are known to lead to a more inflammatory milieu [91] than the more scarring
lesions of higher disease stages [95].
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8
The Etiology of the Antiphospholipid Syndrome
Sara De Carolis, Giuseppina Monteleone, Cristina Garufi,

Rotem Inbar, Miri Blank, and Yehuda Shoenfeld
Introduction
Phospholipids (PL), the basic components of all cell membranes, consist of two layers. The inner layer
contains negatively charged anionic alcohol groups facing the cytoplasm, and the outer layer contains
neutral or zwitterionic alcohol groups facing the extracellular fluid or bloodstream. In certain conditions
such as ischemia, cell injury, or autoimmunity, negatively charged PLs can be exteriorized. The
exteriorized PLs may be an antigenic stimulus for the production of antiphospholipid antibodies (aPL) or
permit a number of serum proteins with procoagulant activity (β2-glycoprotein I [β2-GP1], prothrombin,
protein C, protein S, and annexin V) to bind PL epitopes and be presented to the immune system in unique
“neoantigenic” conformations, which may induce aPL formation [1]. aPL may recognize either the PL
region of the complex or an epitope consisting of the portion of the PL and neighboring aminoacyls on
the protein carrier, or may react with the protein alone.
In pregnancy, placental tissues are continuously remodeled resulting in the externalization of inner
surface PLs such as phosphatidyl serine (PS) [2].
aPL require a cofactor (apolipoprotein H or β2GP1), a negatively charged phospholipid binding protein,
to exert their effects. β2GP1-dependent aPL are thought to recognize their antigen on placental tissue,
inhibit growth and differentiation of trophoblasts, and cause inflammation, defective angiogenesis, and
thrombosis, leading to impaired placentation.
Molecular Mimicry
Molecular mimicry between β2GP1 bacterial and viral epitopes is the principal mechanism by which
infectious agents may induce aPL or antiphospholipid syndrome (APS) in genetically prone individuals.
The organisms most commonly associated with APS are parvovirus B19, cytomegalovirus (CMV),
toxoplasma, rubella, varicella, human immunodeficiency virus (HIV), streptococci, Staphylococci Gram-
negative bacteria, and Mycoplasma pneumoniae [3].
A molecular resemblance between β2GP1 epitopes and infectious pathogens (Haemophilus influenzae,
Neisseria gonorrheae, HP, CMV, and tetanus toxoid) have been described [4]. Moreover, proteins found
in infectious agents can cause polyclonal activation of a subset of T-lymphocytes, or polyclonal-B-cell
activation. Super-antigens may also induce a nonspecific immune response. Various organisms can
modulate the release of cytokines and chemokines which are involved in growth, differentiation, and
chemotaxis of the Th-cell population and regulate MHC class 1–2 molecule expression [5].
aPL are often associated with false-positive serological tests for toxoplasmosis, rubella, CMV, herpes,
HIV, Lyme disease, and syphilis. Indeed, the serological diagnosis of infection may be confounded by
changes in several components of the immune response leading to false-positive results [6]. Several studies
observed that in autoimmune diseases, the presence of TORCH IgM is associated with a worse outcome
of autoimmune diseases [7]. CMV IgM is more frequent in APS pregnancies than in healthy controls. In
primary APS (PAPS) and secondary APS, a poorer pregnancy outcome was observed between women
70
The Etiology of the Antiphospholipid Syndrome 71
with CMV IgM false-positivity compared to women without CMV IgM false-positivity [8]. In addition, in
healthy pregnancies the obstetric outcome is affected by the presence of false-positive TORCH antibodies
when associated with aPL [8].
The β2GP1 molecule seems to be the most significant antigen in APS. Passive transfer of anti-β2GP1
antibodies induced experimental APS in naïve mice [9]. Immunization of BALB/c, PL/J mice, or New
Zealand white rabbits with β2GPI resulted in the generation of anti-β2GPI antibodies. High titers of
mouse anti-β2GP1 antibodies have been associated with increased proportion of fetal resorption,
thrombocytopenia, and a prolonged activated partial thromboplastin time (aPTT), indicating that lupus
anticoagulant may be active in experimental APS [10].
Pathogenic anti-β2GP1 autoantibodies directed against the TLRVYK epitope have been found in mice
that were immunized with H. influenzae or N. gonorrhoeae that exhibit the TLRVYK sequence, or
with tetanus toxoid that does not present the sequence TLRVYK but could still serve as a mimotope.
Anti-β2GP1 autoantibodies have been shown to be pathogenic for experimental APS, inducing fetal loss,
thrombocytopenia, and a prolonged aPTT [11]. The pathogenic effect of monoclonal antibodies to β2GP1
is inhibited by the addition of synthetic peptides including the TLRVYK sequence. Synthetic peptides
prevented the development of APS in mice injected with monoclonal antibodies to β2GP1, or decreased
the degree of endothelial cell activation, monocyte adhesion, and the expression of adhesion molecules in
vitro [12]. A CMV-derived synthetic peptide (TIFI) with specific affinity to β2GP1 phospholipid binding
site has been shown to inhibit the adhesion of the aPL molecule to the trophoblast cell membrane in vitro
in a dose-dependent manner [13]. These findings correlated with the protective effect of TIFI observed in
animal models, in which injection of aPL at pregnancy day 0 caused increased fetal loss rate and growth
restriction [13].
Other infections, such as syphilis and Lyme disease, induce aPL that directly recognize phospholipids
without involving β2GP1, and hence do not lead to APS.
Alteration of microbiome “dysbiosis” can induce antiphospholipid syndrome in people with genetic
predispositions [14]. Segmented filamentous bacteria (SFB) influence T cells-phenotype, T-dependent,
and T-independent antibody production [15]. If homeostasis is disrupted (by infections or drugs),
proinflammatory interactions could occur with local and systemic effects on the immune system. These
effects include breaches of the mucosal barriers and generation of commensal specific memory T cells
and autoantibodies. Therefore, commensal bacteria may promote breaks in tolerance and the induction
of persistent aPL in predisposed individuals. Roseburia intestinalis, which is prevalent in the intestine of
APS patients, has many homologous sequences to both the major B and T cell epitopes and thus could
stimulate lymphocytes [14].
Recently, a novel syndrome has been described: autoimmune syndrome induced by adjuvants (ASIA),
including vaccines [16], infectious agents, silicone [16], pristane, and aluminum salts. Infectious agents
and vaccines have many similarities in facilitating antibody production, immune reactions, and a wide
spectrum of autoimmune phenomena. Many adjuvants have been found to trigger autoimmunity by
themselves [17]. Several vaccines have been correlated to the onset of APS, e.g., tetanus toxoid and
seasonal influenza [16]. In addition, induction of experimental APS by immunization with tetanus toxoid
added to different adjuvants has led to different effects on fertility [18]. Furthermore, immunization with
Complete and Incomplete Freund’s adjuvant induced specific pathogenic β2GP1-dependent autoantibodies
in heterozygous factor V Leiden mice. The intriguing finding in this study was the induction of high levels
aPL following adjuvant immunization alone [16].
Mechanisms of Reproductive Failure in APS

Genetic Predisposition
A higher prevalence of aPL has been seen in the serum of patients of similar descent, and in mouse models
[19]. The most consistent association is with HLA-DR4 and DRw53 [19]. Genetic association studies have
shown a significant correlation of the polymorphisms involved in blood coagulation and proinflammatory
states (TLR4) with thrombotic APS [20]. C1D (DNA binding and apoptosis-inducing protein) is a risk
factor for obstetric APS by preventing extravillous trophoblast differentiation in some early miscarriages.
C1D might be a possible cause of pregnancy complications in APS [21].
APS may require two “hits”; the initial hit may lead to the production of anti-β2GP1 antibodies, and
infectious agents may be the second hit, leading to APS by activating Toll-like receptors or complement
[22]. However, the two-hit hypothesis does not explain why some people with aPL have no features of
the disease. Pathogenicity may depend on structural differences in epitope specificity or glycosylation of
the antibodies that may cause modifications of effector functions. There is hyposialylation in the glycans
terminate portion of anti-β2GPI IgG determining a pro-inflammatory action.
The majority of circulating β2GPI contains unpaired free thiols which constitute the reduced form of
β2GPI [23]. The free thiols exposed on β2GPI are involved in the interaction with platelets and endothelial
cells. This pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules
from oxidative stress. Post-translational modifications of cysteines include the addition of oxygen or
nitrogen oxide (NO) or glutathione, and are enhanced under oxidative or nitrosative stress (e.g., infections).
The oxidation of β2GPI may increase the immunogenicity of the molecule through a TH1 mechanism,
inducing the maturation of dendritic cells. Mature cells secrete interleukins, such as IL-12, IL-1, IL-6,
IL-8, IL-10, and tumor necrosis factor alpha (TNF-α) [19].
Thrombosis
The hypercoagulable state in APS involves all three major components governing hemostasis: platelets,
fibrinolysis, and the coagulation cascade. aPL inhibit both protein C activation and the function of
activated protein C (APC), thereby preventing the inactivation of activated factor V and VIII [24].
Inhibition is dependent on the presence of β2GP1, which is a prerequisite for the binding of aPL to
protein C. Autoantibodies directed against protein C, protein S, and thrombomodulin have been reported
in some APS patients [25].
Other mechanisms may be responsible for thrombogenesis. aPT antibodies may induce TF expression
and improve the binding between prothrombin (PT) and the surface of endothelial cells, leading to
thrombogenesis. Other studies have shown that thrombotic activity is related to the presence of
antiphosphatidylserine-prothrombin complex (aPS/PT), rather than aPT itself. This complex is more
frequently found in patients with lupus anticoagulant (LA), but its association with thrombosis seems to
be independent of the presence of LA [26]. APT might also bind thrombin, preventing its inactivation
by antithrombin (AT) [27]. Thrombin activation can lead to platelet activation. Tissue factor−related
procoagulant activity and tissue factor mRNA levels in monocytes are increased in PAPS with thrombosis
when compared to those without thrombosis.
Tissue factor pathway inhibitor (TFPI) antibodies may impair TFPI activity and contribute to
hypercoagulability due to the coexistence of protein C IgG antibodies that indicate an increased
disposition to thrombosis [28]. Further studies have suggested that anti-β2GP1 antibodies increased the
thrombotic response in animal models, particularly the antibodies directed against the first domain of
β2GP1 [29]. The anti-β2GP1/β2GP1 complex can induce expression of TF in monocytes by the activation
of mTOR protein kinase [30].
Potentiation of the procoagulant activity of human umbilical vein endothelial cells (HUVEC) by
aPL is strongly decreased after depleting IgG from the serum [31]. Human anti-β2GP1 IgM monoclonal
antibodies and polyclonal anti-β2GP1 antibodies induce tissue factor at both protein and mRNA levels in
HUVEC monolayers in vitro [32]. aPL can further upregulate adhesion molecules (E-selectin, ICAM-1,
and VCAM-1) expression and secretion of the proinflammatory cytokines IL-1b and IL-6. Increased
plasma levels of soluble VCAM-1 have been found in PAPS patients with recurrent thrombosis.
Decreased endothelial cell prostacyclin2 (PGI2) and increased thromboxane A2 (TXA2) production
by platelets may predispose to thrombosis. aPL enhance platelet TXA 2 production and allow platelet
activation [33].
A minor degree of platelet activation can lead to exposure of phospholipids, which can potentially be
amplified in the serum of APS patients [34]. β2GP1 initially binds to these phospholipids and then binds
aPL to form β2GP1-phospholipid complexes. These complexes can further activate platelet aggregation
by allowing the interaction between the Fc portion and the platelet surface FcγRII receptors [34,35].
Furthermore, aPL may influence the placental circulation by attacking certain placental epitopes such
as Annexin A5, a potent anticoagulant protein. Annexin V, found on the apical surface of placental
syncytiotrophoblast, forms a protective shield on the phospholipid surface, blocking phospholipids from
becoming available for coagulation reactions. The annexin-V shield could be damaged by either binding
to anti-annexin-V or preventing its binding to the PL membrane, or by blocking autoantibodies against
annexin-V/PL [36]. Anti-annexin-V autoantibodies have been detected in patients with systemic lupus
erythematosus (SLE) and APS associated with pregnancy loss, while reduced levels of annexin-V have
been observed on the placental villi of women with aPL, recurrent pregnancy loss, and a thrombogenic
background [37].
Arachidonic Acid and Prostacycline

aPL inhibit arachidonic acid release [38] (an essential prerequisite for prostacycline production) and
increase the concentration of TXA2, thus altering the thromboxane/prostacycline balance [39]. The
alteration in the PGI2/TXA2 balance leads to vasoconstriction, which impedes the blood supply to the
fetus, and platelet activation with the procoagulant effects.
In a mouse model of experimental APS, Shoenfeld and Blank [40] infused aCL to pregnant mice in
order to induce APS. Mice that were co-treated with a thromboxane receptor antagonist had a significant
reduction in the fetal resorption rate from 45% to 19.8% and an increase in mean placental and embryo
weights. There was also an increased platelet count in treated mice, indicating the effect of thrombocyte
aggregation in APS.
Inflammatory Responses
During pregnancy, an imbalance in the maternal immune response toward a proinflammatory response
(involving complement, TNF, and chemokines) has been linked to aPL-induced fetal loss in animal
models [41]. Following fetal resorption due to injection of IgG with aPL activity to pregnant naïve mice,
histological examination of the decidua revealed deposition of human IgG with mouse complement,
neutrophil infiltration, and local TNF secretion.
In animal models, Pierangeli et al. [42] have shown that inhibition of the complement cascade in
vivo, using C3 convertase inhibitor, blocks aPL-induced fetal loss and growth retardation and inhibits
aPL-mediated thrombosis. Mice deficient in complement C3 and C5 showed resistance to thrombosis,
endothelial cell activation, and fetal loss. Hence, complement activation may be critical in the pathogenesis
of thrombosis and fetal loss associated with aPL [42].
Factor H, a complement inhibitor, has structural similarity to β2GP1. A significant increase in levels
and frequencies of Factor H autoantibodies was found in cohorts of patients with APS compared with
matched healthy controls in PAPS and secondary antiphospholipid syndrome (SAPS). Factor H interacts
with various types of cells, particularly when these are damaged or contain deposits of C3b resulting from
activation of the complement cascade. [43]
In the placentas of APS patients, there was increased deposition of complement products C4d and C3b
compared with normal subjects [44]. Mild hypocomplementemia and low C3, C4 levels were reported
in some studies including patients with APS with no other associated systemic autoimmune diseases
[45–48]. Lower C3 and C4 levels at the baseline and at the end of pregnancy have been reported to
significantly correlate with poor pregnancy outcome [48]. Moreover, increased plasma levels of the
activation products Bb and C5b-9 have been reported in women with aPL and adverse pregnancy
outcomes. Activation products are considered to be a more sensitive marker of complement activation
and may promote leukocyte recruitment/activation and the release of proinflammatory and antiangiogenic
mediators responsible for placental damage [49].
Hypocomplementemia may be a prognostic factor for poor pregnancy outcome in APS patients, which
could be used to identify APS pregnancies at higher risk of obstetric complications [6]. The protective
role of heparin in APS in a mouse model has even been related to the anticomplement effect rather than
anticoagulant activity [50].
It has been reported that aPL, by activation of toll-like receptor 4 (TLR4), induce uric acid production in
response to human trophoblast, which in turn activates the Nalp3/ASC (apoptosis-associated speck-like protein)
inflammasome complex, leading to IL-1β and IL-8 secretion with a strong inflammatory response. [51].
The anti-inflammatory cytokine, IL-3 is important for the maintenance of normal pregnancy. IL-3
enhances placental and fetal development while increasing the number of megakaryocytes. The serum
level of IL-3 in pregnant patients with PAPS or APS secondary to SLE has been found to be lower than
in controls. In vitro studies revealed that low dose-aspirin stimulates IL-3 production [52].
Other cytokines may be involved in the etiology of APS. The level of the proinflammatory and
prothrombotic cytokine TNF-α was shown to be significantly higher in patients with APS than healthy
controls. This mediator links complement C5a-C5aR interactions and pathogenic aPL to fetal damage
[53]. aPL that target decidual tissue cause a rapid increase in decidual and systemic TNF-α levels. Studies
on mice have suggested that miscarriages induced by aPL are less frequent in the presence of TNF-α
deficiency or TNF-α blockade. In humans, TNF-α increases throughout pregnancy and has been related
to miscarriages, fetal losses, PE, and preterm birth as well as IL-10 reduction [54].
Trophoblast cells expressing the surface antigen CD1d bear phosphatidylserine (PS). Anti-β2GP1
antibodies have been shown to interact with the PS-bearing CD1d, causing release of IL12 and induction
of IFNα production, thus providing additional evidence that APS-related pregnancy loss involves an
inflammatory mechanism [55].
Defective Endometrial Angiogenesis

Endometrial angiogenesis, decidualization, trophoblast invasion, and uterine vessel remodeling are all
crucial for successful pregnancy. Matrix metalloproteinases (MMP) have an essential role in the process
of basement membrane and matrix degradation, which enables trophoblast invasion. aPL-mediated
inhibition of trophoblast invasion is one possible mechanism leading to recurrent pregnancy loss [56].
aPL may affect the maternal side of the placenta by directly binding human endometrial endothelial cells
(HEEC), modulating VEGF and MMP activity, and thus leading to a significant reduction in angiogenesis
both in vitro and in vivo, and consequently reducing the number and total length of the capillaries formed
in HEEC. aPL reduce both VEGF and MMP production and NFKB DNA (a promoter gene for several
MMP) binding activity in a dose-dependent manner [57].
Quao et al. [62], using an aPL that binds to domain V of β2GPI, found that HEEC proangiogenic (VEGF,
PlGF) and antiangiogenic-factor production (Flt-1) was augmented, while basal chemokine secretion
(MCP-1, G-CSF, GRO-α) was inhibited [58].
Flt-1 contributes to poor placentation by impairing endothelial function, impacting uterine vessel
remodeling, and blocking the action of VEGF and PlGF, which promote trophoblast differentiation,
invasion, and angiogenesis. In addition, aPL suppress HEEC secretion of the chemokines required for
recruiting the macrophages and natural killer cells necessary for spiral artery remodeling [59]. Hence,
aPL may contribute to shallow trophoblast invasion and disrupted spiral artery remodeling seen in
obstetric APS by directly impacting the uterine endothelium [60]
A complementary study [61] demonstrated the beneficial effect of low molecular weight heparin
(LMWH) on aPL inhibited HEEC angiogenesis in an in vitro model. The addition of LMWH restored
VEGF secretion and MMP activity explaining the improved pregnancy outcome in aPL patients treated
with LMWH.
Abnormal Placentation
During the course of pregnancy, trophoblast fragments are shed into the maternal circulation. In
normotensive pregnancy, trophoblast debris may be the result of apoptosis. In preeclampsia, the process
may be more necrotic [62]. aPL may increase the amount of necrotic trophoblast debris from placental
explants, which may activate endothelial cells. In a study by Pantham et al. [63], RNA from first trimester
placentas treated with aPL was extracted and genomic data analyzed using microarrays. Changes in the
transcriptome of placentas explants were observed, including the mRNA of multiple genes involved in
the regulation of apoptosis [63].
Viall et al. [64] summarized the involvement of placental cells in APS due to the following features:
placental infarction, hypovascular villi, impaired spiral artery remodeling, decidual inflammation,
increased syncytial knots, decreased vasculosyncytial membranes and substantially more fibrosis, and
infarcts [64].
• Placental infarction is caused by the impairment of uteroplacental blood supply due to spiral
artery occlusion by an intraluminal thrombus. However, placental infarction cannot explain all
cases of obstetric morbidity in women with aPL.
• Decidual inflammation is stimulated by the invasive extravillous trophoblasts, as it has been
demonstrated that macrophages cluster around extravillous trophoblasts. The interaction of
aPL with TLR4 on extravillous trophoblasts may result in the production of proinflammatory
chemokines and cytokines.
• Syncytial knots may represent structures that are destined for extrusion from the surface of the
syncytiotrophoblast into the maternal blood.
• Vasculosyncytial membranes are thin regions of the syncytiotrophoblast that are specialized for
maternofetal exchange. Fetal nutrient and oxygen supply may be limited in the third trimester
when these structures are usually abundant but decreased in the presence of aPL.
Moreover, decidual vasculopathy is characterized by histopathological change such as atherosis of the

spiral arteries, that can be found in association with intraluminal thromboses or can itself occlude a spiral
artery if severe.
From a clinical point of view, uterine artery Doppler velocimetry provides a noninvasive indirect
method of studying the uteroplacental circulation and strongly suggests impaired trophoblastic invasion
of the placental bed, before any clinical sign of preeclampsia or fetal growth restriction [6]. The abnormal
results of uterine artery Doppler velocimetry are strongly associated with poor pregnancy outcomes. A
normal uterine artery velocimetry has been shown to be a negative predictive factor for maternal-fetal
complications [6], while abnormal uterine artery velocimetry indicates a high risk of maternal-fetal
complications.
Triple, Double, and Single aPL Positivity

The negative role of multiple aPL positivity has been confirmed by several studies [65]. The combination
of triple aPL positivity with a previous history of thrombosis represented the worst risk profile for adverse
pregnancy outcome. The presence of triple positivity for aPL is due to antibodies directed to the first
domain of β2GPI (DmI). DmI is strongly associated with APS, because the recombinant deletion mutants
of β2GPI are able to interact with β2GPI-autoantibodies only if DmI is present [66]. High-risk patients
with APS and triple laboratory positivity (compared to double and single positivity) had a significantly
higher titer of circulating IgG anti-DmI antibodies. Double and single positivity correlate with a low titer
or absence of IgG anti-DmI antibodies. Single positivity for the aPL profile seemed to be associated with
positive tests for anti-domain 4/5, considered as nonpathogenic antibodies [67].
As regards the significance of double positivity and single positivity for aPL, the double positivity
(aCL and anti-β2GPI) was seen to be a risk factor for pregnancy loss, and anti-β2GPI antibody was a
better prognostic marker for pregnancy loss than aCL [68]. Moreover, positive tests for two or more
antiphospholipid antibodies and low complement levels have been associated with adverse pregnancy
outcomes [46].
For the role of single aPL positivity, the PROMISSE study on pregnancy outcome in women with
APS and/or SLE underlined the adverse prognostic value of LAC, in addition to the well-known adverse
role of triple aPL positivity [69]. Otherwise, the presence of anti-β2GPI IgG was the antibody associated
with the lowest live-birth rate and highest incidence of preeclampsia, IUGR, and stillbirth, compared to
anticardiolipin antibodies or lupus anticoagulant alone [70], while aCL is the most common sole aPL. The
presence of single positivity for aPL was associated with a good response to treatment [71].
Conclusions
APS is a systemic syndrome whose etiology involves both environmental and genetic factors.
Infections may play an important part in the etiology by using different mechanisms, predominantly
molecular mimicry, to induce aPL. Other environmental factors include vaccines and other adjuvants,
as demonstrated by the ASIA syndrome. aPL exert their pathogenic effects via various mechanisms,
including the induction of a hypercoagulable state, inflammatory processes, defective angiogenesis with
abnormal placentation, and alterations in placental cell death patterns.
Complement involvement is crucial in the pathogenesis of APS and it has been demonstrated by its
genetic inhibition in mice models. In addition, triple positivity is associated with recurrent miscarriage
and fetal loss/stillbirth so may indicate the need for nonstandard treatment.
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9
Defects in Coagulation Factors Leading
to Recurrent Pregnancy Loss
Aida Inbal and Howard J.A. Carp
Introduction
The evidence for pregnancy loss having a thrombotic basis is due to the association between
antiphospholipid antibodies (aPL) and recurrent pregnancy loss. Due to the assumption that aPL induce
thrombosis causing pregnancy loss, it has been assumed that any prothrombotic state may also increase the
chance of pregnancy loss due to a thrombotic mechanism. Hereditary thrombophilias have been classified
as (i) defects in coagulation inhibitors (antithrombin, protein C, protein S, tissue factor pathway inhibitor,
and thrombomodulin deficiency); (ii) increased levels or function of pro-coagulation factors (factor V
Leiden [FVL], prothrombin gene mutation G20210A, dysfibrinogenemia and hyperfibrinogenemia, and
increased levels of factors VII, VIII, IX, and XI); (iii) hyperhomocysteinemia, mainly due to C677T
homozygosity for the methylenetetrahydrofolates reductase (MTHFR) gene; (iv) defects of the fibrinolytic
system, involving plasminogen, tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI),
thrombin-activatable fibrinolysis inhibitor (TAFI), factor XIII, and lipoprotein A; and (v) altered platelet
function (platelet glycoproteins GPIb-IX, GPIa-IIa, and GPIIb-IIIa).
Deficiencies of coagulation factors (F) such as FI, FII, FV, FVII, FX, FXI, and FXIII are general
bleeding disorders and pose unique problems for women due to their impact on reproductive health
[1]. Gynecological and obstetric manifestations include miscarriage, bleeding during pregnancy, and
postpartum hemorrhage (PPH). During pregnancy, monitoring the levels of clotting factors determines
the need for prophylactic therapy; hemostatic cover can minimize PPH [1]. Deficiencies of factor XIII
(FXIII) and fibrinogen are associated with pregnancy loss. Both these bleeding diatheses are associated
with impaired wound repair in addition to pregnancy loss and excessive bleeding. This chapter deals with
the association between decreased or increased levels of coagulation factors and pregnancy loss. The
various factors and their association with the trophoblast are shown in Figure 9.1.
Bleeding Diatheses Leading to Pregnancy Loss

Hereditary Factor FXIII Deficiency
Coagulation factor XIII (FXIII) is plasma transglutaminase that participates in the final step of the
coagulation cascade. Following activation by thrombin the active form—FXIIIa—cross-links fibrin
chains through γ-glutamyl-ε-lysine bonds, creating a stable clot resistant to fibrinolysis. In plasma, FXIII
circulates as a heterotetramer (A2B2) composed of two catalytic A subunits (FXIII-A) and two carrier
B subunits (FXIII-B) [2].
The concentration of plasma FXIII decreases during pregnancy, reaching 50% of normal at term.
Likewise, the activity of FXIII-A is significantly decreased at the time of miscarriage [3]. FXIII
deficiency is a hereditary bleeding disorder characterized by severe bleeding manifestations, delay in
wound healing and recurrent miscarriages in homozygous women [2,4]. Women who are homozygous
for FXIII deficiency will have obstetric issues including bleeding diatheses, PPH, and miscarriages.
79
Procoagulant effect
FXIII HCY FNG ↑TG FVL FII CYT, IL-6 TNF-α MP
Syncytiotrophoblast
Cytotrophoblast
PC PS AT TFPI FS CYT, IL-4 IL-10
Anticoagulant effect
FIGURE 9.1 Procoagulant and anticoagulant balance of trophoblast. Key: AT = antithrombin, FII = prothrombin
gene mutation (G20210A), FNG = fibrinogen, FS = fibrinolytic system, FVL = factor V Leiden, HCY = homocysteine,
PC = protein C, PS = protein S, TFPI = tissue factor pathway inhibitor, ↑TG = increased thrombin generation,
MP = microparticles.
In fact, homozygous women will have up to 66% miscarriage rates [5] and will not carry the pregnancy
to term unless treated with FXIII concentrate throughout pregnancy [4]. The minimal level of FXIII-A
required for normal pregnancy is unknown; however, only 0.5%−2% of FXIII-A is required for normal
hemostasis [6].
The mechanism by which FXIII supports normal pregnancy is unknown. FXIII is essential for
implantation, placental attachment, and further placental development by cross-linking not only between
fibrin chains but also between fibronectin and collagen, the major components of connective tissue
matrix [6]. Hence, FXIII seems to play an essential role in the interaction between the blastocyst and
the endometrium at implantation. FXIII-A also cross-links fibrin(ogen) and fibronectin, both of which
maintain the attachment of the placenta to the uterus [7]. FXIII deficiency may result in periplacental
hemorrhage and subsequent spontaneous fetal loss. Pregnant FXIII-A-subunit knockout mice have
excessive uterine bleeding followed by embryonic demise [8]. FXIII-A is present in the extracellular
space of the extravillous cytotrophoblast shell adjacent to Nitabuch’s layer [9] and has been colocalized
with fibrinogen and fibronectin at Nitabuch’s layer [10]. FXIII-A has been reported to be absent from
the placenta bed in women with FXIII deficiency, leading to deficient cytotrophoblastic shell formation
[10]. Thus, deficiency of FXIII-A at the site of implantation will adversely affect fibrin-fibronectin cross-
linking resulting in detachment of the placenta from the uterus and subsequent pregnancy loss [8,10].
FXIII-A has been shown to have proangiogenic activity both in vitro and in vivo [11]. Since embryo
implantation requires adequate angiogenesis, the supportive role of FXIII in implantation may be partly
due to its proangiogenic activity.
In FXIII-deficient women, administration of FXIII throughout pregnancy results in successful
outcomes [3,6]. Concentrates are available with a half-life of 10–12 days. However, the timing and dose of
FXIII replacement and the optimal level of FXIII remain unknown. The level of plasma FXIII generally
achieved for successful pregnancy is 10% in women with FXIII deficiency. We treat pregnant women
prophylactically with 20 IU/kg of FXIII concentrate every 4 weeks to achieve a FXIII level of above 3%.
A booster dose of 1000 IU is also given before amniocentesis or labor.
Other Alterations in Factor XIII

It is unknown if there is an association between normal or decreased levels of FXIII and recurrent
pregnancy loss. Whereas plasma FXIII-B concentrations increase during pregnancy, FXIII-A tends to
Defects in Coagulation Factors Leading to Recurrent Pregnancy Loss 81
decrease, resulting in an overall steady reduction in plasma FXIII reaching approximately 50% of normal
at term [14]. The A subunit rises with the onset of labor and falls postpartum [12]. This is in contrast to
the progressive increase in levels of fibrinogen and factors VII, VIII, IX, and X during pregnancy [13]. In
a cohort of non-FXIII-deficient women with a history of two or more first trimester miscarriages, plasma
FXIII levels were not found to be predictive for subsequent pregnancy loss [14]. A substitution of Tyr by
Phe at position 204 in exon 5 of the FXIII-A gene was found in one study to be more prevalent in women
suffering three or more miscarriages [15]. Pasquier et al. [16] measured FXIII-A and FXIII B-subunit
antigen levels in 264 women with two or more unexplained consecutive miscarriages at or before 21
weeks of gestation, or at least one later pregnancy loss. The control group consisted of 264 women with
no history of miscarriage and at least one living child. Overall, there were no differences in FXIII-A
and FXIII-B levels between patients and controls. Hence, in the general population, pregnancy loss does
not seem to be associated with reduced plasma FXIII levels. Whether locally reduced FXIII-A levels or
impaired FXIII function in the placenta may contribute to an increased risk of pregnancy loss remains
to be investigated.
Fibrinogen Deficiency
Thrombin cleaves fibrinogen to its fibrin monomer, which then polymerizes and is stabilized by FXIII.
Fibrin(ogen) is also a target for fibrinolytic factors that dissolve excess fibrin to maintain vascular patency
and integrity. Fibrinogen is also a primary bridging molecule, linking activated platelets together via their
glycoproteins IIbIIIa [17].
The three overlapping hereditary abnormalities of fibrinogen—afibrinogenemia, dysfibrinogenemia,
and hypofibrinogenemia—have been associated with recurrent pregnancy loss. Afibrinogenemia—a
defect in hepatic fibrinogen secretion or release—is inherited as an autosomal recessive trait and is
associated with bleeding diathesis, impaired wound repair, and recurrent pregnancy loss. A related
form of this disorder is hypofibrinogenemia. Hereditary dysfibrinogenemias are characterized by the
biosynthesis of structurally and functionally abnormal fibrinogen.
Brenner [18] has reported that women with dysfibrinogemia may be predisposed to miscarriage. Of
64 pregnancies in women with dysfibrinogemia, 39% terminated in miscarriage. The mechanisms have
been reviewed by Mosesson [19].
Hypofibrinogenemic women [20] and experimental afibrinogenemic mice [21] have bleeding tendencies,
miscarriage, and abnormal scar formation. Based on the mouse model, absence or a significant decrease in
maternal fibrinogen is sufficient to cause rupture of the maternal vasculature, thereby affecting embryonic
trophoblast infiltration and leading to hemorrhage and subsequent miscarriage.
Cryoprecipitate, fresh-frozen plasma, and fibrinogen concentrate are the sources of fibrinogen
commercially available. Replacement therapy throughout pregnancy is feasible for patients with pregnancy
losses [22]. It has been suggested that the minimal level of normal fibrinogen to maintain pregnancy
is about 60 mg/100 mL [23]. A cryoprecipitate infusion of 0.2 bags/kg body weight (approximately
250 mg/bag) will raise the fibrinogen concentration to 100 mg/dL. Since the half-life of fibrinogen is
approximately 4 days, two weekly infusions of cryoprecipitate during the gestational period should be
sufficient to keep the fibrinogen level above 60 mg/dL and prevent pregnancy loss.
The benefits of substitution therapy should be weighed against the possibility of inducing thrombosis.
Catastrophic thrombosis has been reported during fibrinogen replacement therapy in patients with
afibrinogenemia and dysfibrinogenemia [24]. Prophylactic heparin or LMWH has been advocated for
the peripartum period in these patients.
Thrombophilias
The hereditary thrombophilias cause increased tendency to venous thrombosis and comprise a number of
conditions such as antithrombin, protein C, protein S deficiency, FVL, prothrombin gene (FII) mutation
G20210A, and increased FVIII. There are also various acquired hypercoagulable states, the most common
of which is antiphospholipid syndrome, which is discussed elsewhere. Proteins C and S and antithrombin
are physiological anticoagulants. Deficiencies of these anticoagulants are uncommon [25]. FVL is the
most common cause of inherited thrombophilia [25]. FVL slows down the proteolytic inactivation of
factor Va, by activated protein C (termed activated protein C resistance [APCR]), which in turn leads
to the augmented generation of thrombin. In the G20210A mutation there is more efficient mRNA
processing of the prothrombin gene, which in turn is associated with an increased level of prothrombin
and generation of thrombin.
Thrombosis in Decidual Vessels

The evidence for pregnancy loss having a thrombotic mechanism rests on three pillars: demonstration of
thrombosis in decidual vessels, increased prevalence of thrombophilias in recurrent pregnancy loss, and
a higher incidence of pregnancy loss in the presence of thrombophilias. The investigation of thrombosis
in decidual vessels has been inconsistent. In severe pregnancy complications, including fetal death,
preeclampsia, preterm labor, intrauterine growth restriction (IUGR), or stillbirth, histopathological
examination has revealed vascular hypoperfusion. There are few studies which describe maternal vessel
thrombosis as such, making it difficult to confirm thrombosis as the mechanism of the hypoperfusion.
Arias et al. [26] evaluated 13 placentae of women with preeclampsia, preterm labor, IUGR, or stillbirth.
Ten of 13 women (77%) had thrombophilias, including aPL, protein C, S, and antithrombin deficiencies,
APCR, and FVL. The authors found fetal thrombotic vasculopathy, histologically characterized by
stem artery thrombosis, which may include occlusive or mural thrombosis sclerotic/avascular terminal
villi, hemorrhagic endovasculitis, and inflammatory damage to vessels [27]. However, these histological
changes are on the fetal side of the placenta, not the maternal side.
The fact that no specific placental lesion has been found in thrombophilia could have a number of
explanations. There may be other thrombophilias as yet unknown, which could explain the high incidence
of placental pathology, or that the lesions are the result of inflammatory changes in the placenta associated
with the underlying pathology and unrelated to thrombophilia. Even in the antiphospholipid syndrome,
thrombosis has not been convincingly demonstrated in decidual vessels, and the histological changes
are seen on the fetal, rather than maternal, side of the placenta. It seems that cell surface−associated
membrane receptors rather than soluble factors (such as thrombophilic factors) are more relevant factors
affecting pregnancy outcome [28].
The maternal spiral arteries become remodeled by pregnancy hormones and trophoblast into
uteroplacental arteries toward the end of the first trimester. In the uteroplacental arteries, the lumen is
larger, and the media is replaced by endovascular trophoblast cells. Hereditary thrombophilias predispose
to venous rather than arterial thrombosis. Even if there were thrombosis of the uteroplacental arteries,
it is unlikely that thrombosis could occur in first trimester spiral arteries which are lined by arterial
endothelium rather than endovascular trophoblast. Genetic polymorphisms of the thrombophilic genes of
the parents have a 50% likelihood of transmission to the fetus potentially affecting trophoblast function.
Thus, to determine the true risk for adverse pregnancy outcome associated with genetic thrombophilias,
it is necessary to test the fetus for these thrombophilias.
Prevalence of Thrombophilias in Pregnancy Loss

Rey et al. [29] carried out a meta-analysis of 31 studies in the literature, and reported a significant
association between hereditary thrombophilias and pregnancy loss. Since Rey et al.’s [29] meta-analysis,
a number of papers have appeared assessing the prevalence of one or more thrombophilias in certain
population groups. The results have been inconsistent.
The prevalence of hereditary thrombophilias has also been assessed in recurrent miscarriage.
Disagreements in the literature have prompted the need for meta-analyses to determine whether the
prevalence is increased. Krabbendam et al. [30] have reported a meta-analysis of eleven studies regarding
the association between thrombophilias and recurrent miscarriage. There were significantly higher serum
homocysteine levels among women with a history of recurrent miscarriage, but no increased prevalence
of the MTHFR C667 T mutation. No relation was observed for the levels of antithrombin, protein C, or
protein S. Nelen et al. [31] have performed a meta-analysis to assess the relationship between recurrent
early pregnancy loss and hyperhomocysteinemia. Overall, the pooled odds ratio (OR) for elevated
homocysteine was 2.7 (1.5–5.2), for afterload homocysteine 4.2 (2.0–8.8) and for MTHFR 1.4 (1.0–2.0).
These data support hyperhomocysteinemia as a risk factor for recurrent early pregnancy loss.
There are publications which separate early and late pregnancy losses and the prevalence of
thrombophilias. Preston et al. [32] reported on hereditary thrombophilias and fetal loss in a cohort of women
with FVL or deficiencies of antithrombin, protein C, or protein S. Of 843 women with thrombophilia, 571
had 1524 pregnancies; of 541 control women, 395 had 1019 pregnancies. The incidence of pregnancy loss
before or after 28 weeks was assessed jointly and separately. The risk of loss after 28 weeks was higher
than for early losses OR 3.6 (confidence interval [CI] 1.4–9.4) versus 1.27 (CI 0.94–1.71), respectively.
The highest OR for stillbirth was in women with combined thrombophilic defects 14.3 (CI 2.4–86.0)
compared with 5.2 (CI 1.5–18.1) in antithrombin deficiency, 2.3 (CI 0.6–8.3) in protein-C deficiency, 3.3
(CI 1.0–11.3) in protein-S deficiency, and 2.0 (CI 0.5–7.7) with FVL mutation. Sarig et al. [33] evaluated
145 patients with recurrent miscarriage and 145 matched controls. Late pregnancy wastage occurred more
frequently in women with thrombophilia compared with women without thrombophilia. A meta-analysis
[34] reported that the odds of pregnancy loss in women with FVL (absolute risk 4.2%) was 52% higher
(OR = 1.52, 95% CI 1.06–2.19) as compared with women without FVL (absolute risk 3.2%).
Prevalence of Thrombophilias in Late Obstetric Complications

Kupferminc et al. [35] first reported that hereditary thrombophilias are more prevalent in pregnant women
with fetal growth retardation, preeclampsia, abruptio placentae, or stillbirth. A later systematic review
of 25 studies by Alfirevic et al. [36], confirmed these findings. In Gris et al.’s [37] case-control study of
232 women with a history of one or more second or third trimester losses, 21.1% of patients and 3.9%
of controls had at least one thrombophilia (P < 0.00001). The OR for stillbirth associated with any
positive thrombophilia was 5.5 (CI 3.4–9.0). The conclusion was that late fetal loss might sometimes
be the consequence of a maternal multifactorial prothrombotic state involving placental thrombosis.
Alfirevic et al.’s [36] systematic review has shown that placental abruption was more often associated with
homozygous and heterozygous FVL, heterozygous G20210A, and hyperhomocysteinemia. Women with
preeclampsia/eclampsia were more likely to have heterozygous FVL mutation, heterozygous G20210A
prothrombin gene mutation, homozygosity for the MTHFR (C677 T) mutation, protein C deficiency,
protein S deficiency, or activated protein C resistance. Stillbirth was more often associated with FVL,
protein S deficiency, and activated protein C resistance. Women with intrauterine growth restriction had a
higher prevalence of G20210A, MTHFR, or protein S deficiency. However, they concluded that “Women
with adverse pregnancy outcome are more likely to have a positive thrombophilia screen but studies
published so far are too small to adequately assess the true size of this association.”
Infante-Rivard et al. [38] were the first to dispute the increased prevalence of hereditary thrombophilias
in late obstetric complications. Silver et al. [39] investigated the prevalence of the prothrombin gene
mutation (G20210A) in a multicenter, prospective, observational cohort of 5188 unselected singleton
gestations. There was no association between the prothrombin G20210A mutation and pregnancy loss,
preeclampsia, abruption, or small for gestational age neonates in a low-risk, prospective cohort. A
similar study by Kjellberg et al. [40] showed that FVL carriership did not influence pregnancy-induced
hypertension, birthweight, or prematurity but raised the risk of venous thromboembolism.
The different results could reflect heterogeneity of study design, inclusion criteria, sample size and
population studied, outcome definition and diagnostic criteria, as well as the prevalence of thrombophilias
studied. Nevertheless, there may be an association between some thrombophilias and some adverse
pregnancy outcomes.
Cohort Studies
Case-control studies can only show associations between thrombophilias and pregnancy losses.
In order to infer cause, cohort studiers are necessary. In the case of miscarriage, Ogasawara et al.
[14] reported that the subsequent miscarriage rate was not different for patients with decreased
protein C or S activity or antithrombin. Carp et al. [41] found the live birth rate to be similar to that
expected in recurrent miscarriage, whether the patient had FVL, G20210A, MTHFR, protein C or S,
or antithrombin deficiencies. Salomon et al. [42] have followed up 191 thrombophilic patients who
attended an ultrasound clinic to prospectively assess obstetric complications. The blood flow to the
fetus was not compromised.
In late obstetric complications, Sanson et al. [43] investigated women with deficiencies of antithrombin,
protein S, and protein C. In the 60 deficient subjects, 22.3% of the 188 pregnancies resulted in miscarriage
or stillbirth as compared to 11.4% of the 202 pregnancies in the 69 non-deficient subjects. The relative
risk of miscarriage and stillbirth per pregnancy for deficient women as compared to non-deficient women
was 2.0 (CI 1.2–3.3). However, Rodger et al. [34] carried out a meta-analysis of 10 prospective cohort
studies that examined the association between FVL and the prothrombin gene mutation (G20210A), and
placenta-mediated pregnancy complications. Neither FVL nor PGM increased a woman’s risk of pre-
eclampsia or of giving birth to a small for gestational age infant.
Treatment
This chapter only gives an outline of the treatment options. The figures are more fully described
in Chapter 24. There are reports that the presence of hereditary thrombophilias warrants
thromboprophylaxis. The presumed benefit of antithrombotic therapy and the absence of side effects
has led many clinicians to prescribe LMWH, aspirin, or both to women with recurrent pregnancy loss
and hereditary thrombophilia. However, the role of treatment can only be determined in well-designed
trials where the effect of treatment is compared to untreated or placebo-treated patients. Carp et al.
[44] have reported a comparative cohort study comparing enoxaparin to no treatment in women with
hereditary thrombophilias and recurrent miscarriage. Twenty-six of the 37 pregnancies in treated
patients (70.2%) terminated in live births, compared to 21 of 48 (43.8%) in untreated patients (OR 3.03,
95% CI 1.12–8.36). The beneficial effect was mainly seen in primary aborters, i.e., women with no
previous live births (OR 9.75, 95% CI 1.59–52.48). This benefit was also found in patients with a poor
prognosis for a live birth (five or more miscarriages), where the live birth rate was increased from 18.2%
to 61.6%. However, the trial was neither randomized nor blinded. Skeith et al. [45] have published a
meta-analysis of randomized controlled trials comparing LMWH versus no LMWH in women with
inherited thrombophilia and either prior late (≥10 weeks), recurrent early (<10 weeks) pregnancy
loss, or previous obstetric complications. Eight trials of 483 patients were included. There was no
significant difference in live birth rates with the use of LMWH compared with no LMWH (relative
risk 0.81; 95% CI 0.55–1.19; P = 0.28), suggesting no benefit. However, only four of the trials assessed
women with recurrent pregnancy losses [46–49]. If the results of these four trials are summarized
together with a subsequent trial by Aynioglu et al. [50], there is a 27% benefit in the live birth rate
in the treated group (Figure 9.2) (OR 4.48, CI 2.82, 8.46). Recently, the ALIFE2 study (http://www.
trialregister.nl, Netherlands Trial Register 3361) has started recruiting, in which women with inherited
thrombophilia and recurrent pregnancy loss will be randomized to either treatment with LMWH plus
standard pregnancy surveillance or standard pregnancy surveillance only.
Treated Control Weight % OR with 95% CI

Study ID births/total births/total
Kaandorp et al. [46] 9/13 20/34 28.74% 1.575 (0.4036 to 6.1457)

Clark et al. [47] 5/6 2/4 3.38% 5 (0.2732 to 91.5179)
Visser et al. [48] 13/19 3/10 10.48% 5.0556 (0.9585 to 26.6641)
Schleusner et al. [49] 27/30 24/25 22.10% 0.375 (0.0365 to 3.8504)
Aynroglu et al. [50] 69/89 16/64 35.31% 10.35 (4.8716 to 21.9893)
META-ANALYSIS: 123/157 65/137 100% 4.8885 (2.8218 to 8.469)
(78%) (47%)
0.01 0.1 1 10 100
OR (log scale)
FIGURE 9.2 Meta-analysis of anticoagulants and live birth rate in hereditary thrombophilias.
Other Prothrombotic Mechanisms of Pregnancy Loss

There are other mechanisms that may induce thrombosis or may allow thrombosis to become apparent
in patients with genetic predispositions to thrombosis.
Cytokines
Cytokines are low molecular weight peptides or glycopeptides, produced by lymphocytes, monocytes/
macrophages, mast cells, eosinophils, and blood vessel endothelial cells. Two cytokines have been
associated with initiation of coagulation in infections; TNFα and IL-6 upregulate the expression of tissue
factor, which initiates the extrinsic phase of the coagulation cascade and subsequent thrombin generation.
In addition, interferon γ has been described as detrimental to thrombus resolution [51].
Cytokine imbalances have been described in recurrent pregnancy loss [52], antiphospholipid syndrome
[53,54], preeclampsia [55], preterm births [56], and IUGR [57]. The predominance of prothrombotic
cytokines may lead to placental thrombosis in genetically susceptible individuals.
Microparticles
Placental apoptosis has been described as a salient feature of pregnancy loss [58]. Following apoptosis
and cell activation, the cell membrane is remodeled with the release of microparticles. The microparticles
express procoagulant phospholipids such as phosphatidylserine on their external surface. These
phospholipids are normally found inside the cell membrane. Microparticles lead to increased expression of
adhesion molecules, thus amplifying the pro-coagulant and/or inflammatory response on the endothelial
cell surface. Microparticles have been found in increased numbers in normal pregnancy, when there is
constant deportation of trophoblast into the maternal circulation.
Shetty et al. [59] analyzed nine papers reporting the prevalence of microparticles in recurrent pregnancy
loss (RPL). The majority of studies have found an increased prevalence. However, it has not been
determined whether endothelial microparticles may cause pregnancy loss through subsequent thrombotic
mechanisms or may be a consequence of embryonic death. Twenty-nine to sixty percent of recurrent
first trimester miscarriages are due to chromosomal aberrations that are incompatible with life, and lead
to miscarriage irrespective of other associations or causes of pregnancy loss, including the presence of
microparticles. Even in missed abortion due to chromosomal aberrations, the trophoblast undergoes
apoptosis with subsequent microparticle formation and thrombosis. Microparticles may by themselves
result in adverse conditions, or they may be additive factors to an already existing prothrombotic state in
addition to the pre-existing hypercoagulable status of pregnancy.
Hormones and Thrombosis

The hormones of pregnancy, estrogen, progesterone, and hCG all affect thrombosis. Estrogen may alter
the concentrations of clotting factors to a prothrombotic profile, e.g., raising FVII [60] and plasminogen
activator (PAI-1) [61] and reducing antithrombin III [61]. In mice, estrogen sulfotransferase (a cytosolic
enzyme that catalyzes the sulfoconjugation of estrogens) has a critical role in modulating estrogen activity
in the mouse placenta during mid-gestation [62]. Inactivation of estrogen sulfotransferase caused local
and systemic estrogen excess and an increase in tissue factor, leading to placental thrombosis and fetal
loss. In addition, estrogen can either stimulate or inhibit the production of IL-1 and TNF cytokines [63].
Progesterone, however, seems to have opposing effects. Progesterone has prothrombotic effects
including upregulation of tissue factor expression [64], but progesterone also induces the production of
cytokines, such as IL-4, which upregulates protein S, which inhibits coagulation [65].
In addition to its endocrine luteotropic role, hCG could also have a local role within the uterine
environment. Specific binding sites for hCG have been shown in various cells of the endometrium
and decidua. The local role of hCG in the endometrium has not been fully elucidated. Uzumcu
et al. [66] have assessed endometrial production of cytokines when stimulated by hCG. Increasing
doses of hCG caused a dose-dependent increase in TNFα and IL-6 secretion, both of which have been
reported to be thrombogenic.
SNPs in Coagulation Factors and Pregnancy Loss

The beta-fibrinogen -455G/A polymorphism (A/A genotype) and homozygosity for plasminogen activator
inhibitor (PAI)-1, -675 4G/5G polymorphism were found to be associated with recurrent pregnancy loss;
however, the association is actually very slight [67–69].
Control of thrombin generation is essential for normal hemostasis and is achieved by the physiological
anticoagulants. One such anticoagulant is tissue factor pathway inhibitor (TFPI), an endothelial-associated
protein that downregulates the initial phase of coagulation by inhibiting tissue factor-factor VIIa and
factor Xa complex [70]. Another anticoagulant is antithrombin, a multifunctional serpin (serine protease
inhibitor) that inhibits essentially almost all the active coagulation factors. A recent study [71] analyzed
the association of SNPs in TFPI and antithrombin genes with RPL in 117 nonpregnant women with three
or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to
viability, and 264 healthy fertile nonpregnant women who had at least two term deliveries and no known
pregnancy losses [72]. The results of the study showed that antithrombin 786G > A variant increases
the risk for RPL, while TFPI T-287C variant is protective. Further studies are required to confirm these
findings.
Fetal Thrombophilia
As placental histology usually shows a fetal vasculopathy rather than maternal thrombosis, fetal
thrombophilia may explain the pathological changes. The hemostatic balance in the placenta may be
determined by both maternal and fetal factors cooperatively regulating coagulation at the feto-maternal
interface [73]. Humans have an almost unique placentation in which trophoblast cells line the maternal
blood lakes rather than endothelial cells. Using genome-wide expression analysis, Sood et al. [28]
identified a panel of genes that determine the ability of fetal trophoblast cells to regulate hemostasis at
the feto-maternal interface. In addition, the trophoblast was shown to sense the presence of activated
coagulation factors via the expression of protease activated receptors. Engagement of these receptors
was reported to result in specific changes in gene expression. Hence, fetal genes might modify the risks
associated with maternal thrombophilia. In addition, coagulation activation at the feto-maternal interface
might affect trophoblast physiology and alter placental function in the absence of frank thrombosis. The
author has seen fetal deaths in utero in which sonograms have shown complete occlusion of the umbilical
blood vessels. However, it is impossible to say whether the thromboses caused fetal death or whether the
changes occurred postmortem.
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10
The Immunobiology of Recurrent Miscarriage
Marighoula Varla-Leftherioti, Theodora Keramitsoglou, and Christina Tsekoura
Introduction
A large proportion of unexplained recurrent spontaneous abortions (URSA) may be due to immunological
causes [1]. Immune-mediated pregnancy loss is characterized by either autoimmune or alloimmune
disturbances. In autoimmune abortions, the development of the placenta and the embryo is affected
by maternal autoantibodies and autoreactive cells, which target decidual and trophoblastic molecules.
In alloimmune pregnancy loss, the maternal immune system reacts against the “semi-allogeneic”
embryo and damages the trophoblast through allogeneic, rejection-type reactions. Clinically, the two
categories of auto- and alloimmune-mediated abortions cannot be distinguished, as both represent a broad
immunological imbalance that leads to pregnancy loss [2].
Autoimmune Pregnancy Loss

Approximately 20% of women with recurrent pregnancy loss (RPL) have increased serum levels
of autoantibodies, with antiphospholipid antibodies (aPL) predominating [1]. The etiology of the
antiphospholipid syndrome is the subject of another chapter and will not be discussed here. Suffice to say,
an aPL-related etiology should be suspected in women with ≥3 consecutive pre-embryonic or embryonic
pregnancy losses or ≥1 unexplained fetal deaths above 10 weeks of gestation [3]. In women where an aPL-
associated etiology is suspected, other autoantibodies may coexist. Such as antithyroid autoantibodies
(ATA) (against thyroglobulin [TG] or thyroid peroxidase [TPO]) may be independent markers of “at-risk”
pregnancy even with euthyroid status. It is possible that the high rate of miscarriage in the presence of
ATA is related to very mild thyroid “underfunction,” with the thyroid gland being less able to adapt to
the increased requirements of pregnancy; thus, these women might benefit from thyroid replacement
therapy [4]. Furthermore, ATA may represent a generalized activation of the immune system. ATA have
been found to coexist with activated T cells in the uterus and with non-organ-specific autoantibodies
as well as with increased and hyperactive cytotoxic natural killer (NK) cells in RPL. Hence, treatment
with intravenous immune globulin (IVIg), may neutralize the antibodies and also provide the required
modulation of immune functioning [5].
Alloimmune Pregnancy Loss

From an immunological point of view, the embryo is a semi-allogeneic graft, as it has genetic and
antigenic contribution from both the mother and father [6]. The maternal immune system senses
the fetal allograft as the trophoblast has direct contact with the decidua. The recognition of paternal
antigens would be expected to provoke maternal allo-responses against the embryo similar to those
that develop after organ transplantation. Normally, this does not happen. However, in some cases
of miscarriage, the embryo is infiltrated by lymphocytes and the placenta exhibits lesions similar to
those characterizing rejected grafts [7], indicating that the embryo has been “rejected” by the mother
(alloimmune miscarriage).
89
Th2-Type Immune Response in Normal Pregnancy

In 1987, Wegmann [9] presented the “immunotrophic” theory, according to which the normal
development of the placenta and possibly the secretion of trophoblast hormones (hCG and human
placental lactogen [hPL]) is the result of the influence of cytokines (placenta immunotrophic cytokines),
such as granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor beta
(TGF-β), and interleukin-3 (IL-3) [8]. Consequently, it was suggested that during pregnancy there is a
change of the repertoire of T helper cells (Th1/Τh2 equilibrium) so that Th2-type cytokines (IL-4, IL-5,
IL-10) predominate over Th1-type cytokines (IL-2, interferon γ [IFN-γ]) and benefit the developing
embryo by enhancing placental growth and function as well as by preventing/blocking inappropriate anti-
trophoblast reactions mediated by cytotoxic antibodies and cells (Figure 10.1). More recent studies have
revealed that before the Th2 response, the maternal innate immune system actively reacts by developing
an inflammatory response. Cytokines/chemokines secreted by the trophoblast (IL-8, GRO-α, MCP1)
recruit and “educate” neutrophils and innate lymphoid immune cells (ILC) (NK cells, macrophages [MΦ]
and dendritic cells [DC]), which infiltrate the decidua, accumulate around the invading trophoblast, and
contribute to successful embryo implantation [10].
The trophoblastic antigenic stimulus, the maternal cells that are stimulated for the initiation of the
enhancing response, and the exact factors modulating the Th2 shift remain unclarified. Nevertheless,
it has become clear that the acceptance of the embryo is regulated through the cumulative effect of
preimplantation factors of maternal, embryonic, and paternal origin, and molecules expressed on
trophoblastic and decidual cells [11]. Changes in metabolic factors, hormones, and cytokines during
ovulation, coitus, and fertilization result in local immunosuppression within the maternal genital tract
and prepare the uterus for the implantation of the blastocyst. Trophoblastic molecules may be specifically
recognized by maternal immune cells or may act as antigen-presenting molecules or have a suppressive/
immunomodulatory function. Decidual immune cells may regulate the immune response not only by
producing cytokines and growth factors, but also by specific recognition of trophoblastic molecules and
suppression of cytotoxic reactions [12].
Recognition
by specific decidual cells IL-4 Th2>Th1
cytokines
Trophoblastic antigens ? ? IL-10
IL-13
Treg cells
Th2 response
Inflammatory
response
TGF-β IL-3 GM-CSF
Hormone-dependent
local Immunotrophism Facilitation
immunosuppression growth, maturation reaction
of the placenta
Blocking
age
Block xic antibodies
to to
of cy
ions
react
FIGURE 10.1 Immunologic mechanisms in normal pregnancy. IL, interleukin; Th, T-helper; TGF-β, transforming growth
factor β; GM-CSF, granulocyte-macrophage colony- stimulating factor; Treg, regulatory T cell.
The Immunobiology of Recurrent Miscarriage 91
The Cytokine and Hormonal Network in Normal Pregnancy

The cytokine network at the feto-maternal interface is complex; the embryo has been described as
“bathing in a sea of cytokines” [13]. Preferential expression of Th1 proinflammatory cytokines is required
in early pregnancy and at the end of pregnancy, Th2 anti-inflammatory cytokines are required in mid-
pregnancy. IFN-γ, a Th1 cytokine that is potentially deleterious for pregnancy, may have a beneficial
role very early in pregnancy by contributing to the vascular development and remodeling of the uterine
spiral arteries required for implantation and successful gestation, and again at late stages of pregnancy
for the activation of myometrial smooth muscle cells associated with uterine contractions in labor [14].
Different cell populations are involved in the production of both Th2 and Th1 cytokines as well as other
cytokines (i.e., IL-12, -15, -18), chemokines, and growth factors that control the differentiation and the
activation of immune cells locally. Cytokines that control the shift to Th1 responses (i.e., IL-12) coexist with
those enhancing Th2 responses (i.e., IL-10). The cytokines are controlled by hormones on a competitive
basis. Progesterone promotes the production of IL-4 and IL-5, whereas relaxin promotes the production
of IFN-γ by T cells [15]. Just as the hormones control the production of cytokines, hormone secretion is
induced by cytokines. For example, Th2-type cytokines induce the secretion of hCG by the trophoblast,
which stimulates the corpus luteum to produce progesterone. Progesterone enhances the production of Th2
cytokines by competing with relaxin, which enhances the production of Th1 cytokines [16].
Th1-Type Immune Response in Alloimmune Abortions

A predominantly Th1 response or defective production of Th2-type cytokines appear to predispose in
spontaneous miscarriage [17]. In response to the conceptus or other antigens, decidual lymphocytes secrete
Th1-type cytokines (IL-2, IFN-γ, TNF-α), which adversely affect the development of the embryo. Fetal
rejection occurs through inflammation and lymphocyte infiltration of the trophoblast, trophoblast damage
by NK cells, cytotoxic antibodies, and vasculitis affecting the maternal blood supply to the embryo [18].
The disruption of one or more of the mechanisms leading to tolerance in normal pregnancy may lead to
miscarriage. These disturbances may include (a) absence of immunosuppressive preimplantation factors
in the genital tract, (b) disturbed innate immune cell function, (c) absence of immunodependent specific
suppression at the feto-maternal interface, and (d) inappropriate expression or defective recognition of
trophoblastic and immunoregulatory molecules by decidual cells (Figure 10.2).
Although the tests for the identification of the Th1 predominance in recurrent spontaneous abortion
(RSA) women are controversial [19], an increase of Th1 cytokine responses has been shown by several
studies [20] and intervention treatments have been used to reduce the Th1/Th2 ratio in the peripheral
blood of women with RPL. The beneficial effect of the above interventions in the outcome of pregnancy
is debated later in this book.
Factors Triggering Th1 Abortogenic Responses

Stress, infection, autoimmunity, and gene variations are among the factors that may trigger Th1 cytokine-
mediated abortions.
Stress
Stress may trigger a Th1 cytokine profile [21]. In the murine CBA/J × DBA/2J model, stress has been
suggested to induce a neurogenic inflammatory response toward a Th1 response via upregulation of
adhesion molecules [22].
Infections
Infections may lead to Th1 cytokine-triggered miscarriages due to the availability or presence of bacterial
endotoxins [23]. Prasad et al. [24] have recently reported increased Th1 cytokines in the serum of RSA
women with Chlamydia trachomatis infection, and Voskakis et al. [25] have suggested that the Th1
Triggering factors:
Stress
Infections
Detected recognition of trophoblastic
Maternal genes
antigens and immunoregulatory molecules TH1 cells
Autoimmunity Th1>Th2
by decidual cells IL-4
cytokines
TGF-α IFN-γ
TH17 cells Detected expression of IL-10 IL-2
trophoblastic antigens
TH17 cell
IL-13
Treg cells
Th1 response
Disturbed Mφ DC Treg cells

TLR function
TGF-β IL-3 GM-CSF
Absence of pre-
implantation Rejection
immunosuppressive Immunotrophism reaction
factors
Blocking
antibodies
ckage
No blo reactions
NK action: oto xic blast Cytotoxic
of cyt g tropho antibodies
Damage of e t in
targ and cells
trophoblast
FIGURE 10.2 Immunologic mechanisms in abortion.
response is induced when chlamydial antigens (possibly heat shock proteins) are recognized by specific
decidual T cells bearing Vδ2 receptors, which secrete abortogenic cytokines when activated.
Maternal Genes
Maternal genes may regulate the response to stress; luteal phase support and paternally inherited trophoblastic
antigens may determine the cytokine balance in pregnancy [18]. In a recent meta-analysis, Shi et al. [26]
found significant associations between RSA and 53 genetic polymorphisms of 37 genes, including genetic
variants of HLA-G, IFN-γ, TNF, IL-6, and IL-10, molecules known to be involved in the Th1 response.
Autoantibodies
Autoantibodies may cause miscarriage by altering the production of cytokines. Buttari et al. [27] have
shown that in vitro oxidized β2-GPI interacts with DCs and stimulating secretion of IL-12, which induces
the production of IFN-γ and favors differentiation of Th1 cells. Furthermore, an increased risk for
miscarriage exists in women with thyroid autoimmunity, who are found to have increased serum levels
of Th1 and Th17-related cytokines [5].
Regulatory T Cells and Th17 Inflammatory Cells in Normal Pregnancy

The regulation of the Th1/Th2 balance is due to role of regulatory T cells (Treg) and Th17 cells, which
act competitively and reciprocally at the feto-maternal interface [28].
Treg Cells
Treg cells (CD4+CD25+) (Foxp3 mRNA+) are a subset of immunoregulatory T lymphocytes deriving
either from the thymus (natural Treg) or by activation of naïve CD4+ T cells following antigen stimulation
under the influence of TGF-β (adaptive Treg). Through IL-10 and TGF-β, which they secrete in a contact-
depended manner, Tregs exhibit anti-inflammatory and immune-suppressive actions [29]. Aluvihare
et al. [30] were the first to demonstrate that during murine pregnancy there is a systemic expansion
of Treg cells, which can suppress aggressive allogeneic anti-fetal responses. Similarly, Somerset et al.
[31] found that Tregs increase in the peripheral blood during early pregnancy, peak during the second
trimester, and decline postpartum. Saito et al. [28] have suggested that tolerogenic DCs take up paternal
antigens from the seminal plasma after coitus, present antigen fragments on their surface in association
with class II MHC molecules, and activate naïve Treg cells of thymic origin, which become paternal
antigen-specific, proliferate, and migrate from the vagina to the pregnant uterus by chemoattractant
mechanisms.
Decidual Tregs (dTregs)

dTregs are considered to act in an antigen-specific manner to control effector cells. By secretion of
inhibitory cytokines (TGF-β, IL-10, and IL-35), and consumption of the γc-family cytokines (IL-2,
IL-4, IL-7, IL-15), dTreg cells may suppress activation and expansion of conventional T lymphocytes,
inhibit the release of proinflammatory cytokines, increase T cell apoptotic rates, and modulate the
functions of decidual DCs. IL-10 inhibits the upregulation of the expression of MHC and costimulatory
molecule on DCs, decreasing their antigen-presenting capacity, suppresses release of proinflammatory
cytokines, and upregulates the expression of inhibitory molecules [32]. Furthermore, Tregs induce
direct cell-to-cell contact suppression by ligation of their surface molecules to target/effector cells.
The ligation of the cytotoxic T lymphocyte−associated antigen 4 (CTLA-4) on DCs is important for
generating an immunosuppressive milieu at the feto-maternal interface, since it induces the expression
of indoleamine 2,3-dioxygenase (IDO) on DCs and controls the balance between Treg and Th1 cell
responses. IDO, a tryptophan-catabolizing enzyme expressed by trophoblasts and macrophages,
exerts a suppressive influence over neighboring T cells, preventing them from activating anti-fetal
responses [33].
Th17 Inflammatory Cells

Th17 inflammatory cells are proinflammatory cells, which are dynamically balanced with Treg cells.
They are characterized by the production of a distinct profile of effector cytokines, including IL-17 (or
IL-17A), IL-17F, IL-6, IL-21, and IL-22, and they can also be induced to produce IFN-γ in the presence
of IL-12 [34].
In pregnancy, TH17 cells can be stimulated by fetal alloantigens and secrete proinflammatory
cytokines to induce fetal rejection [35]. Conversely, they may contribute to the prevention of pathological
infections through an array of proinflammatory cytokines and may contribute to embryo protection
through the production of IL-17, which can increase progesterone secretion from the trophoblast [28].
Thus, TH17 cells may be beneficial for pregnancy and become detrimental only when they antagonize
Treg cells.
Treg-Th17 Balance
Th17 expansion is a barrier to establishing maternal tolerance because of mutual antagonism and plasticity
between Treg and Th17 cells. These two cell subsets appear to share a common lineage with their relative
abundance influenced dramatically by the cytokine environment (particularly the ratio of IL-6 to TGF-β)
in which T cell priming occurs. In the absence of IL-6, TGF-β suppress the conversion of naïve T cells
to Th17 cells, while in the presence of IL-6, naïve T cells are converted to Th17 cells, and existing Treg
cells can function as inducers of Th17 cells and themselves convert to Th17 cells [36].
Treg Cells and Th17 Cells in Women with RSA

Studies in women with RSA have shown the following.
Decrease of Treg Cells

Decidual CD4+CD25bright T cells, which can inhibit the proliferation of autologous CD4+ T cells, are
significantly lower in specimens from spontaneous miscarriages than induced abortions [37]. Furthermore,
the proportions of Treg cells in the decidua and peripheral blood are significantly lower in RSA women
than in control women [38]. The low levels of circulating Treg cells in newly pregnant women with a
history of fetal losses has been suggested as marker to predict miscarriage risk [39].
Increase of TH17 Cells and Th17 Cytokines

Increased TH17 cells and Th17 cytokines (e.g., IL-17 and IL-23) are found in the decidual tissue and
the blood of RSA women [40]. Nakashima et al. [41] have suggested that IL-17 expression might be the
mechanism of expulsion of the fetus rather than the cause of miscarriage. TH17 activity may be stimulated
in RSA patients by IL-6 produced during subclinical uterine infections [42], and women with RSA are
genetically predisposed to TH17-mediated fetal losses (decreased frequency of the IL-17F genotype
rs763780) [43].
Treg/Th17 Imbalance
The imbalance between Treg and TH-17 cells and a related elevation in serum IL-6 levels in RSA may result
in insufficient regulation of inflammatory immune responses [44]. Adoptive transfer of CD4+CD25+
regulatory T cells from normal pregnant or expanded in vitro has been shown to prevent pregnancy
resorption in mice, possibly by increasing the expression of progesterone receptors on decidual cells [45].
Coitus has been suggested as a factor to expand the pool of inducible Treg cells that react with paternal
alloantigens, since seminal fluid contains Treg cell-inducing agents (i.e., TGFβ and prostaglandin E) [46].
In the clinical setting, the detection of Tregs- and Th17-producing cells as well the measurement of
IL-6 and IL-17 in the peripheral blood of aborting women may be useful to interpret the beneficial effect
of immunotherapy in women with RSA.
The Role of NK Cells

Decidual NK (dNK) Cells
CD56bright/CD16dim NK cells are the predominant decidual cell population (60%–90% of decidual immune
cells) from the first stages of pregnancy through the first trimester [47]. During the secretory phase of the
menstrual cycle, uterine stromal leukocytes increase highly (from 5%−25%) as a result of NK cell influx
from the blood or other tissues or of reprogramming and differentiation of endometrial stromal cells to
NK cells. In pregnancy, NK cells increase rapidly and are distributed broadly throughout the decidua in
close proximity to the extravillous trophoblast. There numbers decrease in mid and late pregnancy [47].
dNK cells exhibit unique phenotypic and functional properties. They specifically express CD69,
CD49a, CD103, CD9, galectin, high levels of CXCR3, a-1 integrin, and other adhesion molecules. They
also express inhibitory and activating receptors: killer immunoglobulin-like receptors (KIR), C-type
lectin-like receptors (CD94/NKG2A), leukocyte Ig-like receptor (LILRB1), and natural cytotoxicity
receptors (NCR) [48]. Through their receptors, dNK cells may recognize selected epitopes on HLA class
I molecules expressed on invading trophoblast. The specific ligands for most of the receptors are the
non-classical HLA class I molecules G and E as well as the classical HLA class I antigen C, which are
the only HLA molecules expressed on extravillous trophoblast.
The specific interaction of the NK cell receptors with trophoblastic antigens led to the concept of an
embryo recognition model through an “NK cell allorecognition system.” High affinity interactions of NK
receptors with their ligands may provide self-signals to either cytotoxic NK activation (Th1 response) or
inhibition of activation and protection of the trophoblast (Th2 response). If the inhibitory dNK receptors
recognize their specific ligands on the trophoblast, they are expected to inhibit dNK activation for
trophoblast damage; otherwise, dNK develop anti-trophoblast activity [49].
Among the different NK receptor interactions with their specific counterparts on the trophoblast, the
interactions between receptors of the KIR family and their ligands HLA-C molecules appear to be those
mainly involved in the function of an NK cell-mediated allorecognition system in pregnancy [50]. Given
the differences in both the KIR repertoire and the HLA-C allotypes among unrelated individuals, each
pregnancy presents a different combination of maternal KIR receptors on dNK and self and non-self
HLA-C allotypes on the trophoblast. This combination is expected to ensure the appropriate receptor-
ligand interactions to favor pregnancy. Nevertheless, the control of the anti-trophoblast activity of dNK
cells is probably the result of the cumulative interaction of several receptors on maternal dNK with
different self and non-self-class-I molecules expressed on trophoblast.
The exact mechanism by which dNK cells exert their immunomodulatory role in pregnancy is not
fully understood. There is evidence that, simultaneously with blastocyst implantation, dNK cells become
activated and produce cytokines and growth factors to regulate uterine vascular remodeling and trophoblast
differentiation and invasion [51]. Furthermore, dNKs may produce Th2-type cytokines and growth factors
that result in placental augmentation and local immunosuppression and immunomodulation [52]. Of
specific importance for the induction of tolerance is the interaction between NK and DC cells occurring
under a balance of activating and inhibitory signals and resulting in Treg recruitment, inhibition of NK
cytoxicity, and inhibition of maturation or apoptosis of DCs [53].
The Role of NK Cells in Miscarriage

dNK cells with the phenotype CD3-CD16+CD56+ (opposite to the CD3-CD16-CD56+ NKs, which
predominate in normal pregnancy) are considered to be the main cell population involved in cases where
the embryo is “rejected” by the mother. In the frame of the Th1 response, these cells increase in the decidua;
they become activated and may damage the trophoblast either by direct cytotoxicity or by the secretion of
proinflammatory cytokines [54]. Apart from their activation through the Th1 response, NK cells may be
directly activated by infectious agents in the genital tract [55] or because of inappropriate interactions of
their inhibitory and activating receptors and their HLA ligands on trophoblastic cells [50,56].
Clinical studies have demonstrated that women who tend to miscarry have increased numbers of NK
cells of the conventional CD3-CD56+CD16+ type in the uterus [57] as well as increased blood NK
subsets and NK cell activity, all of which have been associated with miscarriage of chromosomally
normal embryos [58,59]. A meta-analysis contacted by Seshadri et al. [60] confirmed the increase of
peripheral NK cell numbers or percentages in RSA women, but not the increase of uterine NK cells.
According to our findings, as well as reports from other authors, miscarrying women have “disturbances”
in the gene repertoire of inhibitory NK receptors, which do not allow sufficient inhibition of NK toxicity
(i.e., limited repertoire of inhibitory KIR receptors or imbalance of inhibitory KIR receptors in favor
of activating KIRs, lack of maternal inhKIR-fetal HLA-C epitope matching, presence of inhKIRs that
do not bind strongly their HLA-C ligands, and dominant expression of the activating CD61 KIR vs. the
inhibitory CD158a and CD158b KIRs on the peripheral NK cells) (reviewed in [61]). Vargas et al. [62] have
reported that women carrying a high content of activating KIR genes have an approximately threefold
increased probability of developing recurrent miscarriage.
Peripheral blood immunophenotyping for the detection of NK cell disturbances is often used for the
identification of women with an alloimmune etiology for the miscarriages. In addition, immunotherapies
such as IVIG administration and soybean-oil-based lipid infusions (discussed in another chapter) are used
to reduce NK cell numbers and increase the live birth rate in RSA women. The authors have recently
presented results on a patented fatty acid−based oral formula, the intake of which was found to decrease
NK cell numbers and toxicity in RSA women, thus providing a promising approach to improve the
possibility of embryo implantation and pregnancy [63].
Specific Factors/Mechanisms Contributing to Fetal Tolerance

Several specific mechanisms have been suggested to contribute to fetal tolerance, and disturbances of
these mechanisms may lead to pregnancy loss.
Sperm
Sperm may promote local immunosuppression via prostaglandin mediation, while TGF-β in seminal
plasma may provide signals for the accumulation of Treg cell in the uterus, the production of growth
factors by the uterine epithelium, and the initiation of an appropriate maternal immune response [46].
A reduced pregnancy loss rate has been reported in women exposed to their partners’ sperm via timed
intercourse before or just after the day of ovum pick-up [64].
Specific Trophoblastic Molecules

Specific trophoblastic molecules and various proteins produced by the trophoblast appear to modulate the
cytokine pattern toward preferential expression of Th2 cytokines. Heat shock proteins, pregnancy-specific
β1-glycoprotein, and increased expression of the non-classical MHC class I HLA-G molecule have been
suggested as stimulants of endometrial macrophages for IL-10 production which enhances a Th2 shift
[18]. Modulation of local placental immunity during pregnancy has mainly been ascribed to HLA-G, with
decreased levels in RSA [65], whereas the 14-bp ins/14-bp del HLA-G genotype, which results in low
membrane-bound and soluble HLA-G expression, has been associated with an increased risk for RSA [66].
hCG
hCG, which is produced by the trophoblast, is crucial for implantation and placentation, and also for the
regulation of maternal innate and adaptive immune responses allowing fetal acceptance. Its modulatory
effects include IDO production by immature DCs, conversion of conventional T cells into fully functional Treg
cells, and generation of suppressive Breg cells. Furthermore, hCG induces the production of progesterone by
the corpus luteum [67]. hCG has been used combined with IVIG in RSA women to increase the suppressive
activity of Treg cells and modulate peripheral blood Th17 and regulatory T cells [68].
Progesterone and Progesterone-Induced Blocking Factor (PIBF)

Progesterone has also a crucial role in the maintenance of pregnancy by modulation of the maternal
immune response and suppression of inflammatory responses. In the presence of progesterone, activated
maternal progesterone receptor−expressing γδT lymphocytes synthesize PIBF, a 34-kD protein. PIBF
is known to mediate the immunomodulatory effects of progesterone during pregnancy via production of
Th2-type cytokines, decrease of decidual NK cell activity, and production of antibodies that may mask
fetal antigens or block anti-fetal responses [69]. The number of maternal progesterone−positive immune
cells and PIBF levels in the urine and serum are decreased in women with threatened miscarriage,
and PIBF expression also decreases in trophoblasts and decidua of RSA women [70]. Progesterone
supplementation increases PIBF concentrations and prevents threatened miscarriage and subsequent
miscarriages in women with RSA [71].
Interaction between Trophoblastic and Maternal Molecules

The interaction between trophoblastic and maternal molecules (signaling pathways) may enhance decidual
and trophoblast development and induce tolerance by preventing maternal T cell activation or apoptosis
of activated cells. Related disturbances have been associated with obstetric complications including RSA.
Related disturbances include dysregulated expression of IDO [72], increased placental expression of
TNF-related apoptosis-inducing ligand (TRAIL) [73], and deficient production of leukemia inhibitory
factor (LIF) by decidual cells [74].
Extracellular Vesicles
Extracellular vesicles deriving from the embryo, the oviduct, the endometrial epithelium, and the decidua are
increased during pregnancy and interact with trophoblast cells to promote their growth and differentiation.
Placental vesicles detected in the maternal circulation may be involved in successful pregnancy by possibly
inducing apoptosis of activated cells. Changes in the release of exosomes and their concentration in maternal
plasma may be associated with pregnancy complications, including recurrent miscarriages [75].
Decidual MΦ and DCs

Decidual MΦ and DCs are immature and have limited antigen presenting capacity. Tolerogenic MΦ (M2),
which produce high levels of IL-10, TGF-β, low levels of IL-12, and may induce maternal tolerance to
fetal antigens, are found in normal pregnancies. MΦ macrophages, which secrete high levels of IL-12 and
low levels of IL-10, are increased in the decidua of spontaneous and recurrent abortions [76]. Similarly,
immature DCs (CD83−), which have tolerogenic action, decrease (or the CD83+ DC increase) in the
decidua of RSA women [77].
γ/δ Τ Lymphocytes
γ/δ Τ lymphocytes, most of which are activated upon recognition of conserved trophoblastic molecules,
constitute the majority of decidual T cells. They are considered the main decidual candidate cell population
for recognition of trophoblastic antigens for the initiation of the pregnancy immune response [69]. γ/δ
Τ cells express receptors for progesterone, produce PIBF under the influence of progesterone, and may
enhance TH2 responses and block cytotoxic reactions. Most of these lymphocytes preferentially express
δ1 TcR chains, which also drive Th2 responses. Barakonyi et al. [78] have shown that peripheral blood
γδ+ T cells from RSA women preferentially express the Vγ9Vδ2 TcR combination. It may be possible
that these T lymphocytes recognize antigenic epitopes of pathogens in the genital tract and develop TH1
antimicrobial responses that might also attack the embryo by molecular mimicry [25].
Matrix Metalloproteinases
Matrix metalloproteinases (MMPs), which are secreted by inflammatory leukocytes and are involved
in intrauterine tissue remodeling, may facilitate embryo implantation and placentation, contributing to
pregnancy success [49]. Dysregulation of the expression and/or of the activity of MMP-9 and MMP-2 has
been observed in spontaneous early pregnancy failure [79].
Inhibitory Checkpoint Molecules

Inhibitory checkpoint molecules are surface molecules that positively or negatively modulate the immune
response. Several inhibitory checkpoint molecules (PD-1, CTLA-4, CD200, LAIR-1, Gal-9, TIM-3, Gal-9,
CD155, and others) are expressed on trophoblast and decidual immune cells, and have been found to be
involved in pregnancy maintenance by regulating maternal-fetal immunity. In a recent study, Xu et al.
[80] presented data on the differences between normal pregnancies and spontaneous abortions for some
costimulatory molecules, such as CD200, TIM-3, LAIR-1, and CTLA-4.
Humoral Factors
Humoral factors (anti-paternal cytotoxic antibodies [APCA] and immunologically specific mixed
lymphocyte reaction blocking factor [MLR-Bf]) may be involved in the pregnancy immune response,
either by covering trophoblast alloantigens or by blocking alloimmune effects of maternal lymphocytes.
Absence of APCA and MLR-Bf has been shown in RSA patients [81].
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11
Immune Testing in Recurrent Pregnancy Loss*
Jeffrey Braverman, Darren Ritsick, and Nadera Mansouri-Attia
Introduction
Although the human immune system has evolved various mechanisms that facilitate immunologic tolerance
of a semi-allogenic conceptus, this tolerance can be broken in various contexts leading to rejection of
the conceptus and reproductive failure. While the previous chapter describes the immunobiology taking
place at the feto-maternal interface, this chapter is devoted to the various types of immune testing, which,
although controversial, may help to select patients for immunotherapy.
The most widely recognized clinical manifestation of failures in development of maternal immune
tolerance for conceptuses is idiopathic recurrent pregnancy loss (RPL). However, it is still relatively
rare that immune testing for reproductive failure is considered. In addition, failure in the development
of maternal immune tolerance for a conceptus can manifest in a full range of clinical outcomes, from
implantation failure/perceived infertility and biochemical pregnancies to early clinical miscarriages,
second trimester miscarriages, and third trimester complications such as preeclampsia, intrauterine
growth restriction, and preterm labor, as well as stillbirth. Some of the most convincing epidemiologic
studies illustrating the effects of adverse maternal immune response to pregnancy relate to preeclampsia
as a “disease of first pregnancies” and the role of increasing inter-pregnancy intervals and changing
partners on the risk for the development of preeclampsia in subsequent pregnancies [1–3]. These results
are now interpreted as consequences of paternal antigen-specific tolerogenic immune memory [4].
Therefore, for several reasons, the population affected by immunological problems of pregnancy is
vastly underestimated in current clinical practice, and testing for immunological etiologies for reproductive
failure is not applied to a patient population with an appropriately diverse set of reproductive outcomes.
Great opportunity exists for improving patient outcomes across the full range of reproductive failure with
increased application of immunological testing to drive proper diagnosis and personalized treatment.
Selection of Patients for Immunological Testing

Immunological testing should be advised for patients experiencing reproductive failure where the
likelihood that failures related to aneuploidy is decreased. Immunological testing is equally justified in
cases where diagnosed or strongly suspected inflammatory or autoimmune conditions are present, which
may lead to a failure in the proper generation of maternal immune tolerance for paternal antigens and are
known to be associated with various forms of reproductive failure.
Immunological testing is warranted in patients with:
1. Failed IVF cycles despite the transfer of good quality embryos

2. Failed IVF cycles with PGS-normal embryos
3. Two or more early miscarriages before a heartbeat was detected (for example, a chemical
pregnancy or a blighted ovum)
* Unfortunately, Jeffrey Braverman passed away after writing, but before the publication of this book. This chapter is now
dedicated to him and the countless patients that he treated before his untimely death.
101
4. Miscarriage after detection of a fetal heartbeat unless testing of the products of conception
(POC) showed a genetic abnormality
5. Miscarriage of a conceptus for which the POC tested genetically normal after dilation and
curettage
6. Stillbirth
7. Significant second/third trimester complications (i.e., preeclampsia, placental abruption, or
preterm labor) in a pregnancy followed by any miscarriage or other reproductive failure
8. Secondary infertility or pregnancy loss following the birth of a son, particularly if the pregnancy
with the son had any complications
9. Endometriosis with more than one miscarriage or IVF implantation failure
10. Under the age of 40 and unexplained poor egg or embryo quality and/or a low AMH or elevated
FSH for maternal age
11. PCOS or strong clinical symptoms of PCOS (i.e., collection of 20 or more eggs with a single
IVF cycle, a history of gestational diabetes, or a strong family history of adult onset diabetes)
and more than one pregnancy loss from pregnancy complications
12. Autoimmune disease and an early pregnancy loss or late pregnancy complication (such as
preeclampsia)
What Can Break Immune Tolerance for a Conceptus

and Lead to Immunological Rejection?
Several mechanisms may lead to failure of one or more of the steps involved in immune tolerance and
result in an inability of the maternal immune system to properly develop or maintain tolerance for paternal
antigens present on a conceptus. Inflammatory conditions, such as endometriosis, PCOS, and autoimmune
disease, are all associated with the production of inflammatory cytokines which can disrupt the tolerogenic
milieu. Maturation and activation of maternal antigen-presenting cells (APCs) by inflammatory cytokines
can bias naïve T cell differentiation away from regulatory T cells (Treg cells) and towards differentiation
of effector T cells (Teff cells), particularly of the Th1 and Th17 lineages [5–7]. This can drive both cellular
and humoral (antibody) responses to paternal antigens present on the conceptus.
Endometriosis, for example, is associated with significantly elevated levels of systemic inflammation,
with many studies demonstrating significant immune system activation at the cellular level and
elevated levels of inflammatory cytokines in follicular fluid, peritoneal fluid, and peripheral blood.
In addition to having negative effects on ovarian reserve and egg quality, this inflammation can
significantly decrease implantation rates and increase the risk for miscarriage and later pregnancy
complications, including preeclampsia [8]. Proper diagnosis and treatment of endometriosis is essential
to optimize the chances for a successful pregnancy. Many common symptoms of endometriosis are
often overlooked however, and many patients with endometriosis have very few or even none of the
typical clinical symptoms associated with endometriosis. It is therefore critical to have genetic and
immune testing that can be effectively and reliably used to identify patients with “occult” or “silent”
endometriosis [9].
Several autoimmune diseases are also associated with increased rates of reproductive failure, including
antiphospholipid syndrome (APS), psoriasis, systemic lupus erythematosus (SLE), and Hashimoto
thyroiditis [10–12]. While many patients with a history of reproductive failure present with a diagnosis of
one or more autoimmune conditions, many others have never been adequately tested. Many autoimmune
diseases also typically present clinical symptoms only later in life that spur the patient to seek a diagnosis.
However, it is known that significant immune system aberrations can be present for many years prior
to clinical symptoms becoming present [13]. These inflammatory changes can also trigger dendritic
cell maturation biasing toward Teff and away from Treg cell responses. In this manner, immunological
reproductive failure may be the first clinical symptoms of a nascent autoimmune condition for many
women of childbearing age.
Immune Testing in Recurrent Pregnancy Loss 103
The initial breakdown of tolerance for paternal antigens can occur at any point of paternal antigen
exposure from initial exposure to sperm and seminal fluid to late in a pregnancy. While skewing
toward Teff (and away from Treg) responses upon exposure to sperm/seminal fluid early in a partner’s
sexual history can lead to early losses or perceived infertility (implantation failure), the initial
failure in tolerance can also occur during a pregnancy which may have no or minimal effect on that
pregnancy, but which generates a memory response that can result in secondary infertility or RPL.
This will be discussed below in reference to secondary infertility/RPL that can occur following the
birth of a son.
Essential Components of a Complete Workup of Reproductive Failure

A detailed and thorough workup of a patient with suspected immunological etiologies for reproductive
failure is critical for proper identification of underlying genetic and immune issues and classification
of the nature and strength of these immunogenetic issues for selection of an appropriate treatment
protocol. For example, autoimmune conditions such as rheumatoid arthritis (RA) and SLE both result
in significant inflammation and increase the risk for reproductive failure. However, these autoimmune
diseases involve different ends of the immunological spectrum, with RA being strongly Th1-dominant
and involving strong cellular immune responses, while SLE is strongly Th2-dominant and involves
a strong humoral immune component. Just as it is important to differentiate these conditions for the
selection of proper treatment, so too is it critical to understand the nature of the immune aberrations
present in a patient with an immunological etiology for reproductive failure. While Th2-deviating
treatments can be effective to treat strongly Th1-dominant conditions, they can have the opposite effect
of exacerbating Th2-dominant conditions, leading to an increase in activation at the cellular level and
in levels of systemic inflammation [14].
As there are many mechanisms for immunological reproductive failure, simple tests that can only lead
to binary conclusions are insufficient and can be misleading. Unfortunately, much of the currently applied
clinical testing in reproductive immunology is based on outdated science and falls into this category.
For example, two of the more widely applied clinical tests are for NK cell cytotoxic activity (NKa)
and leukocyte antibody detection (LAD; equivalent to flow cytometry crossmatch). Testing for NKa is
often the only cellular testing performed in a misguided attempt to identify a single variable universally
associated with immunological reproductive failure. It is based on assumptions made about uterine NK
(uNK) cell function prior to the current knowledge of the differences in phenotype and function of uNK
cells compared with blood NK cells. We now know that (i) uNK cells are poorly cytotoxic, (ii) uNK
cell activity cannot be inferred from activity of peripheral blood NK cells, and (iii) uNK cell activation
is not detrimental to embryo implantation but is actually required to facilitate this process. In our
experience, while NKa testing can be useful as one piece of cellular data to help determine the nature
of a possible underlying immune issue in a patient, NKa has no independent diagnostic or prognostic
value. LAD testing is based on a principle of “blocking” antibodies that can bind to paternal antigens
present on a conceptus and protect against allogenic effector responses. Therefore, the lack of binding
of paternal antibodies to maternal lymphocytes is interpreted as a deficiency in need of correction.
While antibodies that can perform “blocking” activity exist (by occupying antigenic sites but failing
to elicit effector functions) the LAD test does not assess the antigenic specificity, structure, or function
of paternal antibodies; for example, whether they can initiate complement cascade activation. These
antibodies are at least as likely to be competent to trigger effector function, and many more recent studies
have determined an overall negative prognostic value of the presence of paternal antigen-specific anti-
HLA antibodies, including an increased risk for preterm labor. Unfortunately, this misinterpretation of
an insufficiently specific test leads some patients to seek out lymphocyte immunotherapy (LIT) in an
attempt to trigger the production of antibodies specific for paternal antigens. Using far more specific
testing (discussed below) we have found that these patients often have high titers of paternal antigen-
specific anti-HLA antibodies that strongly fix complement. Therefore, in many cases LAD testing is
not useful and may lead some patients to take steps that further decrease their chances for a successful
pregnancy.
Detailed Personal and Family History

In the author’s experience, many indicators of possible immune issues related to reproductive failure are
simply not uncovered or are overlooked in a patient’s history. These include many clinical symptoms
of endometriosis and PCOS. It is also important to ascertain a patient’s familial history of autoimmune
disease, as autoimmune diseases cluster in families due to inherited genetic predispositions, mostly within
the MHC/HLA region of chromosome 6.
Maternal and Paternal Genetic Testing

Full maternal and paternal haplotypes of classical HLA class I (HLA-A, -B, -C) and class II (HLA-DQA1,
-DQB1, -DRB1, -DRB3/4/5) loci are a critical component of testing that allow for analysis of a number
of variables that can affect maternal immune tolerance to paternal antigens.
KIR Haplotype and Maternal and Paternal HLA-C Allotypes

The number and identity of activating and inhibitory killer immune-like receptors (KIRs) expressed
by uNK cells (categorized as KIR A and KIR B haplotypes) and the HLA-C allotypes expressed by
uterine stromal cells determine the threshold for uNK cell activation which is critical for angiogenic
cytokine secretion and spiral artery remodeling. Specific combinations of maternal KIR haplotypes,
maternal HLA-C allotypes, and embryonic HLA-C allotypes have been found to inadequately activate
uNK cell cytokine secretion and are associated with increased risk for poor placentation, which results in
increased rates of spontaneous miscarriage and preeclampsia as well as low birth weight [15–18]. These
combinations include maternal KIR haplotypes lacking activating KIRs (A haplotypes and B haplotypes
lacking the KIR2DS1 gene), particularly when the embryonic HLA-C2 allele content is greater than the
maternal HLA-C2 allele content (by virtue of a paternally-contributed C2 allele).
Autoimmune-Predisposing Alleles and Haplotypes

Genetic predispositions have been identified for almost all known autoimmune conditions which largely
cluster within the MHC/HLA region of chromosome 6. These HLA alleles and haplotypes only predispose
to the development of autoimmune conditions, which in most cases require additional environmental
triggers and/or or additional genetic elements for the development of autoimmunity. However, while the
presence of these predisposing alleles/haplotypes by themselves are not diagnostic of the presence of
autoimmunity, they are frequently enlightening in view of the immune data to assist in the characterization
of an underlying immune condition.
HLA Class II Homozygosity

Paternal peptide antigens are presented by maternal APCs to naïve T cells bound to maternal HLA class
II molecules. Peptide antigens are bound within peptide-binding grooves that are restrictive in an allelic
fashion for which peptides can be bound. Therefore, homozygosity of maternal HLA class II alleles
restricts the repertoire of paternal antigens that can be presented and thereby also limits the ability to
trigger paternal antigen-specific Treg cell differentiation. Studies have shown that homozygosity at the
DRB1 locus is associated with an increased risk for the development of preeclampsia [19].
Lack of Class II Allele Mismatching

MHC genes, and class II genes in particular, have been implicated in both pre- and post-copulatory
mate choice in animals. Several mechanisms for post-copulatory mate choice (cryptic female choice)
based on MHC selection have been described in animals, including selective fertilization, selective
implantation, and selective abortion. The role of MHC (HLA) incompatibility in human reproductive
outcome was extensively studied in the 1980s and 1990s with conflicting results. These studies included
a large diversity of methodological approaches that likely significantly contributed to the incongruent
results. Despite the highly divergent approaches taken to study the effect of HLA (in)compatibility,
a meta-review of this literature showed that there was a modest although significantly increased risk
of recurrent miscarriage in couples sharing at least one allele at the HLA-DRB1 locus [20]. Several
more recent studies have provided further evidence for a significant inverse correlation between
histoincompatibility at HLA class II loci and the occurrence of recurrent miscarriage and preeclampsia
[21,22]. The significance of this effect is further increased when DRB supertypes (which mark ancestral
lineages and therefore groups of antigenically related DRB1 alleles) are considered in place of individual
DRB1 alleles [23].
Class II HY Restricting HLA (HYrHLA) Alleles

In addition to the highly polymorphic class I and class II HLA antigens, peptides of polymorphic proteins
encoded outside of the HLA region can also stimulate allogenic T cell responses. These antigens, referred
to as minor histocompatibility (minor H) antigens can lead to chronic rejection of an allograft through
indirect allorecognition. Minor H antigens include those encoded from diallelic autosomal genes and well
as by genes on the Y chromosome, termed HY antigens.
HY antigen presentation is restricted to a small subset of class I and class II HLA molecules. These
HYrHLA alleles include the class II DQB1*05:01, DQB1*05:02, DRB1*15, and DRB3*03:01 alleles.
Possession of one or more of these class II HYrHLA alleles allows the maternal immune system to
generate an immune response (tolerogenic or effector) to HY antigens present on a male conceptus. These
alleles are found at an increased frequency in women suffering secondary recurrent miscarriage following
the birth of a son, and the presence of anti-HY antibodies is associated with a high rate of preclinical loss
of male embryos, although loss of female embryos is also increased, likely through epitope spreading to
additional paternal antigens. The risk for development of anti-HY immunity is increased by the presence
of more than 1 class II HYrHLA allele and by the occurrence of complications during the pregnancy
with the son [24–26].
HLA-G Polymorphisms
HLA-G is a nonclassical class I HLA molecule that is abundantly expressed on the surface of EVTs and
binds to inhibitory receptors on leukocytes, including ILT2. Isoforms of HLA-G can also be secreted and
provide a tolerogenic signal to APCs as well as function as an activating ligand for KIR2DL4 on uNK
cells. Low levels of soluble HLA-G are associated with a decreased implantation rate, and increased risk
for miscarriage and preeclampsia. Although HLA-G is significantly less polymorphic than classical HLA
genes, a 14 base pair insertion in the 3′ untranslated region results in decreased levels of HLA-G protein,
and homozygosity of this polymorphism is associated with increased risk for recurrent miscarriage and
preeclampsia [24,27].
Cellular Analysis
Lymphocyte Lineage Profiling
Naïve CD4+ T cells can differentiate into one of several lineages upon priming by APCs, depending
on the nature of the APCs involved and the profile of soluble molecules secreted by the APCs during
priming. These lineages include Th1, Th2, Th17, and Treg cells which are distinguished by unique
cytokine expression profiles. Cells expressing these cytokines (IFNγ for Th1, IL-4 for Th2, IL-17 for
Th17, and IL-10 for Treg) can be identified by flow cytometry, and ratios of these cells can be determined
to characterize the CD4+ T cell lineage profile for an individual. Analogous lineages also exist for
CD8+ T cells, NKT cells, and NK cells, which can be similarly characterized. Levels of TNFα-positive
cells can also be used as a general marker of cellular activation. The relative balances of these lineages
within each of these cell types can be used to help characterize the nature of any underlying immune
conditions.
NKa
As discussed above, NKa has no independent diagnostic or prognostic value. It is, however, an additional
cellular variable that can provide information about the nature of underlying immune conditions when
taken together with the rest of the genetic and immune context.
Immunophenotyping
Flow cytometry immunophenotyping can be used to identify relative proportions of various cell types
using combinations of cell surface markers. This can be used to detect total levels of various lymphocyte
populations (including CD4+ T cells, CD8+ T cells, CD4+ NKT cells, CD8+ NKT cells, total B cells,
CD5+ B cells) as well as their activation state using markers of cellular activation (i.e., HLA-DR+ T cells).
Treg cells can be specifically identified using a combination of cell surface markers. Given the
prominent role of these cells in the early immunological response to a conceptus, they are a critical
cell type to accurately and reliably test for. Early clinical studies on levels of Treg cells in pregnancy
used only a very limited set of markers to identify Tregs; CD4 and CD25 with Treg cells identified as
CD4+CD25high[28]. This phenotype, however, does not specifically identify Treg cells but rather a broader
set of T cells, including many non-Treg T cells with no suppressive function. These studies concluded
that there is an increase in Treg cells in peripheral blood in early pregnancy. More recent studies using
a set of markers that more specifically identify Treg cells, however, indicate that Treg cells decrease
in the peripheral blood during the first trimester as they are recruited to decidua [29]. Our data using
CD4+CD25+CD127lowFoxP3+ to identify Treg cells [29] has identified changes in levels of Treg cells
during early pregnancy as an independent factor in pregnancy outcome.
Soluble Factors
Maternal Serum Cytokines
Elevated serum levels of proinflammatory cytokines are found in patients with autoimmune and
inflammatory conditions and are also found in patients with a history of recurrent miscarriage. Lymphocyte
lineage profiling and immunophenotyping are very useful to help characterize the nature (e.g., Th1- or
Th2-dominant) of underlying immune conditions, although they are not as useful in determining the
extent of systemic inflammation that is present. Elevated serum levels of cytokines require significant
levels of tissue inflammation involving recruitment and activation of additional cell types, including
macrophages and neutrophils which can produce relatively larger levels of cytokines.
Paternal Seminal Fluid Cytokines

A tolerogenic milieu of factors present in seminal fluid, including TGFβ, PGE2, and HLA-G, is critical
to maintain maternal APCs in a tolerogenic state and bias naïve T cell differentiation in response to
paternal antigens toward the Treg cell lineage. Paternal inflammatory conditions leading to elevated levels
of inflammatory cytokines in seminal fluid can disrupt this tolerogenic milieu and lead to maturation of
maternal APCs and activation of Teff responses to paternal antigens [30].
Anti-HLA Antibodies
Donor-specific anti-HLA antibodies are capable of mediating acute and chronic rejection of allografts
and are a significant barrier to successful organ transplantation [31]. As discussed above with regard to
the LAD test and LIT, the role of anti-HLA antibodies in pregnancy outcome has been controversial. This
controversy is at least in part due to methodological inconsistencies and deficiencies. Specifically, many
studies have failed to determine the paternal antigen specificity of anti-HLA antibodies, their specific
levels, and their ability to elicit effector functions that mediate tissue damage, such as complement cascade
activation [32]. Several more recent studies have established a clear association of paternal antigen-
specific anti-HLA antibodies with a significantly increased risk for fetal rejection and the development
of pregnancy complications, including preterm birth [33,34]. The presence of antibodies specific for
paternally-derived HLA-C antigens (the only classical HLA locus expressed by early stage embryos) has
also been specifically linked to an increased risk for recurrent miscarriage [35,36].
The Luminex single-antigen bead (SAB) assay is a highly sensitive and specific method for detecting
anti-HLA antibodies for individual HLA antigens. Combined with paternal HLA haplotyping, the
presence and relative levels of maternal antibodies specific for individual paternal HLA antigens can
be determined. Further, the ability of individual anti-HLA antibodies to elicit complement cascade
activation can be assessed by determining their ability to fix C1q in this assay [37]. In addition to treatment
modalities to decrease levels of preformed HLA antibodies and inhibit their effector functions, in many
cases (where an antibody is present which is specific for one paternal allele at an HLA locus which is
heterozygous) it is possible to select embryos lacking the offending paternal antigens.
Other Soluble Factors

Various serological markers for a range of autoimmune conditions can also be tested. APS, an autoimmune
condition that can mediate recurrent pregnancy loss, is covered in detail elsewhere in this book. Notably,
although treatment for obstetric APS is focused on prevention of thrombosis, it is becoming clear that
triggering inflammatory pathways, rather than triggering formation of microclots, is the mechanism
through which antiphospholipid antibodies mediate placental damage [38].
Additional useful serological markers for autoimmune disease include thyroid autoantibodies (anti-
TPO, anti-thyroglobulin, anti-TSH receptor), antinuclear antibodies (ANAs), rheumatoid factor, and anti-
CCP antibodies. In addition to an ANA screen for titer and staining pattern, testing for specific species
of ANA (i.e., ANA-Sm, ANA-Ro, ANA-La, ANA-dsDNA, etc.) can be useful to more specifically define
underlying autoimmune conditions.
The biologically active form of vitamin D (1,25(OH)2D3) strongly modulates dendritic cells to a
tolerogenic phenotype, and deficient levels of vitamin D are associated with increased incidence of
infertility and recurrent pregnancy loss [39, 40].
Determination of a Diagnosis
The above testing can uncover clear markers of conditions such as antiphospholipid syndrome, SLE,
and Hashimoto thyroiditis, for which specific serological markers exist. In other cases, synthesis of
several aspects of the genetic and immune data together with careful consideration of patient’s personal
and family history as well as findings of ultrasound investigations (including Doppler analysis) reveal
important aspects of the nature of the underlying immune conditions present. For example, our experience
in evaluating the above-described set of testing allows us to reliably identify patients with “silent”
endometriosis as confirmed by laparoscopy.
Monitoring the Maternal Immune Response to the Conceptus

A thorough initial workup is essential to establish a personal immunological baseline against which
testing during pregnancy can be compared in order to monitor the immune response to a conceptus. This
monitoring can be effectively used to evaluate the maternal immune response to a conceptus and the
efficacy of immunotherapy.
A shift toward Th2 dominance is characteristic of early pregnancy, and the paradigm that the Th1/Th2
balance of early pregnancy regulates the nature of the alloimmune response to the conceptus has been
prominent in the field of clinical reproductive immunology. In recent years, there has been an increasing
recognition that the Th1/Th2 paradigm of the immunological response to pregnancy is insufficient and the
importance of Treg cell responses has become more prominent [40,41]. Concurrently, the opposing roles
of Treg cells and Th17 cells in many conditions, including autoimmunity and transplant tolerance, have
become increasingly recognized. Indeed, our data indicate that the strongest prognostic factors in early
pregnancy for a successful outcome (live birth after an uncomplicated pregnancy) are efficient Treg cell
recruitment from the peripheral blood to the decidua (as measured by the percent decrease of Treg cells
in the peripheral blood) and a lack of a significant increase in levels of IL-17 producing cells, including
Th17 cells, IL-17 positive CD8+ T cells (Tc17 cells), IL-17 positive NKT cells (NKT17 cells), and IL-17
positive NK cells (NK17 cells).
The Future of Testing in Clinical Reproductive Immunology

As discussed above, SAB testing can be used to detect humoral responses to specific paternal HLA
antigens. Reliable assays for the detection of antigen-specific cellular (T cell-mediated) responses have
also been developed and have been applied in the setting of transplantation. However, these assays detect
allorecognition through direct antigen presentation which is not relevant to pregnancy. Although more
technically difficult, similar assays to detect allorecognition through indirect antigen presentation are
also being developed. The ability to sensitively and reliably detect both cellular and humoral responses
specifically to paternally-derived antigens will significantly advance the field of clinical reproductive
immunology.
The clinical relevance of maternal immune responses to paternally derived minor H antigens, including
HY antigens, is also increasingly being recognized, and testing to detect immune responses to minor H
antigens will further advance the power and specificity of reproductive immune testing.
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12
Uterine Anomalies and Recurrent Pregnancy Loss
Daniel S. Seidman and Mordechai Goldenberg
Introduction
It is frustrating to realize how little is known about the pathophysiology prevalence and impact of uterine
malformations [1]. Reported prevalences range from 0.2% to 10.0% [2]. Newer imaging modalities
currently estimate that the incidence in the general population is approximately 1%, and about threefold
higher in women with recurrent pregnancy loss (RPL) and poor reproductive outcomes [2]. In addition to
pregnancy loss, uterine malformations such as uterine septum, intrauterine adhesions, polyps, or fibroids
predispose to infertility, preterm labor, and abnormal fetal presentations. These malformations are
amenable to surgical correction. Therefore, an accurate diagnosis is essential in order to offer appropriate
treatment.
In this chapter we review the common congenital and acquired uterine anomalies associated with
recurrent pregnancy losses, and discuss contemporary diagnosis and treatment options.
Development and Classification of Müllerian Tract Defects

The female reproductive tract develops from the two Müllerian ducts that develop in the 6-week embryo.
The cephalic ends of the Müllerian ducts form the fallopian tubes, and the caudal portions fuse to
form the uterus, cervix, and the upper two-thirds of the vagina. The ovaries and lower one-third of the
vagina have separate embryologic origins. The Müllerian ducts grow caudally and become enclosed in
peritoneal folds that later develop into the round and ovarian ligaments. Female sexual differentiation is
marked by degeneration of the Wolffian ducts due to the absence of testosterone and Müllerian-inhibiting
substance. At 9 weeks of gestation the uterine cervix is recognizable, and by 17 weeks the formation of
the myometrium is complete. Vaginal development begins at approximately 9 weeks. The uterovaginal
plate forms between the caudal buds of the Müllerian ducts and the dorsal wall of the urogenital sinus.
The uterovaginal plate elongates to form the lower third of the vagina, whereas the upper two-thirds of
the vagina derives from Müllerian ducts. Complete formation and differentiation of the Müllerian ducts
into the female reproductive tract depend on completion of organogenesis, fusion both laterally and
vertically, and resorption.
In failure of organogenesis, one or both Müllerian ducts may not develop fully, resulting in uterine
agenesis or hypoplasia (bilateral) or unicornuate uterus (unilateral). Failure of fusion of the Müllerian
ducts may result in bicornuate or didelphys uterus. Vertical fusion refers to fusion of the ascending
sinovaginal bulb with the descending Müllerian ducts (i.e., fusion of the lower one-third and upper two-
thirds of the vagina). Complete vertical fusion forms a normal patent vagina, while incomplete vertical
fusion results in an imperforate hymen.
After the lower Müllerian ducts fuse, a central septum is present, which is subsequently resorbed to
form a single uterine cavity and cervix. Failure of resorption results in a septate uterus.
The most commonly used classification of Müllerian anomalies is that of the American Society for
Reproductive Medicine (ASRM) [3], which is shown in Table 12.1.
110
Uterine Anomalies and Recurrent Pregnancy Loss 111
TABLE 12.1
Classification of Müllerian Duct Anomalies
1. Class I—Uterine agenesis or hypoplasia
2. Class II—Unicornuate uterus
3. Class III—Didelphys uterus
4. Class IV—Bicornuate uterus
5. Class V—Septate uterus
6. Class VI—Arcuate uterus
7. Class VII—Diethystilbestrol (DES)-exposed uterus
Subseptate Uterus
Subseptate uterus is the most common uterine anomaly in women with RPL and recurrent first trimester
pregnancy loss [4], and may predict poor pregnancy outcome if incidentally diagnosed in the early stage
of a viable intrauterine pregnancy [5]. The association between RPL and a subseptate uterus has been
attributed to decreased connective tissue in the septum, resulting in poor decidualization and placentation
and local uncoordinated myometrial contractility. The septum has been said to have a poorer vascular
supply than the rest of the uterus, subsequently restricting the blood supply to the embryo [6,7]. However,
a systematic review of the literature by Rikken et al. [8] found the intrauterine septum to consist of
endometrium and myometrium similar to the uterine wall. All imaging studies evaluating vascularity
have found the majority of intrauterine septa to be vascularized. Histological studies have found the
intrauterine septum to consist of myometrium covered by endometrium [8]. The degree of distortion of
the uterine cavity has been shown to be higher in women with RPL [9] (mainly due to reduced length of
unaffected cavity, rather than increased septum length). The greater degree of uterine cavity distortion
in RPL supports the hypothesis of septal implantation as a potential cause of miscarriage, since the
likelihood of septal implantation increases with an increasing ratio of septal size to functional cavity.
Arcuate Uterus
An arcuate uterus (intrauterine indentation of <1 cm) is found in 17% in women with recurrent
miscarriage [9] compared to 3.2% in the general population. The diagnosis is difficult when conventional
diagnostic methods are used such as hysteroscopy or laparoscopy [10]. Consequently, little is known about
the prevalence and clinical significance. Although many believe that the arcuate uterus has little or no
impact on reproduction and obstetrical outcomes [11], some studies have reported an increase in adverse
reproductive outcomes, mostly second trimester loss [10,12,13]. Gergolet et al. [13] followed women with
at least one early miscarriage and a subseptate or arcuate uterus undergoing hysteroscopic metroplasty.
The miscarriage rates after metroplasty were similar between the women with subseptate and arcuate
uterus (14.0% and 11.1%, respectively). Before metroplasty, the miscarriage rates were significantly higher
in subseptate uterus group as well as in the arcuate uterus group. The authors therefore concluded that
the arcuate uterus had a similar effect on reproductive outcome as the subseptate uterus both before and
after surgical correction [13].
Unicornuate Uterus
A unicornuate uterus is the result of complete, or almost complete, arrest of development of one of
the Müllerian ducts (Figure 12.1). When the arrest is incomplete (in 90% of patients with unicornuate
uterus), a rudimentary horn with or without a functioning endometrium may be present. The incidence
of unicornuate uterus has been estimated to be 6.3% of uterine anomalies and may be associated with
urinary tract and renal anomalies. Approximately one-third of all pregnancies result in miscarriage
[5,14,15]. The high miscarriage rate is mostly attributed to abnormal uterine vasculature and decreased
muscle mass.
There are no surgical procedures to correct the unicornuate uterus. Prophylactic cervical cerclage has
been suggested for the prevention of miscarriage in patients with unicornuate uterus, although there is no
FIGURE 12.1 Three-dimensional (3D) transvaginal ultrasound of a unicornuate uterus using volume contrast imaging in
plane C (VCIC). (Courtesy of Prof. Yaron Zalael MD, Sheba Medical Center, Tel Hashomer, Israel.)
clear evidence of cervical incompetence [15]. However, with little data to support the use of cerclage, most
clinicians prefer to use careful follow-up with frequent clinical and sonographic evaluation of cervical
length. Resection of a cavitated rudimentary horn is often recommended in symptomatic patients with a
unicornuate uterus suffering from dysmenorrhea and hematometra.
Uterus Didelphis
A double uterus results from the complete failure of the two Müllerian ducts to fuse (Figures 12.2 and
12.3). Therefore, each duct develops into a separate unicornuate uterus. The two uteri may each have a
cervix or may share a cervix. In 67% of cases, uterus didelphis is associated with two vaginas separated
by a thin wall. Didelphic uteri are relatively uncommon, with an estimated incidence of 6.3% of uterine
anomalies [6,9]. The two uteri do not always function normally and are associated with a miscarriage
rate of 20.9% and a preterm delivery rate of 24.4% [9,16]. A long-term follow-up of 49 Finnish women
FIGURE 12.2 Two-dimensional (2D) transvaginal ultrasound of a didelphys uterus with obstructed right vagina
(hematocolpus). (Courtesy of Prof. Yaron Zalael MD, Sheba Medical Center, Tel Hashomer, Israel.)
FIGURE 12.3 Two- and three-dimensional (2D and 3D) transvaginal ultrasound of a didelphys uterus (using volume
contrast imaging in plane C [VCIC]). (Courtesy of Prof. Yaron Zalael MD, Sheba Medical Center, Tel Hashomer, Israel.)
with didelphic uterus and a longitudinal vaginal septum reported an obstructed hemivagina in nine
women (18%). Eight of these nine women also had ipsilateral renal agenesis [16]. Cesarean section rates
are higher due to uterine dystocia and malpresentations [17]. In addition, didelphic uterus is commonly
associated with a patent or obstructed vaginal septum. Fertility is not notably impaired, but endometriosis
is commonly present, possibly because of retrograde menstruation [16].
Bicornuate Uterus
A bicornuate uterus results from partial non-fusion of the Müllerian ducts (Figure 12.4). The central
myometrium may extend to the level of the internal cervical os (bicornuate unicollis) or external
cervical os (bicornuate bicollis). The latter is distinguished from uterus didelphys as there is some
degree of fusion between the two horns, while in uterus didelphys, the two horns and cervices are
separated completely. In addition, the horns of the bicornuate uteri are not fully developed; typically,
they are smaller than those of didelphys uteri. Bicornuate uteri are probably the most common uterine
anomaly after septate and arcuate uterus [17]. The reproductive outcome seems to be directly correlated
with the severity of fundal indentation. It is generally considered that the bicornuate uterus does not
directly affect infertility but may be linked with RPL. Bicornuate uterus can be corrected surgically
by metroplasty.
T-Shaped or Dysmorphic Uterus

T-shaped uterus is characterized by an excess of myometrium in the uterine walls giving rise to a
subcornual constriction ring which causes dysmorphism and hypoplasia of the uterine cavity [18]. The
new classification of uterine anomalies by the European Society of Human Reproduction and Embryology
and the European Society for Gynaecological Endoscopy working group of experts [19] introduced a new
category defined as dysmorphic uterus. Dysmorphic uterus incorporates all cases with a normal uterine
outline but with an abnormal shape of the uterine cavity excluding septa. Class U1a or T-shaped uterus
is characterized by a narrow uterine cavity due to thickened lateral walls with a correlation 2/3 uterine
corpus and 1/3 cervix. Class U1b, or uterus infantilis, is characterized by a narrow uterine cavity without
lateral wall thickening and an inverse correlation of 1/3 uterine body and 2/3 cervix. Class U1c, or others,
was added to include all minor deformities of the uterine cavity including those with an inner indentation
at the fundal midline level of <50% of the uterine wall thickness [20]. In the ASRM classification, these
anomalies are included in Class VII and are mainly related to in utero diethylstilbestrol (DES) exposure
[3]. However, dysmorphic uteri are also found in RPL without DES exposure [20,21].
FIGURE 12.4 Three-dimensional (3D) transvaginal ultrasound of a bicornuate uterus. (Courtesy of Prof. Yaron Zalael
MD, Sheba Medical Center, Tel Hashomer, Israel.)
Myomas
Submucous myomas distort the uterine cavity, the overlying endometrium is usually thin and inadequate for
normal implantation, and hence submucous fibroids can be associated with pregnancy loss [22]. The case
is less clear with intramural and subserous fibroids. In these locations, the size and the number of fibroids
may be significant. Significantly lower implantation and pregnancy rates have been found in patients with
intramural or submucosal fibroids undergoing in vitro fertilization and intracytoplasmic sperm injection
(IVF/ICSI) even without uterine cavity deformation [22]. The pregnancy rate observed within 1 year after
myomectomy is higher than that observed in couples with unexplained infertility and no treatment [23]. A
large retrospective study reaffirmed the observation that while non-cavity-distorting fibroids did not affect
IVF/ICSI outcomes, intramural fibroids greater than 2.85 cm in size significantly impaired the delivery rate
of patients undergoing IVF/ICSI [24]; however, there is little information available for RPL.
Polyps
Polyps are benign hyperplastic endometrial growths that have also been associated with adverse pregnancy
outcomes. It is postulated that polyps and fibroids with intracavitary extension may act as foreign bodies
within the endometrial cavity [25]. In addition, polyps and fibroids might induce chronic inflammatory
changes in the endometrium that make it unfavorable for pregnancy. A case-control study suggested a
molecular mechanism to support the clinical findings of diminished pregnancy rates in women with
endometrial polyps [26].
Since the presence of polyps has been associated with a worse prognosis for pregnancy, polypectomy
is usually considered if no other explanation for the recurrent loss is found [25,27].
Intrauterine Adhesions
Intrauterine adhesions develop as a result of previous surgical procedures, typically curettage, or subsequent
endometritis. Intrauterine scars can probably interfere with normal implantation and may be responsible for
pregnancy loss. A systematic review estimated that intrauterine adhesions are encountered in one in five
women after miscarriage [28]. However, in more than half of these women, the severity and extent of the
adhesions was mild, with unknown clinical relevance. Although the authors have failed to identify studies
associating intrauterine adhesions and long-term reproductive outcome after miscarriage, Hooker et al. [28]
have reported similar pregnancy outcomes subsequent to conservative medical or surgical management.
Investigation of Uterine Integrity

Ultrasound
Transvaginal sonography (TVS) is usually the initial investigation but can be enhanced by three-
dimensional (3D) ultrasound. TVS allows accurate and rapid characterization of the uterus, including
its size and position as well as the presence of anomalies such as a duplicated cervix, duplex uterus,
septum, or unicornuate uterus. TVS is also useful in determining the size and location of uterine myomas,
intrauterine polyps, and endometrial irregularities that might suggest adhesions. The ability to visualize
both the uterine cavity and the fundal uterine contour on a 3D scan facilitates the diagnosis of uterine
anomalies and enables differentiation between septate and bicornuate uteri. The additional use of color
Doppler ultrasound may also allow visualization of intraseptal vascularity and may help in distinguishing
the avascular from the vascular septum.
3D ultrasound is an accurate and reproducible means of diagnosing congenital uterine anomalies [29]
(Figures 12.1 and 12.5), with a clear advantage over hysterosalpingography (HSG), hysteroscopy, and
laparoscopy, since it is noninvasive. The results of 3D ultrasound have been shown to concur with HSG
in major congenital anomalies [29]. It has been suggested that the ability to visualize both the uterine
cavity and the myometrium on a 3D scan facilitates the diagnosis of uterine anomalies and enables easy
differentiation between subseptate and bicornuate uteri.
Woelfer et al. [12] assessed the reproductive outcomes in women with congenital uterine anomalies
detected incidentally by 3D ultrasound in 1089 women with no history of infertility or RPL. Of these, 983
women had a normally shaped uterine cavity, 72 an arcuate, 29 a subseptate, and 5 a bicornuate uterus.
Women with a subseptate uterus had a significantly higher proportion of first trimester loss compared
with women with a normal uterus. Women with an arcuate uterus had a significantly greater proportion
of second trimester loss and preterm labor. Woelfer et al.’s [12] study demonstrated the potential value of
3D ultrasound and contributed evidence to the proposed association between congenital uterine anomalies
and adverse pregnancy outcomes.
FIGURE 12.5 Three-dimensional (3D) transvaginal ultrasound of a septated uterus (3D rendering). (Courtesy of Prof.
Yaron Zalael MD, Sheba Medical Center, Tel Hashomer, Israel.)
Sonohysterography
Transvaginal sonohysterography (SHG) is carried out by the intrauterine infusion of an isotonic saline
solution. The sensitivity and specificity of SHG is similar to hysteroscopy. With the proper setup and
training, transvaginal SHG is a low-cost, easy, and helpful method of diagnosing uterine malformations.
SHG detected all uterine anomalies found in a study of 54 patients with primary or secondary infertility
or RPL and a clinically or sonographically suspected abnormal uterus [30].
It is now possible to combine 3D ultrasound with SHG. Sylvestre et al. [31] carried out a study of 209
infertile patients suspected to have an intrauterine lesion on 3D SHG. Ninety-two patients with a lesion
underwent hysteroscopy. In these 92 patients, polyps were found in 48 women, submucous or intramural
myomas in 35 cases, both polyps and myomas in 3 cases, 4 Müllerian anomalies, 1 thick endometrium,
and 1 patient had intrauterine synechiae. As 3D SHG allowed precise recognition and localization of
lesions, it was suggested that if 2D and 3D SHG are normal, invasive diagnostic procedures such as
hysteroscopy could be avoided.
Alborzi et al. [32], performed a prospective study to determine whether SHG can differentiate septate
from bicornuate uterus, in 20 patients with a history of RPL and an HSG diagnosis of septate or bicornuate
uterus. SHG effectively differentiated septate and bicornuate uterus and may eliminate the need for
laparoscopy in order to differentiate between these anomalies.
Hysterosalpingography
Hysterosalpingography (HSG) has long been used to evaluate the contour of the uterine cavity, cervical
canal, and fallopian tube. The radio-opaque contrast medium fills the cavity, allowing the accurate
identification of filling defects, scarring, or a septum. However, HSG cannot differentiate between a
septate uterus and a bicornuate uterus. Furthermore, HSG cannot determine the myometrial extension or
the size of intrauterine lesions. Therefore, HSG is primarily used to assess tubal patency and has a limited
role in the imaging of uterine malformations.
Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is an accurate noninvasive technique for the evaluation of uterine anomalies.
MRI has been shown to be a valuable tool in the diagnosis of selected cases of Müllerian duct anomalies [33].
Although most anomalies will be initially diagnosed at HSG and SHG, further imaging will often be required
for definitive diagnosis and elaboration of secondary findings [34]. At this time, MRI is justified only in special
cases where its high accuracy and detailed elaboration of uterovaginal anatomy is needed.
The use of MRI remains limited due to its cost. However, in selected cases careful use of MRI to
delineate the pelvic soft tissues may greatly aid in precise definition of the anomaly and in planning the
most appropriate corrective surgery [34].
Diagnostic Hysteroscopy
Hysteroscopy offers the best and the most direct assessment of the uterine cavity. During the procedure
intracavitary structures can be directly visualized and directed biopsies can be obtained when indicated.
A retrospective study by Zupi et al. [35] found an association between the hysteroscopic findings in 344
women with recurrent spontaneous abortion and major or even minor uterine anomalies. The anomalies
were shown to correlate with an increased risk of recurrent miscarriage [35].
The intramyometrial extension of fibroids cannot be assessed, however, and therefore the estimate of
size remains imprecise. Hysteroscopy alone cannot differentiate between a septate uterus and a bicornuate
uterus; laparoscopy or SHG is required to complete the evaluation.
Diagnostic Laparoscopy
Laparoscopy allows the surgeon to assess the outer surface of the uterus and other pelvic structures. It is
used to establish the precise diagnosis of the various congenital and acquired anomalies. Laparoscopy is
also used for the removal of subserous and intramural fibroids [36]. Currently laparoscopy is rarely used
to clarify uterine anatomy and is generally reserved for women in whom interventional therapy is likely
to be undertaken.
Choice of Method for Imaging Uterine Morphology

Ultrasonography is the most readily available and least invasive mode of imaging in suspected uterine
abnormalities (Table 12.2). 2D sonography allows excellent assessment of myometrial morphology and
is especially useful for determining the number, size, and location of myomas. SHG allows accurate
delineation of intrauterine polyps and improves the accuracy of identifying submucous myomas and for
assessing the size of uterine septa. 3D sonography greatly enhances our ability to differentiate between
a uterine septum and a bicornuate uterus (Figures 12.2–12.5). Hysterosalpingography can help delineate
the integrity of the uterine cavity, but due to its invasive nature and the associated exposure to radiation,
is rarely used for RPL.
Hysteroscopy can be performed with 2−3 mm scopes without the need for speculum, tenaculum, or
anesthesia [37]. This simple outpatient procedure provides an accurate assessment of the uterine cavity.
It remains the method of choice for assessment of the presence and extent of intrauterine adhesions. It is
also the optimal method to evaluate the size and extension of polyps and submucous myomas. However,
hysteroscopy cannot fully differentiate between a uterine septum and a bicornuate uterus.
The role of MRI is limited due to its cost. However, in selected and complicated cases MRI may
help to clarify the details of soft tissue anatomy and may be especially useful when planning surgical
correction [33,34].
TABLE 12.2
Imaging Modalities for Assessing Uterine Anomalies in Women with Recurrent Pregnancy Loss
Imaging Modalities Advantages Disadvantages Cost
Ultrasonography Readily available Poor demonstration of uterine Low
Least invasive contour
Excellent assessment of the myometrial Uterine cavity not clearly
morphology demonstrated
Hysterosalpingography Shows the contour of the uterine cavity, Exposure to radiation Moderate
cervical canal, and tubal lamina Iodine sensitivity risk
Painful
Pelvic inflammatory disease risk
High false-positive rates
3D Sonography Allows visualization of both uterine Equipment not readily available Moderate
cavity and myometrium Requires experienced operator
Enables easy differentiation between
subseptate and bicornuate uteri
Sonohysterography Good evaluation of uterine cavity Time consuming Low
Tubal patency assessed High false-positive diagnosis rate
for intrauterine adhesions
Diagnostic Most accurate assessment of the uterine Limited efficiency of Moderate
Hysteroscopy cavity differentiating between uterine
Simple outpatient procedure septum and bicornuate uterus
No information on tubal patency
Invasive
Risk of infection, perforation
MRI Useful in clarifying details of soft No information on tubal patency High
tissue anatomy Not easy to interpret results
Diagnostic Accurate for differentiating between a Invasive High
Laparoscopy uterine septum and a bicornuate uterus Requires general anesthesia
Low postoperative morbidity
Treatment
As stated above, little evidence can be found in the current literature demonstrating that uterine factors are
causally linked with reproductive loss. However, there are reports suggesting that treatment may improve
the fertility outcome [38,39]. The published evidence includes several observational series that demonstrate
successful fertility, with term pregnancy rates ranging from 32% to 87% following hysteroscopic division
of intrauterine adhesions. The evidence supporting a direct link between a septate uterus and reproductive
loss is derived from the results of metroplasty. Several case series have demonstrated a reduction in
the spontaneous abortion rate, from 91% to 17%, after hysteroscopic metroplasty. However, there are
no prospective controlled trials that have provided conclusive evidence that the correction of uterine
anatomic abnormalities benefits the next pregnancy.
Endoscopic surgery is the main course of treatment offered to patients with uterine anomalies (Table
12.3). Operative hysteroscopy currently allows a technically straightforward method of correcting
intrauterine pathology such as septum, fibroids, or polyps. However, not all anatomic defects can be
surgically corrected and not all anomalies require surgical intervention. The most crucial step before
making any treatment decision is accurate imaging in order to determine the exact anomaly.
There are many questions regarding the optimal management of patients with RPL and uterine
anomalies. The following section discusses various questions in light of currently available data.
Should Intrauterine Polyps Be Excised?

Although the association between endometrial polyps and pregnancy loss has not been proven, polyps
are more common in patients with recurrent spontaneous miscarriage [40]. Surgical excision is usually
recommended, since there are data suggesting that hysteroscopic polypectomy can increase fertility
[25,27,38,41]. However, there is little information regarding RPL.
Hysteroscopy resection is the optimal method of performing polypectomy. Hysteroscopic polypectomy
can be performed by excision with forceps or gentle curettage. A study that assessed 240 cases of
hysteroscopic polypectomy concluded that resectoscopic polypectomy required more operating time,
had more glycine absorption and complications, but had a lower recurrence rate than other hysteroscopic
techniques [42]. The resectoscope had a 0% recurrence rate and grasping forceps had a 15% recurrence
rate [42]. The introduction of bipolar electrodes may increase the safety of hysteroscopic endometrial
polypectomy in an outpatient setting [43].
TABLE 12.3
The Role of Surgical Intervention in Women with Uterine Anomalies and Recurrent Pregnancy Loss
Postoperative Technical Likelihood
Study Morbidity Difficulty of Benefit Cost
Hysteroscopic polypectomy + + ++ +
Hysteroscopic adhesiolysis + + - ++ +++ +
Hysteroscopic myomectomy + - ++ ++ - +++ ++ + - ++
Hysteroscopic metroplasty for septate uterus + + ++ + - ++
Hysteroscopic metroplasty for hypoplastic/ + ++ + ++
DES-exposed uterus
Abdominal metroplasty +++ +++ ++ +++
Cervical cerclage ++ ++ + ++
Interruption of a fallopian tube with ++ ++ ++? ++
hydrosalpinx
Low +, High +++.

Does the Resection of a Uterine Septum Improve Pregnancy Outcome?

Septate uterus is more prevalent in women with repeated pregnancy loss [44]. However, it may be difficult
to differentiate between a “normal” arcuate uterus and a septate uterus (Figures 12.6 and 12.7). In order
to justify metroplasty, reliable diagnosis is required.
Although no randomized controlled studies are available, observational studies have reported
impressive results following incision of a septum in patients with recurrent miscarriage [45,53].
Fedele et al. [44] studied the reproductive outcome in 102 patients with a complete (n = 23) or partial
septate uterus (n = 79) and infertility or recurrent miscarriage. Following hysteroscopic metroplasty
the cumulative pregnancy and birth rates at 36 months were 89% and 75%, respectively, in the septate
uterus group and 80% and 67% in the subseptate uterus group. Dalal et al. [45], reported on 72 women
with unexplained primary infertility who underwent hysteroscopic septal resection. Thirty-three women
FIGURE 12.6 Two-dimensional (2D) transvaginal ultrasound of a septated uterus. (Courtesy of Prof. Yaron Zalael MD,
Sheba Medical Center, Tel Hashomer, Israel.)
FIGURE 12.7 Two- and three-dimensional (2D and 3D) transvaginal ultrasound of a septated uterus of the same patient in
Figure 6 (using volume contrast imaging in plane C [VCIC]). (Courtesy of Prof. Yaron Zalael MD, Sheba Medical Center,
Tel Hashomer, Israel.)
(45.8%) conceived within one year of surgery. Only four women (12%) miscarried, and only five (15%)
had preterm delivery. Sugiura-Ogasawara [39] published a comparative cohort study on 109 women with
two or more miscarriages who underwent septotomy (hysteroscopic or by open surgery) and compared
the live birth rates to 15 women who did not undergo surgery. Although the study was underpowered
to show a statistically significant effect, there was a 20% benefit from surgery (81% live births after
surgery compared to 61.5% without surgery) [39]. However, hysteroscopic metroplasty is associated with
a substantial and as yet non-quantified increased risk of uterine rupture during subsequent pregnancies
[46–48]. Uterine perforation and/or the use of electrosurgery increase this risk but are not considered
independent risk factors [47].
Pang et al. [49] have suggested that a septate uterus per se is not an indication for surgical intervention,
because it is not always associated with a poor obstetric outcome. Heinonen [50] retrospectively analyzed
the results of 67 patients with a complete septate uterus including the cervix and a longitudinal vaginal
septum. There was no association with primary infertility, and pregnancy was reported to progress
successfully without surgical treatment. In women with one miscarriage, the situation remains
controversial, and a conservative approach has been suggested since it is expected that after a single
miscarriage 80%−90% of women will have a live birth in the next pregnancy. A recent Cochrane review
[51] concluded that hysteroscopic septum resection in women of reproductive age with a septate uterus
is widely performed to improve reproductive outcomes, in spite of the complete lack of evidence from
randomized controlled trials to support the surgical procedure in these women.
Should the Cervical Portion of the Septum Be Spared in

Patients with a Complete Septate Uterus?
It was previously believed that in patients with a complete septate uterus, the cervical portion of the
septum should be spared and the dissection started the level of the internal os to avoid secondary cervical
incompetence. However, a multicenter, randomized, controlled clinical trial by Parsanezhad et al. [52]
examined whether division of the cervical portion of a uterine septum is associated with intraoperative
bleeding, cervical incompetence, or secondary infertility. Twenty-eight women with complete uterine
septum and a history of pregnancy wastage or infertility were randomized to undergo metroplasty
including division of the cervical portion of the septum, or the same procedure with preservation of the
cervical portion. Resection of the cervical portion was reported to make the procedure safer, easier, and
less complicated than preservation of the cervical septum [52,59]. However, neither cervical incompetence
nor subsequent live birth rates were examined.
Management of Myomas in Recurrent Pregnancy Loss

Although myomas are more prevalent in women with recurrent spontaneous miscarriage [53], the causal
association remains poorly established. It is therefore still undetermined which women will benefit most
from myomectomy. Evidence, mostly from the in vitro fertilization (IVF) literature, suggests that only those
myomas that distort the endometrial cavity impair fertility [24,54]. Patients with distorted uterine cavities
due to submucous fibroids of more than 2 cm have higher pregnancy rates following hysteroscopic resection.
The location and size of the myomas are the two parameters that influence the success of a future
pregnancy [53,54]. It seems that subserosal myomas have little, if any, effect on reproductive outcome,
especially if they are less than 5−7 cm in diameter. The impact of intramural myomas on the outcome of
pregnancy is still disputed [53]. However, intramural myomas that do not encroach upon the endometrium
also can be considered to be relatively harmless to reproduction if they are smaller than 4−5 cm in
diameter. Hysteroscopic myomectomy is the gold standard for the treatment of submucous myomas and
intramural myomas that distort the uterine cavity. The removal of larger fibroids may require a two-stage
procedure in order to avoid intraoperative complications.
Conservative myomectomy is the gold standard for the removal of most intramural and subserosal
uterine myomas in women who desire to preserve their uterus. Pregnancy rates following myomectomy
are in the 50%−60% range, with most having good outcomes [24]. However, spontaneous uterine rupture
has been reported in pregnancy following laparoscopic myomectomy [55].
Laparoscopic-assisted myomectomy (LAM) is another approach that is often a very convenient and
less invasive form of surgery [56]. In carefully selected patients, LAM is a safe and efficient alternative to
both laparoscopic myomectomy and myomectomy by laparotomy. Indications include numerous large or
deep intramural myomas. LAM allows easier repair of the uterus and rapid morcellation of the myomas.
In women who desire a future pregnancy, LAM may be a better approach because it allows meticulous
suturing of the uterine wall in layers and eliminates excessive electrocoagulation [56].
Uterine fibroid embolization is a minimally invasive technique that has been successfully used in
the management of symptomatic myomas [57]. This procedure is not without risk, as after uterine
fibroid embolization, transient ovarian failure has been reported, as has permanent amenorrhea
associated with endometrial atrophy. The pregnancy rate has not been established following uterine
artery embolization. However, higher rates of pregnancy complications have been reported following
uterine artery embolization compared to myomectomy [36]. These complications include preterm
delivery (OR 6.2%, 95% CI 1.4–27.7), malpresentations (OR 4.3%, 95% CI 1.0–20.5), spontaneous
miscarriage, abnormal placentation, and postpartum hemorrhage. A prospective cohort study of 66
women who desired a future pregnancy and were treated with uterine artery embolization has resulted
in an alarming observation [57]. Although uterine artery embolization was effective in improving
bleeding, bulking, and pain symptoms, and in sparing the ovarian reserve, no woman in this study
delivered successfully after uterine artery embolization [57]. The poor reproductive outcomes indicate
that uterine artery embolization should not be performed routinely in young women of childbearing age
with extensive fibroids [57]. There are no trials of any type of myomectomy and subsequent pregnancy
outcome in RPL.
Is Cervical Cerclage Indicated in Women with Uterine Anomalies?

Seidman et al. [58] have studied the effect of cervical cerclage on the survival rate of the fetus in 86
pregnancies in women with congenital uterine anomalies and a random group of 106 pregnancies in
women with normal shaped uteri. Sixty-seven and 29 pregnancies were managed with cervical cerclage in
each group, respectively. The proportion of live births was significantly higher in women with malformed
uteri who underwent cerclage (88%) compared to those without cerclage (47%). No statistically significant
beneficial effect of cerclage was found for normal uteri, even when only patients with RPL were
considered [58]. However, the precise indications for cervical cerclage remain controversial. The Cervical
Incompetence Prevention Randomized Cerclage Trial (CIPRACT) found that therapeutic cerclage with
bed rest reduces preterm delivery before 34 weeks of gestation and compound neonatal morbidity in
women with risk factors and/or symptoms of cervical incompetence and a cervical length of <25 mm
before 27 weeks of gestation [59]. Risk factors for cervical incompetence included DES exposure and
uterine anomaly.
Cervical incompetence is a challenging clinical diagnosis and is an infrequent cause of pregnancy loss
even in patients with gross structural abnormalities of the genital tract. Prophylactic cerclage for patients
with uterine anomalies and DES exposure should be recommended only when other risk factors, such as
three or more midtrimester pregnancy losses or preterm deliveries, are present [56].
Does Strassman Metroplasty Still Have a Role in Patients with a Bicornuate Uterus?
The Strassman procedure surgically unites the two horns of a bicornuate uterus. This procedure often
leaves a small cavity with scarring. The postmetroplasty reproductive capacity of women with a bicornuate
uterus has been reported to be good [60,61]. Furthermore, the role of abdominal metroplasty has been
suggested as a valid approach [61] (using Jones or Strassman techniques) in patients with bicornuate,
T-shaped, or septate uteri, when associated with other pelvic lesions not amenable to transcervical
hysteroscopic surgery. However, surgical correction of a bicornuate uterus is poorly supported by data and
rarely seems warranted for pregnancy maintenance. In a comparative cohort study by Sugiura-Ogasawara
et al. [39], 14 patients with two or more miscarriages were treated by the Strassman operation, and the
subsequent live birth rates compared to 32 women not undergoing surgery. The proportion of live births
was 66.6% (8/12) compared to 78.65% (22/28), respectively.
Does Hydrosalpinx Affect Pregnancy Outcome after Early Recurrent Miscarriage?

Hydrosalpinx is known to have a detrimental effect on the outcome of IVF. A Cochrane systematic review
identified 5 randomized controlled trials involving 646 women [62]. Although no trial reported on live
births, the odds of ongoing pregnancy (OR 2.14%, 95% CI 1.23−3.73) and of clinical pregnancy (OR 2.31%,
95% CI 1.48−3.62) were increased with laparoscopic salpingectomy for hydrosalpinges prior to IVF. In
RPL, a prospective randomized controlled trial [63] enrolled 13 patients with a unilateral hydrosalpinx
diagnosed by sonography or HSG and in whom other causes of miscarriage had been excluded. The patients
were randomized to undergo laparoscopic unilateral tubal fulguration or no surgical intervention. Six of
the seven patients in the treatment group and five of the six in the control group conceived. Five patients in
the treatment group and none in the control group had a pregnancy progress beyond the first trimester. The
progressing pregnancies in the treatment group reached 36–40 weeks’ gestation, a statistically significant
difference. The authors concluded that laparoscopic tubal fulguration improves pregnancy outcome in
selected patients with previous recurrent early miscarriage and a unilateral hydrosalpinx. This study clearly
needs further confirmation in a larger patient sample [63].
Is Hysteroscopic Metroplasty Indicated in the Dysmorphic Uterus?

Management of the dysmorphic uterus is controversial due to subjectivity of the diagnostic methods,
different operative techniques, and the lack of studies comparing surgical treatment with expectant
management [20]. Di Spiezio Sardo et al. [21] showed that hysteroscopic metroplasty is apparently safe
and effective in expanding the uterine cavity volume and normalizing the appearance of the uterine cavity.
Additional retrospective studies have shown that hysteroscopic metroplasty can improve the outcome of
patients with dysmorphic uteri [20,64]. A recent small series reported by Boza et al. [20] found that 8 of 10
patients with RPL conceived either spontaneously or with assisted reproduction following hysteroscopic
metroplasty. In the case of DES exposure, older series used endoscopic metroplasty to expand the
uterine cavity. Metroplasty has shown anatomical correction in 23 of 24 cases [65]. The final result was
considered to be excellent in terms of anatomical correction in 15 patients. DES exposure is no longer
seen today. However, T-shaped uteri are often diagnosed in patients with RPL, and surgical correction
remains controversial [66]. Giacomucci et al. [66] assessed the results of hysteroscopic metroplasty in
patients with a T-shaped uterus and RPL. Before surgery, the overall term delivery rate was 5.5%. After
surgery, the overall term delivery rate was 59% and correlated with the number of previous miscarriages
(P = 0.0008). The authors concluded that the term delivery rate was approximately tenfold higher after
surgery. However, as Giacomucci et al.’s [66] study used a “before and after” model in which the same
women acted as their own controls, it is difficult to draw any conclusions.
At present it seems that hysteroscopic metroplasty, with its simplicity and minimal postoperative
sequelae, seems to be the operation of choice in women with a hypoplastic malformed uterus and a history
of severe infertility and/or RPL [21,22,64]. However, larger series with a better study design are necessary
before hysteroscopic metroplasty can be recommended for all women with DES-exposed, T-shaped or
hypoplastic malformed uterus and recurrent miscarriage.
Conclusions
The prevalence and impact of uterine malformations on reproduction are still not clearly established
despite the wide use of modern imaging modalities [13]. Consequently, the investigation of most women
with recurrent miscarriage could probably be completed by screening with ultrasonography, preferably
utilizing 3D techniques, and in selected cases, hydrosonography (Table 12.2) [26]. More invasive and
expensive imaging modalities, including hysteroscopy, laparoscopy, and MRI, may be required for
inconclusive cases with a suspected uterine deformity, or women with a higher number of miscarriages.
Surgical intervention for uterine malformations remains poorly supported by randomized controlled
trials (Table 12.3). It is generally agreed that adhesions, polyps, and protruding submucous myomas should
be hysteroscopically resected. However, the need for hysteroscopic division of a uterine septum remains
debatable but may be indicated in a patient with two or more pregnancy losses. Abdominal metroplasty
for the bicornuate uteri is even more difficult to support in light of its significant associated morbidity and
lack of controlled data. Abdominal metroplasty is currently recommended only in selected rare cases with
recurrent severe problems in the second and third trimesters. Cervical cerclage is only indicated in women
with uterine anomalies in the presence of a clinical diagnosis of cervical incompetence or additional risk
factors. In women with hydrosalpinges and early recurrent miscarriage, laparoscopic salpingectomy or
proximal tubal occlusion should be considered.
Miscarriages seem to be an inevitable byproduct of human reproduction and are not always correctable.
Thus, surgical intervention should be carefully considered and based on the patient’s clinical history, and
not merely as an attempt to correct all anatomical uterine defects.
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13
The Male Factor in Recurrent Pregnancy Loss
Catherine F. Ingram, Nannan Thirumavalavan, Marc Goldstein, and Dolores J. Lamb
Introduction
Traditional thinking suggested that a male’s role in reproduction ended upon fertilization, after which
maternal factors predominate and carry an embryo until birth. As such, evaluation of the couple with
recurrent pregnancy loss (RPL) has focused on the female, with the American Society for Reproductive
Medicine (ASRM) recommending a thorough history, physical exam, laboratory evaluation (karyotype,
antiphospholipid syndrome testing, thyroid function testing, hemoglobin A1c, and prolactin), and imaging
(hysterosalpingography and ultrasound) [1]. The last few decades have uncovered that male factors
play a significant role in successful live births. As such, couples with “unexplained” RPL in which no
female factors are identified may in fact have a male factor. This is an active area of research, and our
understanding of the male contribution to RPL continues to evolve. In this chapter, we review the current
evidence regarding a male’s contributions to unexplained spontaneous RPL, with a focus on laboratory
evaluation and patient outcomes.
Role of the Paternal Genome in Fertilization and Embryogenesis

Fertilization begins when the male gamete contacts the zona pellucida of the mature oocyte. Thereafter,
the acrosomal reaction occurs in which hydrolytic proteases released from the sperm head break down the
zona for the male gamete to approach the oocyte plasma membrane, or oolemma. Fusion of sperm with
the egg first triggers rapid depolarization of the oolemma to prevent polyspermy. Soon after, increased
intracellular calcium activates the cortical reaction, during which the oocyte releases granules to react
with the zona and make it impenetrable.
After fusion with the oolemma, the entire sperm, head to tail, is absorbed into the egg, after which
the haploid nuclei of both sperm and egg are dubbed pronuclei. Because mature oocytes lack centrioles,
this essential organelle must be donated from the sperm for syngamy, and for the first mitotic division,
to be achieved [2]. Thus, the human zygote inherits its mitotic apparatus paternally, and defects in the
centrosome complex can prevent the embryo from progressing beyond the one-cell stage [3]. While
the spermatic spindle mediates the first mitotic division of the embryo, transcriptional products of
the maternal genome regulate blastomere function for the first cleavages. In fact, utilization of the
embryonic genome, a mixture of both paternal and maternal DNA, does not occur until at least the
four-cell stage [4].
The paternal genome is not necessary for oocyte activation. In an enlightening study conducted in
1991, using a calcium ionophore, Winston et al. were able to activate half of fresh and aged human
oocytes to begin cell division [5]. However, none of those human parthenotes developed beyond the
eight-cell stage, highlighting the importance of the male contribution for embryonic development. Even
when mammalian sperm DNA is damaged beyond repair, fertilization occurs successfully. Ahmadi
et al. showed no significant difference in fertilization rates between hamster spermatic DNA irradiated
with 100 GY of gamma rays and the non-irradiated control group [6]. However, irradiated sperm had
significantly poorer embryogenesis than untreated sperm and had no resulting live fetuses.
126
The Male Factor in Recurrent Pregnancy Loss 127
Although fertilization occurs successfully with a damaged sperm genome, how does the rest of
the pregnancy proceed? Zini et al. examined the relationship between sperm DNA denaturation and
reproductive outcomes after intracytoplasmic sperm injection (ICSI). Participants included infertile men
(n = 60) divided into three groups with varying fractions of DNA denaturation (DD) in their sperm. No
significant difference was found among the three groups with respect to fertilization. However, men with
>30% of sperm with DD produced lower quality embryos [7]. While the study by Zini and colleagues
found no difference in pregnancy rates, this study was conducted using ICSI and only the best-quality
embryos, those without multinucleation, were implanted. The authors argue that if embryos containing
multinucleated blastomeres had randomly been implanted, it is reasonable to hypothesize that pregnancy
rate would have been deleteriously affected. Further research exploring the role of DD on live birth rates,
particularly in men attempting natural conception or conception using intrauterine insemination (IUI)
where embryo quality cannot be assessed, is warranted.
Examination on a molecular level seems to agree with these clinical conclusions. Sperm chromatin
is notable for its three-part structure. Most sperm chromatin is condensed within toroid coils bound
to protamines [8–11], with other regions bound by histones [15] and intervals with nuclear matrix
attachment [13,14]. While the sperm protamine is replaced by histones shortly after fertilization,
some research has suggested that the histone-bound and matrix-associated regions are passed to the
embryonic DNA and are important for development [15–17]. For example, one study found that embryos
fertilized by sperm with disrupted matrices cannot divide past the one-cell stage [18]; another study
showed that retained nucleosomes were significantly enriched with epigenetic modifications at loci
important in developmental regulation [19]. Protamines themselves are crucial for fertility, and the
ratio of sperm protamine RNA has been used to counsel patients regarding chances of success with
assisted reproductive technology (ART) [20,21]. Rogenhofer et al. evaluated protamine mRNA content
from sperm of 25 men with unexplained RPL compared to 32 healthy volunteers (with normal semen
parameters, but not confirmed fertility). Elevated levels of both protamine 1 and 2 mRNA were found in
men with RPL, suggesting that tight regulation of protamine levels may be responsible for appropriate
initiation of paternal gene expression. Thus, while the specific roles of the sperm genome in embryonic
development
Laboratory Evaluation of the Male with Recurrent Pregnancy Loss

Semen Parameters
Semen analysis is considered the staple of workup for the infertile male despite many limitations of this
technique [22–27]. Semen analysis cannot routinely predict fertility or the ability to produce a live birth,
and it is fraught with significant lab-to-lab variability [25,26]. Similarly, standard semen analysis has
shown limited utility in identifying causative factors for men with RPL. In fact, current ASRM guidelines
state that “standard semen parameters, including sperm morphology, do not appear to be predictive or
recurrent pregnancy loss” [1]. Eisenberg et al. studied 340 couples during their pregnancy from the LIFE
study, a prospective study designed to assess couples from pre-conception onwards [27]. The study design
is important as they were able to capture any early pregnancy loss because enrollment was performed
pre-conception. The authors found that 28% of couples who became pregnant suffered a pregnancy loss.
However, there were no differences in any semen parameter between couples who suffered a pregnancy
loss and those who did not, including sperm concentration, total count, semen volume, sperm viability,
motility, or morphology. In addition, no relationships were found in couples experiencing more than one
pregnancy loss. Poor sperm DNA quality, defined as a DNA fragmentation index (DFI) greater than
30%, was the only parameter predictive of pregnancy loss, when adjusted for smoking and alcohol use
[28–30]. Multiple analyses of couples with RPL have demonstrated no difference in standard semen
analysis compared to a control group [30]. However, some studies have found that men with RPL have
decreased motility and morphology [31–34]. It is important to stress that this association is not replicated
across all studies and at the current time there appears to be no role for semen analysis in uncovering an
underlying factor in male factor RPL.
Chromosomal Anomalies
Structural chromosomal abnormalities in both men and women are an identifiable cause for RPL. These
structural abnormalities include reciprocal translocations (24%–50%), Robertsonian translocations (17%–
24%), X-chromosome mosaicisms (4%–12%), and inversions. The ASRM currently recommends that both
partners in a couple experiencing RPL undergo somatic karyotyping [1]. Approximately 50%–70% of
conceptions that result in pregnancy loss have chromosomal abnormalities, which are most commonly
trisomies, followed by monoploidies and polyploidies [35–37]. The frequencies of certain chromosomal
abnormalities are dependent on the gestational age of the fetus [36]. Chromosomal anomalies in pregnancy
loss can arise in two major ways—either random errors in germ cell proliferation or nonrandom
chromosomal anomalies. Errors in germ cell proliferation are more common and are often random. Thus,
these abnormalities are equally likely to occur in couples with and without a history of RPL [36]. The
meiotic errors often result from non-disjunction and lead to aneuploidies [36,38]. Unlike random errors in
germ cell proliferation, non-random chromosomal abnormalities are considered one of the few undoubted
causes of RPL [36]. Specifically, male contribution to RPL has often been credited to karyotype anomalies.
Approximately 2%–4% of couples with a history of RPL have structural parental chromosomal
rearrangements, compared to a much lower (∼0.2%) rate in the general population [36,39,40]. The most
common karyotypes observed in either partner suffering from RPL are reciprocal translocations (24%–
50%), Robertsonian translocations (17%–24%), and X-chromosome mosaicisms (4%–12%), with greater
rates of chromosomal abnormalities seen in females at a 2:1 ratio [41,42]. Less commonly associated
observed parental abnormalities include chromosomal inversions and insertions. The exact type and
location of the cytogenetic abnormality noted could help predict the likelihood of the couple having
a live birth. Surprisingly, transmission of parental cytogenic abnormalities occurs at lower rates than
expected [36]. In a large series of couples with RPL, Carp et al. presented karyotypes from both parents
and the products of conception. Of the 39 fetuses of parents with a chromosomal anomaly, only 12 (30%)
had an abnormal karyotype [38]. This aligns with data from Stephenson et al., in which 36 specimens
from RPL where one parent was a carrier of a structural rearrangement, 33% had euploid embryos [44].
Fortunately, after appropriate counseling and interventions, live birth rates improved. Couples with a
reciprocal translocation had an improved live birth rate from 14%−63%; Robertsonian translocations
from 27%−69%, and inversions 31%−100% [43]. However, it is important to note that concomitant
conditions such as antiphospholipid syndrome were also diagnosed and treated. Given its potential to
improve outcomes, standard evaluation of RPL includes karyotyping in both the male and female partner
followed by thoughtful, individualized genetic counseling [44].
Cytogenetic analysis using karyotyping is limited by the size of the structural change. Karyotyping
often cannot detect structural changes less than 5 megabases (Mb). However, other cytogenetic
techniques, such as comparative genomic hybridization (CGH), are able to detect microdeletions and
microduplications of 500 bp or greater but lack the ability to detect the balanced translocations such as
reciprocal translocations or inversions. Still, this methodology enables for the detection of Y chromosome
microdeletions that have also been associated with male factor RPL. Y chromosome microdeletions
have been observed at increased frequencies (16%–82%) in male partners of RPL compared to controls
with no history of miscarriages [45–47]. Wang et al. compared 507 couples with RPL who had a normal
female endocrine evaluation and a normal semen analysis to 465 “control” couples using G banding of
the Y chromosome [48]. The RPL group had significantly more Y chromosome polymorphisms than the
control group (12% vs. 2.2%, p < 0.05). All Y chromosome polymorphisms were detected at significantly
greater rates in the RPL group than the control group. While some studies have shown no association
between Y chromosome microdeletions and RPL, it has been proposed that the lack of association may
be due to decreased overall fertility rates [44,49]. A meta-analysis by Pereza et al. evaluated nine trials
assessing Y chromosome microdeletions and RPL—two trials had a positive result, while seven had a
negative result [49]. As such, further investigation is required to understand the true role of Y chromosome
microdeletions in RPL. Some reports suggest that molecular tests such as CGH could detect greater rates
of chromosomal abnormalities and perhaps uncover causative factors in patients with unexplained RPL
[50,51]. However, the benefit and cost effectiveness of using these techniques for diagnosis in RPL are
currently a topic of debate [40].
Sperm Aneuploidy
Sperm aneuploidy, defined as an increase or decrease from the normal haploid state, is increased in
couples with RPL. The concept of sperm aneuploidy originated in the 1990s, when Giorlandino et al.
performed sperm fluorescence in situ hybridization (FISH) analysis of chromosomes X, Y, 12, 13, 15, 18,
and 21 in two men with RPL and reported increased rates of nullisomy, specifically in chromosome 15
(12% and 17% vs. normal of 0.5%) [52]. In 2001, Rubio et al. assessed 40 men with RPL for chromosomes
X,Y, 13, 18, 21, X, and Y with sperm FISH and found increased disomy of sex chromosomes in 17.5%
of patients with RPL [53].
In a larger retrospective review, Ramasamy et al. compared semen from 140 couples with RPL (defined
as “recurrent miscarriage or inability to achieve pregnancy via ICSI”) to 140 control samples [54]. Men
with RPL had significantly lower sperm density (36.5 vs. 116.9 million/mL, p < 0.001) and sperm motility
(46.7% vs. 62.2%, p < 0.001). Men with RPL also had greater rates of sex chromosome disomy (1.04%
vs. 0.38%, p = 0.015), chromosome 18 disomy (0.18% vs. 0.03%, p < 0.001) and chromosome 13 and 21
disomy (0.26% vs. 0.08%, p = 0.002). In this study, no relationship between aneuploidy rate and DNA
fragmentation was found. Interestingly, 40% of men with RPL, with normal sperm density and motility
had increased sperm sex chromosome and autosomal aneuploidy, highlighting the importance of FISH
as an adjunct to the standard semen analysis in couples with RPL. Others have found similar results, with
varying rates of aneuploidy depending on the specific probes used [55,56].
More recently, Esquerre-Lamare et al. conducted a prospective study of 33 cases with RPL and
compared them to 27 controls [57]. The authors found a significantly higher BMI in the RPL group (BMI
25 vs. 24, p = 0.025) and a higher likelihood of having a family history of infertility (53% vs. 24%,
p = 0.031). No differences were found in DNA fragmentation index between the two groups, but sperm
from the RPL group displayed increased aneuploidy (1.07% vs. 0.65%, p < 0.001), specifically with
respect to disomy 18 (0.08% vs. 0.04%, p = 0.003) [57].
Sperm FISH assays generally assess chromosomes 13, 18, 21, X, and Y, as aneuploidies in these
chromosomes are compatible with life. The step-by-step details performing FISH are beyond the scope
of this chapter. Though quantitative and qualitative interpretation of results are possible, the qualitative
method allows easier identification of patients who are at risk for aneuploidy. Abnormal sperm FISH results
usually fall in one of two categories—aneuploidy could be increased globally in all tested chromosomes
or restricted to a single chromosome [58]. Aneuploidy in multiple chromosomes likely represents a defect
in meiotic division. Unfortunately, FISH is a terminal assay and it is not possible to choose euploid
sperm identified by this method for ART [59]. In addition, there exists no consensus level of aneuploidy
that makes a live birth impossible or that necessitates ICSI. Despite this limitation, performing FISH to
assess sperm aneuploidy in men with RPL can provide significant prognostic information. For example,
sperm FISH can help quantify the likelihood of transmitting aneuploidies and other chromosomal
rearrangements to the offspring. Though no intervention currently can reduce sperm aneuploidy, with
appropriate genetic counseling the couple may elect to pursue preimplantation genetic screening (PGS)
to select and transfer only euploid embryos during in vitro fertilization (IVF)/intracytoplasmic sperm
injection (ICSI) [31,44,58]. If patients happen to conceive naturally, more stringent prenatal testing to
ensure the fetus in euploid can be offered [58]. Other options, depending on the couple’s preferences, could
include use of donor sperm or even avoiding ART and pursing adoption.
Sperm DNA Quality

DNA damage is another sperm characteristic associated with RPL. Unlike in diploid cells, DNA in haploid
sperm cells requires compact packaging to facilitate sperm head hydrodynamics and to shield DNA from
damage. As mentioned previously, DNA in sperm is tightly bound to dense proteins called protamine [60].
Some regions are not as tightly bound and are more exposed, leaving them more vulnerable to DNA damage.
Many factors could contribute to DNA damage either during spermatogenesis or during transport of sperm.
These include apoptosis during spermatogenesis, DNA strand breaks during spermiogenesis, damage by
endogenous caspases and endonucleases, oxidative stress and sheering forces during transit, a failure to
repair DNA mutations, or environmental toxins such as smoking, radiotherapy, or chemotherapy [61,62].
In 2003, Carrell et al. compared semen parameters and DNA fragmentation among 21 men with
RPL, 42 men from the general population, and 26 fertile donors [63,64]. The authors reported decreased
percentage of normal morphology in the RPL group compared to both the general population and fertile
donors, as well as decreased sperm viability compared to fertile donors. Using terminal deoxynucleotidyl
transferase–mediated dUTP nick-end labeling (TUNEL), which assesses DNA fragmentation, Carrel
and colleagues identified that sperm from men with RPL had significantly greater rates of DNA damage.
Absalan et al. evaluated 30 couples with RPL (defined as three or more spontaneous miscarriages at less
than 20 weeks of gestation) and compared them to 30 fertile couples from Iran [32]. The RPL group had
significantly lower sperm motility (64.23% vs. 56.31%, p < 0.05) and sperm morphology (26.73% vs.
51.56%, p < 0.05).
Zidi-Jrah et al. found increased sperm DNA fragmentation (17.1% vs. 10.2%, p = 0.016) when comparing
sperm from 22 men with RPL to 20 fertile men [64]. Men with RPL also had a greater percentage of
sperm with abnormal nuclear chromatin decondensation (23.6% vs .11.8%, p < 0.001). Most recently, a
meta-analysis by McQueen et al. compared 517 men with RPL to 384 fertile men. Men with RPL had
significantly greater rates of sperm DNA fragmentation (mean difference: 10.7%, CI 5.82–15.58) [65].
Despite the heterogeneity of the 15 prospective trials included, subgroup analyses revealed that this
relationship persisted when analyzing either two or three pregnancy losses as inclusion criteria, or when
analyzed by the type of assay used. In addition to decreased DNA quality, increased semen reactive
oxygen species has been identified in semen from men with RPL [66,67]. Two very recent reports also
indicate that high DNA fragmentation can be a useful predictor of RPL [28,29].
While emerging evidence over the past two decades demonstrates that impaired sperm DNA quality
contributes to RPL, assessing and acting on abnormal sperm DNA quality remains a challenge. Primarily,
there are multiple assays used to assess sperm DNA quality, including sperm chromatin condensation
assay (SCCA), TUNEL assay, sperm chromatin dispersion assay (SCD), and the comet assay, each with
their strengths and weaknesses. Because of the various assays used, no agreed-upon cutoffs exist for
what are considered “abnormal” levels of DNA damage, and variability leads to difficulty in appraising
the literature. The expertise needed to perform these tests may also limit their availability to specialized
centers, prohibiting some patients and providers from utilizing these techniques. Nonetheless, measuring
sperm DNA damage has become a useful tool to help counsel couples with RPL, as some interventions,
discussed below, may improve sperm DNA quality.
Interventions traditionally used in infertile men have been investigated for couples with RPL. Ghanaie
et al. performed a prospective study to evaluate varicocele repair for couples with RPL [68]. Including
only men with normal semen parameters, the authors randomized a group of 136 couples with RPL evenly
into a control group and an intervention group that underwent varicocele repair. The intervention group
had a higher pregnancy rate (44.1% vs. 19.1%, p = 0.003), a higher live birth rate per pregnancy rate
(86.7% vs. 30.8%, p = 0.002), and a lower miscarriage rate (13.3% vs. 69.2%, p = 0.003). The mechanism
by which varicocele repair improved outcomes is likely related to improved sperm DNA quality, as
multiple studies have demonstrated that varicocele repair improves sperm DNA and decreases reactive
oxygen species [69–73]. Baccetti et al. found that after varicocele repair, men have an improvement in
sperm morphology as assessed by electron microscopy, and improvement in sperm FISH findings [74].
However, varicocele repair in the setting of male-factor RPL has not been thoroughly investigated and
further work is needed.
Conclusions
Though significant strides in our understanding of the male contribution to RPL have been made during
the past two decades, we remain unable to confidently identify contributing factors in men with RPL.
Many avenues of evaluation are ripe for further investigation and hold great potential for improving
patient outcomes in the future. Several areas currently being investigated include gene-level mutations,
micro-RNA abnormalities, and sperm epigenomics. For example, certain polymorphisms in the gene
for MTHFR, an enzyme involved in methionine synthesis, may be associated with RPL in women and
men [75]. In another example, Asadpor et al. found men with RPL had an increased mutation rate in an
X-linked gene, ubiquitin-specific protease (USP26), an enzyme involved in removal of histones, germ
cell apoptosis, mitotic proliferation, and more [76]. Regarding epigenetics, altered DNA methylation
and altered histone retention in sperm has also been associated with RPL, and this is an active area of
investigation [21,75,77,78] The role of micro-RNAs in RPL have been studied in women and are now
actively being investigated in men as well [79].
The interplay between environment and the inherent genome also continues to be researched. It is
hoped that with further investigation we will be able to not only improve our counseling of couples who
suffer from RPL, but also improve their outcomes by helping them achieve parenthood.
Funding Support
This work is supported in part by NIH grants K12 DK0083014, the Multidisciplinary K12 Urologic
Research (KURe) Career Development Program awarded to DJL (NT is a K12 Scholar) from the National
Institute of Kidney and Digestive Diseases to Dolores J. Lamb. DJL is also supported in part by the
Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust. The content is solely
the responsibility of the authors and does not necessarily represent the official views of the National
Institutes of Health.
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14
Ultrasound Follow-Up in Early Pregnancy
Akhila Vasudeva and Pratap Kumar
Introduction
The early pregnancy scan is an essential part of contemporary routine antenatal care. In patients with
recurrent pregnancy loss (RPL), a normal early pregnancy scan can be highly reassuring. At the same
time, abnormal sonological findings may herald a nonviable pregnancy, detect chromosomal or structural
malformations which are more common among these women, or forecast higher risk of poor pregnancy
outcome. The most commonly used transducers are linear array or sector transducer (3–5 MHz for
abdominal examination), and the transvaginal probe (5–10 MHz). In first trimester ultrasound, transvaginal
sonography (TVS) is necessary up to approximately 10 weeks, and thereafter a transabdominal probe
is mostly used. However, a transvaginal probe is complementary to abdominal ultrasound in order to
complete the anatomical evaluation.
With modern ultrasound machines, there is only a negligible rise in tissue temperature, usually less
than 1°C. It is unlikely that there is any deleterious effect of ultrasound in the first trimester during
embryogenesis with routine gray-scale ultrasound [1]. Although the potential for embryonic effects from
Doppler imaging exists, there is little evidence that it is teratogenic as long as pulses are applied at low
level with minimal usage of the Doppler.
Normal Sonological Findings in the First Trimester

Knowledge of the “normal” development of the embryo and fetus is essential when scanning in the first
trimester. The primary goal of ultrasound examination in the first trimester is to determine whether
the pregnancy is intrauterine, embryonic/fetal number, viability by ruling out missed abortion/molar
pregnancy, gestational age assessment by gestational sac (GS) size or crown-rump length (CRL),
aneuploidy screening, and to rule out structural abnormalities. It is also important to rule out uterine
anomalies, evaluate fibroids (if any), and to rule out adnexal pathology. In cases of multiple pregnancy,
assessment of chorionicity is of paramount importance.
4 to 5 Weeks
The GS can first be imaged sonographically at about 4.4–4.6 weeks from the last menstrual period (LMP),
when the sac is 2–4 mm in size. The intradecidual sign and the double decidual sac sign are specific for
intrauterine pregnancy and rule out the possibility of ectopic pregnancy [1]. The serum β hCG (human
chorionic gonadotrophin) level, at which an intrauterine GS should be seen with modern high-resolution
vaginal probe, is called the discriminatory zone, usually between 1000–2000 IU/L. When β hCG is
above the discriminatory zone, absence of an intrauterine sac significantly raises the possibility of ectopic
pregnancy. When hCG is below this level, one cannot be certain and the incremental rise of β hCG
indicates the location/viability of pregnancy. In recurrent biochemical pregnancy losses, ultrasound is not
very useful as there is no sonological evidence of pregnancy in the presence of very low β hCG.
The yolk sac is a circular structure located between the chorion and the amnion, and is first visualized
at the fifth postmenstrual week. The size of the embryo ranges from 2–3 mm in size and appears as a
134
Ultrasound Follow-Up in Early Pregnancy 135
linear structure attached to the yolk sac and close to the uterine wall. Although embryonic cardiac activity
can be visualized at this time, rates of less than 100 beats per minute (bpm) are not predictive of a poor
outcome, and follow-up scanning is imperative [2].
Week 6
Ultrasonographically, the embryo appears as an undifferentiated structure at this time, except for the
heartbeat. An average heart rate of 130 bpm can be seen using M-mode scanning. If the embryo is less
than 4 mm, the absence of cardiac activity is nondiagnostic. Once a fetal heartbeat is visualized, the risk
of miscarriage decreases, as most miscarriages are blighted ova. Toward the end of the sixth week, the
embryo is seen separately from the yolk sac. After fetal cardiac activity, the next anatomical structure to
become visible is the primitive neural tube. Sonographically, this appears as a hypoechoic longitudinal
structure running the length of the embryo, visible in the form of two parallel lines [2].
Weeks 7 to 9
The head and trunk can be visualized separately. Within the head, an intracranial cystic structure is
visualized corresponding to the fourth ventricle (rhombencephalon) [2]. The cerebral hemispheres can
be visualized in some embryos at this gestation. The initial sign of normal herniation of the gut can be
seen as an echogenic area at the abdominal insertion of the cord.
Week 8
The choroid plexus becomes visible and grows correspondingly with the cerebral hemispheres, developing
into a crescent shape traversing the roof of the fourth ventricle (Figure 14.1). The third ventricle (diencephalon)
is wide. The stomach can first be visualized at this gestation as a small hypoechogenic area on the left side
of the upper abdomen and should be seen in all embryos by 11 weeks [2]. It is possible to identify the atrial
and ventricular walls of the heart moving reciprocally at the end of week 8 [2], with the atrial component
appearing larger than the ventricular component. Clear identification between the thoracic and abdominal
contents is possible by the ninth week. The cerebral hemispheres should be visualized in all embryos by
week 9. At 9 weeks, the size of the lateral ventricles increases rapidly and the third ventricle narrows. The
spine is still characterized by two echogenic parallel lines. Normal midgut herniation can be seen as a large
hyperechogenic mass. The long bones, hands, and feet can be first imaged at this time.
FIGURE 14.1 An 8-week TVS image showing a developing embryo and the yolk sac.
The Early Anomaly Scan at 10 to 14 Weeks

This scan needs a systematic approach to view the fetal anatomy, similar to that of a second trimester
targeted scan. The aim is to obtain a transverse section of the head to demonstrate the ossified cranial
bones, a midline echo, and the choroid plexuses should be seen in the ventricles (Figure 14.2); a mid-
sagittal view of the face should be obtained to demonstrate the nasal bone, orbits, and a normal profile;
sagittal section of the spine should be determined to view the presence of intact skin over the back, and
transverse and longitudinal planes of the spine from neck to sacrum; a transverse section of the thorax
should be sought to demonstrate the four-chamber view of the heart with a normal axis, and also a three-
vessel view (Figures 14.3 and 14.4); transverse and sagittal sections of the trunk and extremities should
be obtained to demonstrate the stomach in the left upper quadrant, kidneys (Figure 14.5), bladder (Figure
14.6), abdominal insertion of the umbilical cord, and all the long bones, hands (Figure 14.7), and feet.
FIGURE 14.2 Developing choroid plexus in the 12-week fetus showing a typical “butterfly sign.”
FIGURE 14.3 A four-chamber view demonstrated in a 12-week fetus.

FIGURE 14.4 A three-vessel view demonstrated in a 12-week fetus.
FIGURE 14.5 Kidneys visualized in an 11-week fetus.
FIGURE 14.6 Bladder seen in an 11-week fetus.

FIGURE 14.7 Open hand with five digits seen in an 11-week fetus.
Diagnosis of Miscarriage/Nonviable Pregnancy in Early Scans

The criteria for diagnosing a nonviable pregnancy or miscarriage by ultrasound has been under constant
debate [3]. The Royal College of Obstetricians and Gynaecologists (RCOG) in the UK has reviewed its
guidance [4] to doctors and revised the ultrasound criteria used to define miscarriage to the following:
(i) a mean GS diameter of 25 mm (with no obvious yolk sac), or with a fetal pole with CRL 7 mm (the
latter without evidence of fetal heart activity); (ii) TVS should be performed in all cases for diagnosing
non viability; (iii) where there is any doubt about diagnosis and/or a woman requests a repeat scan, this
should be performed at an interval of at least 1 week from the initial scan before medical or surgical
measures are undertaken for uterine evacuation. Also, it is a good practice that second observer confirms
the findings. No growth in GS size or CRL is strongly suggestive of a nonviable pregnancy in the absence
of embryonic structures on a repeat scan.
Preisler et al. performed a prospective multicenter observational trial attempting to validate these
guidelines on diagnosis of nonviable pregnancy [5]. They also aimed to examine the influence of
gestational age on interpretation of mean GS diameter and CRL values and determine the optimal
intervals between scans and findings on repeat scans that definitively diagnose pregnancy failure. This
study confirmed the usefulness of the above-mentioned diagnostic criteria. In addition, gestational age
at initial TVS was found to be an important factor. They found that beyond 70 days from LMP, mean GS
diameter ≥18 mm for GS without an embryo and embryo with CRL ≥3 mm without visible heart activity
were both indicative of early pregnancy failure with a fair degree of accuracy. After a gap of 7 or more
days, no cardiac activity in the embryo in both scans, or poor growth of GS (not doubling in 14 days when
GS is <12 mm), and pregnancies without an embryo and mean GS diameter ≥12 mm showing no embryo
heartbeat after 7 or more days all indicated early pregnancy failure.
The routine use of TVS has led to improvements in the management of early pregnancy loss [3,6,7].
Once a certain diagnosis of miscarriage has been made, a proportion of women (up to 70%) will elect
for expectant management [6]. Other women will choose medical or surgical management to deal with
the miscarriage. However, expectant or medical management precludes genetic testing of the embryo in
recurrent miscarriage. Whichever method is chosen, the diagnosis of a complete miscarriage is generally
accepted as an endometrial thickness <15 mm with no evidence of retained products of conception. TVS
is a sensitive tool for detecting residual trophoblastic tissue. Blood flow in the intervillous space in cases of
first trimester miscarriage using color Doppler imaging predicts higher success of expectant management.
The success of expectant management varies between 80%–96% within 2 weeks in women with
incomplete miscarriage, 59%–62% in missed miscarriages, and 52% in “anembryonic pregnancies”[6].
It is generally accepted that evacuation of the retained products of conception should be offered after
2 weeks. Expectant management of miscarriage, using ultrasound parameters to determine eligibility,
could significantly reduce the number of surgical evacuation procedures unless accurate genetic testing
is required. In the absence of a previous ultrasound scan documenting the presence of an intrauterine
pregnancy, women with ultrasound features suggestive of a complete miscarriage should be managed as
having a pregnancy of unknown location and have serum β hCG levels taken to check resolution of the
pregnancy. This is needed so as not to miss a diagnosis of ectopic pregnancy [3].
Threatened Miscarriage, Subchorionic Hematoma, and Its Significance

Vaginal bleeding in very early pregnancy does not seem to be associated with any immediate or long-term
consequences. Conversely, vaginal bleeding at 7–12 weeks, even in the presence of detectable fetal cardiac
activity, is not only associated with a 5%–10% miscarriage rate before 14 weeks of gestation but also with
adverse pregnancy outcome [6]. The incidence of intrauterine hematoma in the first trimester in a general
obstetric population is approximately 3.1%. The presence of a retroplacental hematoma (especially below
the cord insertion) is significantly correlated with an increased risk for adverse pregnancy outcomes,
such as miscarriage, pregnancy-induced hypertension, placental abruption, preterm delivery, fetal growth
restriction, fetal distress, meconium-stained amniotic fluid, operative delivery, neonatal intensive care
unit admission, and also fetal demise/perinatal mortality [8]. The presence of a hematoma may be
associated with a chronic inflammatory reaction in the decidua, resulting in persistent myometrial activity
and expulsion of the pregnancy. The development of a hematoma may be the first sign of incomplete
placentation and be associated with acute oxidative stress, which may impair subsequent placental and
membrane development.
Predicting the Risk of Early Pregnancy Failure

Based on Ultrasonographic Parameters
Gestational Sac
Once a GS has been documented on ultrasound, subsequent loss of viability in the embryonic period
remains around 11%. A smaller than expected GS and a slower rate of growth (<1 mm/day) can predict
poor pregnancy outcome, even in the presence of embryonic cardiac activity [6]. Small gestation sac size
(before 9 weeks) has been associated with chromosomal abnormalities such as triploidy and trisomy 16.
At the same time, a large, empty, slowly growing, and irregular GS lying low in the endometrial cavity
(Figure 14.8) is suggestive of pregnancy failure [9].
Crown-Rump Length
If an embryo has developed up to 5 mm in length, subsequent loss of viability occurs in 7.2% of cases.
Loss rates drop to 3.3% for embryos of 6–10 mm and to 0.5% for embryos over 10 mm. A smaller than
expected CRL has been associated with subsequent miscarriage, aneuploidy, fetal demise, and poor
pregnancy outcome, including fetal growth restriction [6,10,11].
Yolk Sac
The predictive value of secondary yolk sac (SYS) measurements in determining the outcome of an early
pregnancy is limited. Most pregnancies that miscarry during the third month of pregnancy have normal
SYS measurements at their initial scan before 8 weeks of gestation. The yolk sac is found to persist
inside the GS after embryonic demise. Thus, variations in SYS size and sonographic appearance in most
abnormal pregnancies are probably the consequence of poor embryonic development or embryonic death
rather than being the primary cause of early pregnancy failure [6]. However, observing the yolk sac is
FIGURE 14.8 Gestational sac irregular in shape, with poor choriodecidual reaction, lying relatively low in the uterine cavity.
important because if the yolk sac is large (>5.6 mm), or not visible when the mean GS diameter reaches
over 13 mm, a follow-up TVS in a week is needed, as these findings are strongly associated with early
pregnancy failure [9].
Fetal Heart Activity

Fetal heart activity is the earliest proof of a viable pregnancy and it has been documented in utero by
TVS as early as 36 days’ menstrual age. From 5−9 weeks of gestation there is a rapid increase in the
mean heart rate from 110−175 bpm. The heart rate then gradually decreases to around 160–170 bpm.
An abnormal developmental pattern of fetal heart rate (FHR) and/or bradycardia has been associated
with subsequent miscarriage. In particular, a slow FHR at 6–8 weeks appears to be associated with
subsequent fetal demise. A single observation of an abnormally slow heart rate does not necessarily
indicate subsequent embryonic death, but a continuous decline of embryonic heart activity is inevitably
associated with miscarriage [6].
Other Sonographic Markers

Abnormal shape of the GS and increased echogenicity/thickness of the placenta have been proposed as
sonographic markers associated with early spontaneous miscarriage [6].
Prediction Models
Stamatopoulos et al. attempted to develop and test a prediction model to assess the risk of subsequent
pregnancy failure among women who are diagnosed to have a viable intrauterine pregnancy using an early
pregnancy scan [12]. They found that the possibility of subsequent pregnancy failure is reduced among
those who had a higher embryonic heart rate in the presence of larger GS-to-CRL ratio.
Aneuploidy Screening in 11–14 Weeks Scan

In this era of noninvasive prenatal screening (NIPS) for aneuploidy, the role of first trimester combined
screening test for aneuploidy screening has been debated. NIPS, being the ideal screening test for
aneuploidy with highest accuracy, has its own limitations considering the cost and the applicability to
general population. In addition, NIPS cannot replace the late first trimester scan, considering that fetal
structural anomalies are much more common than aneuploidy among the majority of young women, of
which almost half could be detected by a trained operator. Also, increased nuchal translucency (NT)
or presence of structural abnormalities may call for a more detailed genetic testing rather than only
aneuploidy [13]. Thus, NIPS can be usefully incorporated into the screening program as a second-line
strategy after high risk/intermediate results in the combined screening test. Thus, the most effective
screening test for Down syndrome (and other aneuploidies) remains to be the combined screening test
performed between 11–14 weeks of gestation [14], with the detection rates as high as 80%–90%. This
test involves the measurement of nuchal translucency and maternal serum estimation of free β hCG and
PAPP-A. NT has now evolved as the single most accurate ultrasonographic screening for Down syndrome.
When the screening results are intermediate, there are other sonologic markers used to refine the risk of
Down syndrome, for example nasal bone, ductus venosus Doppler blood flow, tricuspid regurgitation,
etc. Wide application of this 11- to 14-week ultrasound for aneuploidy screening has improved our
understanding of fetal anatomy and physiology.
A recent prospective multicenter study performed in the Netherlands confirms that fetal chromosomal
abnormities can be detected with a high degree of accuracy following a combined screening test
performed at late first trimester [13]. USGs were performed by operators trained and certified for NT
scans. Of the total 34 chromosomal anomalies in the study population, 33 (97%) were detected by first
trimester screening, either because of increased NT or because of high risk combined screening results.
Detection of Structural Abnormalities in the First Trimester Scan

In women with RPL, structural anomalies and aneuploidies are more common than in the general
population. Many anomalies can be detected in the early scan, although not all are associated with RPL.
Some anomalies are incompatible with intrauterine life. If fetal demise occurs in the first trimester, the
patient will present with missed abortion. If demise occurs later, there may be midtrimester fetal death.
Other anomalies are compatible with intrauterine life but not extra-uterine life, presenting as stillbirth if
there is no intervention prior to birth (e.g., anencephalus or renal agenesis). Others are compatible with life
but are associated with severe disability (e.g., open meningomyelocele). In such circumstances, the patient
may elect to terminate the pregnancy. Therefore early detection should be the aim. The majority (80%)
of common fetal malformations develop before 12 weeks’ gestation. Advances in ultrasound technology
and the improvement of high-resolution transvaginal equipment have enabled detailed anatomical
investigation of the fetus earlier than the classical mid-pregnancy scan [15]. However, the detection
rate varies widely between studies, ranging from 26%–70% [15–19]. There are several limitations to
the detection of malformations in first trimester scanning. The resolution of ultrasound equipment is
around 1 mm. Consequently, the small size of fetal anatomical features is still a pivotal limiting factor
before 12 weeks. Furthermore, many fetal anomalies develop at the end of organogenesis over a variable
period of time, and many anomalies may not be apparent before the end of the first trimester, such as
agenesis of the corpus callosum. Some anomalies have sonographic features that are different from those
usually seen during the routine midtrimester anomaly scan (i.e., anencephalus). By contrast, normal fetal
developmental features, such as midgut herniation, have the same features as pathological exomphalos,
hence confirmation of the exact gestational age is crucial for early diagnosis.
Some malformations will almost always be detected, such as anencephaly, and some will never be
detected, such as microcephaly. There are also abnormalities that are potentially detectable depending
on a number of factors—the objectives set for such a scan and consequently the time allocated for the
fetal examination, the expertise of the sonographer, and the quality of the equipment used; second,
the presence of an easily detectable marker for an underlying abnormality, and third, the evolution in the
phenotypic expression of the abnormality with gestation.
It is outside of the scope of this chapter to summarize all the anomalies that can be diagnosed, and
for a full review, the reader is directed elsewhere. However, multiple organ scanning is possible. First
trimester scanning can detect defects of the cranium (i.e., anencephaly [Figure 14.9]) and brain (i.e.,
holoprosencephaly [20]), spine [21,22], face and palate, heart [23,24], congenital diaphragmatic hernia,
abdominal wall defects (Figure 14.10), bladder, kidneys, choledochal, hepatic, and omental cysts,
anorectal malformations, bowel atresia, skeletal dysplasias [25], kyphoscoliosis, cystic hygroma, and fetal
FIGURE 14.9 The “Mickey Mouse sign” in an anencephalic fetus at week 13.
hydrops (Figure 14.11). Protocols have been published detailing how early anatomical survey should be
done, indicating views that should be obtained, structures that should be investigated, and measurements
that should be taken in order to exclude or detect all anomalies that should be seen at an early scan [13].
A recent multicenter prospective observational study summarized the accuracy of the late first trimester
scan (12–13 weeks) in detection of structural anomalies, in comparison to a second trimester targeted scan
[13]. This study reiterates the important role of late first trimester scan in detection of fetal abnormalities
in the era of NIPS for aneuploidy. In this study, all sonographers were certified for NT measurements. In
addition, they were given training in first trimester detection of anomalies. Overall, 23/51 (45%) structural
anomalies were detected at the early scan. Detection rate was 100% for all particularly severe and lethal
anomalies; however, 33.3% of cardiac defects could be detected at first trimester. After detection of fetal
anomaly in first trimester, 83% parents opted for termination of pregnancy.
When an anomaly is discovered, it is often difficult for a patient to decide on which course of action to
take. In the case of RPL, the problem is compounded, as the pregnancy with anomalies may be the first
pregnancy to have survived until the early scan. It may also be the last pregnancy to survive.
FIGURE 14.10 Liver and stomach herniating outside the abdominal wall, gastroschisis.
FIGURE 14.11 A hydropic fetus at 9 weeks, also showing abnormal morphology for gestation.
Advances in Genetics
In the majority of cases with ultrasound abnormalities, the fetal karyotype is normal when banding
techniques are used. However, advances in genetic testing have introduced high-resolution testing which has
enabled additional genetic anomalies to be diagnosed to explain the anomalous ultrasound findings. Newer
molecular genetic techniques such as single-nucleotide polymorphism (SNP), next-generation sequencing
(NGS), and microarray and array comparative genomic hybridization (CGH). Array CGH can be applied
to detect copy number variations (CNVs) down to a resolution as low as 1 Kb [26]. By applying array CGH
in prenatal diagnosis in conjunction with chromosomal analysis, approximately 3.6% additional clinically
significant genomic imbalances can be detected when the karyotype is normal, regardless of the indication of
the referral [27–30]. This detection rate increases to 5.2% when the pregnancy has a structural malformation
on ultrasound. Array CGH is a useful tool for the detection of submicroscopic CNVs and for identifying
candidate genes for euploid miscarriages [31]. Array CGH can be performed on the uncultured cells. Thus
results are quicker, and it also overcomes the problem of culture failure, maternal contamination, and poor
chromosome morphology associated with conventional karyotyping. The American College of Obstetrics
and Gynecology and Society for Maternal and Fetal Medicine, in their recent committee opinion (Number
581, December 2013), have recommended array CGH as a preferred technique of prenatal diagnosis when
there are fetal structural anomalies on the ultrasound. Specifically, CGH is preferred in cases of fetal demise/
stillbirth, as it is more likely to yield results with improved detection of causative abnormalities. However,
committee opinion does not recommend CGH on first/second trimester pregnancy losses as of now, since
limited data are currently available on the clinical utility in this setting.
Clinically relevant CNVs may be identified explaining the cause of euploid miscarriages. In addition,
maternal cell contamination (MCC) can be identified, and parental origin of chromosomal aberration can
be traced. These tests can also be applied on archival tissue stored from prior miscarriage. Comparing three
methods of genetic evaluation of products of conception—conventional cytogenetics, SNP microarray,
and array cGH, the performance characteristics and interrater agreement of these techniques are similar,
and each platform has its respective advantages and disadvantages.
Conclusions
This chapter summarizes the role of first trimester sonography in the diagnosis and prognosis of pregnancy.
Visualization of normal fetal anatomy in the first trimester, along with a low risk of aneuploidy screening,
affords patients reassurance and reduction in anxiety. Earlier detection of lethal or severe fetal structural
abnormalities allows for earlier decision making for pregnancy termination or earlier referral to a tertiary
center and coordination of care among the appropriate specialists.
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15
Threatened Miscarriage and
Howard J.A. Carp
Introduction
Threatened miscarriage is defined by the National Library of Medicine, Medical Subject Headings (2012
MeSH), as bleeding during the first 20 weeks of pregnancy while the cervix is closed. It is the most
common complication in pregnancy, occurring in 20% of all pregnancies. The condition may progress to
miscarriage in approximately one half of cases [1,2], or may resolve. There are problems of definition, as
the bleeding may include anything from spots of blood to potentially fatal shock. Bleeding is particularly
worrying in recurrent pregnancy loss (RPL) where the patient assumes that another miscarriage
is imminent. In RPL, vaginal bleeding is a common complication occurring in 50 of 162 women in
Reginald’s series [3] and 50 of 102 patients in the author’s series [4] of women with RPL. The reason for
this bleeding remains unclear. Of recurrent miscarriages, 75% are blighted ova [4]. However, when the
pregnancy succeeds and there is a live embryo within the uterus, bleeding still occurs in 40%–50% of
patients. The treating physician is faced with the question of whether any treatment can effectively prevent
the pregnancy from being miscarried. In some cases of RPL, the patient may be under some form of
treatment to prevent another miscarriage, and the question arises as to whether supplemental treatment is
indicated. Many factors can affect the decision to intervene: the natural history, presence of a heartbeat on
ultrasound, whether the heartbeat is bradycardic, the size of the embryo, low βhCG levels and insufficient
rise in serial βhCG levels, possibly low progesterone levels, and high CA-125 levels. All the above factors,
which are used to determine the need for intervention, also attempt to determine viability, as treatment
can only affect a live embryo or an embryo at a stage prior to 5.5 weeks, (usually the earliest that a fetal
heart can be detected). However, a thorough search of the literature failed to find any reports of therapy to
prevent threatened miscarriage developing to miscarriage in women with previous recurrent miscarriage,
except the recent PRISM trial [5].
Initial assessment should include a speculum examination to exclude bleeding from the cervix or
vagina. Physical examination is also required to exclude extragenital causes of bleeding, and ectopic
pregnancy.
Natural History
If threatened miscarriage is assumed to be a homogeneous condition, miscarriage may ensue in
approximately 50% of cases [1,2], or may resolve. However, in the older literature, there was no ultrasound
performed to detect the fetal heartbeat. In many cases, bleeding may have occurred after fetal death.
After detection of a fetal heartbeat, the prognosis is good. A number of observational studies have quoted
the subsequent miscarriage rate to be 3%–4% [6] to 15.4% [7] with a mean of 8.7%. Weiss et al. [8]
enrolled patients into a database on presenting with a viable embryo at 10–14 weeks. If the patient reached
10–14 weeks, the chance of miscarrying prior to 24 weeks was 1%–2%. In addition, the likelihood of a
pregnancy loss after the detection of a fetal heartbeat was 69/359 (14.2%) in Li et al.’s series [9] and 22.7%
of 185 study patients with multiple spontaneous abortions in Laufer et al.’s [10] series.
145
Prognostic Factors
Ultrasound
A number of factors can help determine the prognosis in threatened miscarriage. However, ultrasound is
the most useful. Ultrasound can first differentiate between an intrauterine pregnancy, a molar pregnancy,
or ectopic pregnancy. An intrauterine sac is visible by 5.5 weeks. At 7 weeks a heartbeat should be
detected. An empty sac with a diameter of at least 15 mm at 7 weeks and 21 mm at 8 weeks has a
diagnostic accuracy of 90.8% in predicting miscarriage [11]. Fetal heart activity should be visible with
a vaginal probe when the crown-rump length is 4 mm. Fetal bradycardia and discrepancy between
gestational age and crown-to-rump length are adverse prognostic factors [12,13].
Progesterone Levels
Serum progesterone levels are often used to make prognoses about the continued development of
pregnancy. The lowest progesterone level to be associated with a viable pregnancy was 5.1 ng/mL
in the series by Stovall et al. [14]. A single progesterone level ≥25 ng/mL was associated with a 97%
likelihood of viable pregnancy. Al-Sebai et al. [15] summarized 358 threatened miscarriages <18 weeks,
a single progesterone level ≤45 nmol/L (14 ng/mL) was reported to differentiate between miscarrying
and ongoing pregnancies (sensitivity 87.6%, specificity 87.5%). Serum progesterone levels of less than
≥12 ng/mL were associated with an increased risk of miscarriage in Arck et al.’s [16] series, and
<35 nmol/L in Lek at al.’s [17] series.
However, there are pitfalls to using serum progesterone levels as a predictive marker of miscarriage or
for determining the need for progesterone supplementation. Progesterone secretion is pulsatile. Blood may
be drawn at a pulse peak or nadir. Hormone levels may be normal but histology abnormal due to deficiency
of progesterone receptors. As with other presumptive causes of miscarriage, low hormone levels may be
a result of nonviability. In the blighted ovum or after embryonic death, there is no villous circulation.
Trophoblastic failure after villous circulatory failure results in low human chorionic gonadotrophin
(hCG) levels. If hCG does not stimulate the corpus luteum, progesterone levels will fall, explaining the
mechanism of expulsion but not necessarily that of embryonic death or the cause of miscarriage.
Human Chorionic Gonadotrophin Levels

Nonviable pregnancies often have lower hCG levels than viable pregnancies [18]. In addition, hCG levels
rise more slowly in pregnancies destined to miscarry. βhCG levels should generally double each 48 hours.
However, as hCG is produced by the trophoblast, blighted ova may have very high levels of hCG.
hCG has a number of isoforms. Hyperglycosylated hCG (hCG-H) has been reported to be the most
dominant form in the first 2 weeks of pregnancy. hCG-H accounts for 90% of total hCG in first 2–3
weeks when invasive trophoblast activity is high [19]. Low levels of hCG-H may be a better marker than
total hCG.
Other Serum Markers

Inhibin A and activin A have been associated with threatened miscarriage failing to develop [20].
CA-125 is constant or increases in miscarriage. In ongoing pregnancy, there are low or steeply declining
CA-125 concentrations [21]. A single CA-125 ≥43.1 IU/mL has been associated with increased risk of
miscarriage [22].
In a systematic review of 1253 women with threatened miscarriage [23], assessing progesterone levels,
hCG, PAPP-A estradiol, and CA-125 as markers of nonviability concluded that CA-125 was the most
sensitive marker (648 women in 7 studies); sensitivity 90% (confidence interval [CI] 83%–94%), specificity
88% (CI 79%–93%), positive likelihood ratio 7.86 (CI 4.23–14.60), negative likelihood ratio 0.10 (CI
0.06–0.20). A negative test was likely to identify those who are likely to continue pregnancy. Estradiol
was the next best marker, with a sensitivity of 45% (CI 6%–90%), specificity 87% (CI 81%–92%), positive
likelihood ratio of 3.72 (95% CI 1.01–13.71), negative likelihood ratio of 0.62 (CI 0.20–1.84).
Threatened Miscarriage and Recurrent Pregnancy Loss 147
Progesterone-Induced Blocking Factor

The progesterone-induced blocking factor (PIBF) is an anti-inflammatory cytokine produced by
T-lymphocytes when treated with progesterone. The production rises with trophoblast invasion. [24].
PIBF blocks NK cell cytotoxic activity [25], increases the production of IL10, IL3, and IL4 (Th2) [26], and
mediates the progesterone-induced suppression of decidual lymphocyte cytotoxicity [27]. Hence PIBF
is related to anti-abortive effects of progesterone. PIBF levels have been reported to be lower in women
with subsequent miscarriage [16]. The problem is that although PIBF was described as long ago as the
1990s, it has not come into clinical use and is not available as a diagnostic test.
Late Obstetric Complications

Ahmed et al. [29] carried out a retrospective case-controlled study on 89 women with threatened
miscarriage who were matched for age and parity to 45 control women in order to evaluate the effect of
threatened miscarriage on early and late pregnancy outcomes.
The overall adverse pregnancy outcome was significantly higher in women with threatened miscarriage
compared to the control group (p = 015). The miscarriage rate was obviously significantly higher in the
study group compared to controls (16.9% vs. 2.2%, respectively, p = 001). Preterm delivery, low birth
weight, and premature rupture of membranes were also significantly higher after threatened miscarriage
(15.7% vs. 2.2%, p = 0.001), (15.7% vs. 2.2%, p = 0.001) and (6.7% vs. 4.45%, p = 0.016), respectively.
There were no significant differences in other pregnancy outcomes such as premature rupture of the
membranes, hypertensive disorders, intrauterine growth restriction, or the cesarean section rate.
Subchorionic Hematoma
Subchorionic hematoma is seen in approximately 18% of all cases of first trimester threatened miscarriages
[30]. There is one observational study on the natural history of subchorionic hematoma in threatened
abortion after detection of the fetal heart [31]. The incidence of miscarriage was 8.9%, similar to other
cases of threatened miscarriage. However, a meta-analysis by Tuuli et al. [32], which assessed trials
in which the presence of a fetal heart was not identified, included 1735 women with a subchorionic
hematoma. Of these pregnancies, 17.6% progressed to miscarriage. However, no series has addressed the
prognosis of subchorionic hematoma in women with RPL.
Various authors have tried to draw implications of the effect of the size of the hematoma. Bennet
et al. [31] claimed that a large hematoma was associated with three times increased risk of miscarriage
(19% vs. 71%), but the size of the hematoma was not found to be significant in other studies [33,34].
However, a retroplacental hematoma of any size may become infected at any stage of pregnancy, leading
to contractions and subsequent pregnancy loss.
Hematoma and Late Obstetric Complications

Nagy et al. [35] carried out a prospective study of 187 pregnant women with intrauterine hematomas
and 6488 control women. Women with retroplacental hematoma were found to have an increased risk of
severe obstetric complications, irrespective of external bleeding.
Hematoma was associated with an increased rate of instrumental deliveries (risk ratio [RR] = 1.9; CI
1.1–3.2) and cesarean deliveries (RR 1.4; CI 1.1–1.8). The risk of pregnancy-induced hypertension and pre-
eclampsia were significantly greater in the hematoma group (RR 2.1; CI 1.5–2.9, and RR 4.0; CI 2.4–6.7,
respectively). In later pregnancy, a retroplacental hematoma is known as placental abruption, and indeed
the incidence of abruption was higher (RR 5.6; CI 2.8–11.1) and placental separation abnormalities were
also significantly more frequent in the hematoma group (RR 3.2; CI 2.2–4.7). Perinatal complications
were also significantly higher in patients with a hematoma, including preterm delivery (RR 2.3; CI 1.6–
3.2), intrauterine growth restriction (RR 2.4; CI 1.4–4.1), fetal distress (RR 2.6; CI 1.9–3.5), meconium-
stained amniotic fluid (RR 2.2; CI 1.7–2.9). Admission to the neonatal intensive care unit was more
common (RR 5.6; CI 4.1–7.6). Furthermore, the frequency of intrauterine demise and perinatal mortality
was increased in the hematoma group, but this difference did not reach statistical significance.
Treatment
As the chance of threatened miscarriage developing to miscarriage has been reported to be as low as
3%–4% [6] to 15.4% [7] with a mean of 8.7%, it is debatable whether any treatment is warranted. However,
antenatal depressive and anxiety symptoms affect one in four women in the first trimester, with even
higher prevalence in threatened miscarriage [36]. In the case of threatened miscarriage after RPL, anxiety
levels are even higher. It is estimated that around 30% of women with RM are depressed and that even a
higher proportion have high levels of state and trait anxiety [37,38]. To determine if treatment is required,
it is necessary to assess the results of treatment against nontreatment while keeping the patient’s mental
state in mind. Various forms of treatment are discussed below.
However, the results of treatment may be confounded, as threatened miscarriage may be due to separation
of the placenta in a normal embryo, or a defense mechanism to prevent the continued development of an
abnormal embryo. The most important confounding factors are embryonic structural malformations or
chromosomal aberrations. These are discussed more fully elsewhere in this book but may have affected
the results of treatment of threatened miscarriage. When patients with embryonic anomalies are included
in a trial, the results would be skewed in favor of a negative effect. Confounding of the results should be
borne in mind in any negative trial. Neither embryonic structural defects nor chromosomal aberrations
were taken into account in any of the trials mentioned below.
Bed Rest
Bed rest is often prescribed for bleeding in pregnancy. However, there is little evidence of efficacy. It is
often said that bed rest prevents the patient having to face the stress of work and daily chores when she
is so stressed about the pregnancy developing. However, many women may be under less stress when
occupying themselves with their normal activities rather than only thinking about their pregnancies while
lying in bed. Harrison et al. [39] carried out a randomized trial of hCG supplementation versus bed rest.
In the bed rest group, 15 of 20 women miscarried. The authors concluded that hCG supplementation was
superior. Bed rest was not found to be effective in a Cochrane systematic review [40]. The systematic
meta-analysis only found two studies for review including 84 women. Neither bed rest in hospital nor bed
rest at home showed a significant difference regarding the prevention of miscarriage. (RR 1.54; CI 0.92–
2.58). Bigelow and Stone [41] in a review quoted four papers. Three of the four papers found no benefit
from bed rest. In the case of retroplacental hematoma, one paper [42] showed that when compliant patients
with bed rest were compared to noncompliant patients, the patients on bed rest had fewer miscarriages
and more term pregnancies (p = 0.0001).
Human Chorionic Gonadotrophin

There are many reasons that hCG supplementation should work in threatened miscarriage. hCG enhances
implantation through the effect of glycosylated hCG [43]. hCG increases the blood supply to the embryo
by affecting angiogenesis and vascular remodeling, which are crucial for implantation and placental
development. hCG promotes angiogenesis via upregulation of VEGF [44]. However, there is little evidence
of hCG having a beneficial effect in threatened miscarriage. Harrison [39] carried out a comparative study
of hCG placebo and bed rest in 61 women with threatened miscarriage. Six out of 20 miscarried on hCG,
10/21 miscarried on placebo, 15/20 miscarried on bed rest. The effect of hCG was significantly better than
bed rest, but no advantage was seen over placebo. Devaseelan et al. [45] carried out a Cochrane database
systematic review of three papers on hCG and threatened miscarriage, including Harrison’s [39] trial. The
meta-analysis [45] showed no statistically significant difference in the incidence of miscarriage between
hCG and “no hCG” (placebo or no treatment) groups (RR 0.66; 95% CI 0.42–1.05).
Progestogens
Wahibi et al. [46] carried out an analysis of two trials of oral dydrogesterone compared to placebo, and
two trials of vaginal progesterone. The overall figures showed a statistically significant benefit (odds
ratio [OR] = 0.53; CI 0.35–0.79) in favor of progestogen supplementation. It is interesting to note that in
the women who were treated with vaginal progesterone the treatment was not statistically effective in
reducing miscarriage when compared to placebo (RR = 0.47; 95% CI 0.17–1.30), whereas oral progestogen
(dydrogesterone) was effective (RR = 0.54; CI 0.35–0.84). Carp [47] published a subsequent meta-analysis
on five randomized studies including 660 patients. The results showed a statistically significant reduction
in the odds ratio for miscarriage after dydrogesterone compared to standard care of 0.47 (CI 0.31–0.7).
The 24% miscarriage rate in control women (78/325) was reduced to 13% (44/335) after dydrogesterone
administration (11% absolute reduction in the miscarriage rate).
Lee et al. [48] published a meta-analysis of progestogens in threatened miscarriage. There is a subgroup
meta-analysis of four trials of vaginal progesterone. Not one had a statistically significant effect, and the
meta-analysis, although showing a trend to a lower miscarriage rate, did not reach statistical significance
(OR = 0.72; CI 0.39–1.34).
Recently, the results of the PRISM trial have been published [5]. The PRISM trial was a multicenter,
randomized, double-blind, placebo-controlled trial to evaluate vaginal micronized progesterone in women
with threatened miscarriage. Treatment commenced at the time of bleeding and continued through 16
weeks of gestation. A total of 4153 women were randomly assigned to receive progesterone (2079 women)
or placebo (2074 women). The incidence of live births after at least 34 weeks of gestation was 75% (1513
of 2025 women) in the progesterone group and 72% (1459 of 2013 women) in the placebo group (relative
rate 1.03; 95% CI 1.00–1.07; p = 0.08). Hence in contrast to the dydrogesterone trials above, there was
no significant effect. However, when a subgroup analysis was performed for women with three or more
previous miscarriages the live birth rates compared to controls was 72% and 57%, respectively (relative
rate 1.28; 95% CI 1.08–1.51). Hence there may be benefit in prescribing vaginal micronized progesterone
in patients with recurrent miscarriage and vaginal bleeding. However, it is difficult to reconcile the results
of the PRISM study with those of the PROMISE study [49] by the same author which did not show any
beneficial effect of vaginal micronized progesterone in recurrent miscarriage.
There is little evidence concerning 17 hydroxyprogesterone acetate or caproate by intramuscular
injection. However, Shearman and Garrett [50] found 17 hydroxyprogesterone caproate to have no beneficial
effect in threatened miscarriage. Considering the pain and discomfort associated with intramuscular
injection and the lack of evidence, it is therefore not recommended for threatened miscarriage.
Progestogens in Subchorionic Hematoma

There are two trials of progestogens in subchorionic hematoma. Both are open-labeled observational
studies. In the first study, Pelinescu-Onciul et al. [51] treated 125 women with micronized progesterone
600 mg/d. Of these pregnancies, 18.7% terminated in miscarriage. In the second study [52], 100 women
with threatened miscarriage and a viable embryo received dydrogesterone. There were 93 live births and
7 miscarriages. The difference in results was significantly better in the dydrogesterone group (RR = 2.04;
CI = 1.05–3.97). However, these results should be treated with caution due to the methodological flaws
of comparing two separate cohorts of patients who were not randomized.
Safety and Side Effects

Safety and side effects should always be taken into account when drugs are used in pregnancy.
Progesterone itself has anti-androgenic effects and has been reported to lead to hypospadias [53,54].
Progesterone-related hypospadias has been seen in 8.4% of case mothers and 2.4% of control mothers,
for intakes from 4 weeks before conception to 14 weeks (OR 3.7; CI 2.3–6.0) [54]. Check et al. [55]
found two cardiovascular malformations, omphalocele, hydrocephalus, and club foot with cleft palate in
382 women exposed to either progesterone or 17-α hydroxyprogesterone. These studies had no control
group. However, progesterone has been reported as safe but is classified by the US Food and Drug
Administration as a category B drug. Maternal side effects include nausea, headache, and sleepiness.
If administered vaginally, there is discomfort in the presence of bleeding and the suppositories may be
washed out if bleeding is severe.
A review of birth defects associated with dydrogesterone use during pregnancy [28] concluded that
clinical experience with dydrogesterone provided no evidence of a causal link between maternal use
during pregnancy and birth defects. It is estimated that between 1977−2005, approximately 38 million
women were treated with dydrogesterone and more than 10 million fetuses exposed. There also seem to
be no major side effects in the mother.
Psychological Support
Approximately 30% of women with RM are depressed and an even higher proportion have high levels of
state and trait anxiety [37,38]. These couples generally do not receive social support and may also face
insensitive attitudes. To lower levels of distress, couples often withdraw from friends and do not receive
the social support they need. While psychological support may not affect the likelihood of threatened
miscarriage developing to miscarriage, psychological support is undoubtedly beneficial. The primary
physician is the most important person to provide psychological support, whether family physician,
gynecologist, or even nurse practitioner. Unfortunately, the pressure of clinical work and lack of training
and experience on the part of the physician do not always allow them to provide the required guidance.
Psychologists may be able to provide the support, but not all patients are willing to undergo support by
a psychologist.
One way to compensate for the lack of social support from family and friends is to seek couples who
share similar experiences. Meeting other couples with recurrent or threatened miscarriage can decrease
the sense of loneliness and reassure couples that their reactions and feelings are normal. Units that treat
recurrent miscarriage should ideally organize support groups for couples willing to attend.
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16
The Role of Cerclage and Pessaries
Israel Hendler and Howard J.A. Carp
Introduction
Cervical insufficiency is defined as the inability of the uterine cervix to retain a pregnancy in the absence
of contractions or labor. It is a clinical diagnosis characterized by recurrent painless cervical dilatation
and spontaneous midtrimester loss of a viable fetus. However, there are other predisposing conditions
for midtrimester loss, such as spontaneous rupture of the membranes, bleeding, or infection, which may
indicate a different origin for midtrimester loss rather than primary cervical insufficiency [1]. Cervical
insufficiency was first described in the English literature in 1678; however, even today the diagnosis is
clinical and made in retrospect after a poor obstetric outcome. The diagnosis is difficult to make and
is solely based upon careful history and review of the medical records rather than accurate diagnostic
imaging studies or other laboratory tools. True cervical insufficiency is probably uncommon; however,
the lack of clear diagnostic criteria makes the incidence unknown.
Cervical cerclage, first introduced by Shirodkar in 1955, is an appropriate and well-designed solution
for true cervical insufficiency. However, due to lack of strict diagnostic criteria, the indications for cerclage
are still far from clear, as are the optimal methods and timing. This chapter focuses on the diagnosis of
cervical insufficiency, the obstetric management of pregnant women at high risk for preterm delivery or
midtrimester loss by ultrasonographic follow-up of cervical length, the particular problems of cerclage
in recurrent pregnancy loss (RPL), the role of transcervical and transabdominal cervical cerclage, and
the optimal timing and method of performing the procedure.
Pathophysiology
The pathophysiology of cervical insufficiency is poorly understood. The cervix develops from fusion and
recanalization of the distal paramesonephric (Müllerian) ducts [2], which is complete by approximately 20
weeks’ gestation and is composed of both muscle and fibrous connective tissue. The fibrous component,
which is responsible for the tensile strength of the cervix, increases in proportion from the external os
toward the body of the uterus. Cervical insufficiency is thought to be related to a defect in tensile strength
at the cervicoisthmic junction [3]. Although several theories of pathophysiology have been considered,
the difficulty in obtaining biopsy samples from the human cervix before, during, and after term and
preterm deliveries has hampered this understanding. In 1996, Iams et al. [4] challenged the traditional
understanding of the cervix as being either “competent” or “incompetent.” Transvaginal ultrasonography
of cervical length was performed in 2915 women at 24 weeks of gestation. Ultrasound revealed that the
association between cervical length and the risk of preterm delivery is evident across the entire range of
cervical lengths. Even among women whose cervical length was above the 10th percentile, the risk of
preterm delivery increased as cervical length decreased. Therefore, the length of the cervix may be an
indirect indicator of cervical competence and should be seen as a continuous rather than a dichotomous
variable. However, Iams et al.’s [4] study demonstrated a normal bell-shaped curve distribution of cervical
length in the general population of women at 24 weeks’ gestation with a mean (± SD) of 35.2 ± 8.3 mm.
Thus a short cervical length could be a normal phenomenon and not necessarily a definite marker for
preterm birth. The length of the cervix is directly correlated with the duration of pregnancy: the shorter
153
the cervix, the greater the likelihood of preterm birth. However, the cervix is a dynamic structure in
pregnancy, occasionally shortening with no apparent relationship to uterine contractions. Iams et al. [4]
have proposed the model of a continuum of cervical compliance (“competence”) similar to the natural
biologic variation in the population in other physical traits, such as height and weight. In this model,
cervical compliance and cervical length vary among women, and these qualities are just some of the
components of uterine function that affect the timing of delivery; many women who have a congenitally
short cervix deliver at term [5–9].
Risk Factors for the Etiology of Cervical Insufficiency

Congenital Factors
A functional defect in the cervix can be caused by an anatomic abnormality (such as congenital Müllerian
anomalies including canalization defects (e.g., septate uterus), unification defects (e.g., bicornuate uterus),
and even arcuate uterus, in utero diethylstilbestrol (DES) exposure, or collagen disorders (e.g., Ehlers-
Danlos syndrome). Congenital defects may explain the familial tendency for cervical insufficiency. As an
example, in one study, 34 of 125 (27%) women with cervical insufficiency had a first-degree relative with
the same diagnosis, but none of the 165 unaffected women had a family history of cervical insufficiency.
Acquired Factors
Obstetric Trauma
A cervical laceration may occur during labor or delivery, including spontaneous deliveries, forceps,
vacuum, or cesarean births. Laceration might weaken the cervix and contribute to cervical insufficiency
[10]. Levine et al. [11] described the effect that a cesarean delivery in one pregnancy has on the risk
of preterm birth (PTB) in a subsequent pregnancy. They found that, when compared with a cesarean
delivery in the first stage of labor, a cesarean delivery in the second stage of labor in one pregnancy
confers sixfold increased odds of spontaneous preterm birth (sPTB) in a subsequent pregnancy. Three
possibilities of intraoperative procedures that may contribute to the sPTB risk are (i) the transverse
hysterotomy incision that is thought to be in the lower uterine segment but is actually at the top of the
cervix, (ii) the unintentional taking up of the cervix into the hysterotomy closure, and (iii) extension of a
tear down into the cervix at the time of fetal delivery. These intraoperative procedures are more common
during a second-stage cesarean delivery and can lead to cervical trauma, which may alter the integrity
and strength of the cervix for future pregnancies.
Mechanical Dilation
Mechanical dilation of the cervix during gynecologic procedures may weaken the cervix. Prior cervical
mechanical dilatation is one of the most common associated risk factors. In a meta-analysis, an increasing
number of voluntary pregnancy terminations was associated with an increasing risk of spontaneous
preterm births.
Treatment of Cervical Intraepithelial Neoplasia

Cervical biopsy, laser ablation, loop electrosurgical excision procedures (LEEP), or cold knife conization
may all weaken the cervix [12]. However, in most cases of presumed cervical insufficiency no known
risk factor can be found.
Diagnosis of Cervical Insufficiency

Unfortunately, there are no reliable prepregnancy tests to confirm cervical insufficiency in “at-risk”
women. In the past, clinicians have suggested a variety of tests, including assessment of the width of
The Role of Cerclage and Pessaries 155
the cervical canal by hysterosalpingography and/or hysteroscopy, ease of insertion of cervical dilators
of various diameters (Hegar test), the force required to withdraw a Foley catheter with its bulb inflated
through the internal os, and different methods to measure force required to stretch the cervix using an
intracervical balloon and vaginal examination on a weekly basis during the second trimester of pregnancy
in high-risk women with RPL to assess softening and shortening of the cervix. None of these has been
validated in rigorous clinical studies. The obvious flaw with these techniques is the failure to account for
the effects of pregnancy on the dynamic capabilities of the cervix.
With the advent of transvaginal ultrasonography and measurement of cervical length, features such
as shortening, effacement, and dilatation with the presence of funneling and prolapse of the membranes
have enabled clinicians to predict outcome long before symptoms occur. However, it is still unclear if
cervical shortening is indicative of a primary cervical problem. Without any reliable, objective method
of distinguishing cervical insufficiency from other causes of premature cervical change, management is
pragmatically based on combining features within the history (e.g., previous painless dilatation, cervical
surgery) with ultrasound findings.
Cervical Cerclage
Transvaginal cerclage in pregnancy was first reported in 30 women by Shirodkar in 1955. These 30
women had 4–11 prior late miscarriages. The need for cerclage was based on diagnosis of weakness
of the internal os by repeated vaginal examinations. Many investigators have reported variations on
the surgical technique of transvaginal cerclage, the most common being the McDonald procedure.
When first described, cerclage was used for two indications: initially for prior second trimester loss
with painless cervical dilation in the current pregnancy (i.e., physical examination indicated), and soon
after for recurrent second trimester loss not attributable to other causes. Seventy years later, cerclage
is performed in 1:54–1:220 deliveries worldwide, although there is still confusion about the diagnostic
criteria for cervical insufficiency and uncertainty regarding the benefits.
Techniques of Cerclage
McDonald Cerclage
The McDonald cerclage is the most commonly performed method of cerclage. The technique is performed
by exposing the cervix with a speculum and inserting a purse-string suture of silk, monofilament nylon, or
braided tape around the exocervix as high as possible to approximate to the level of the internal os. The
suture is usually placed at the junction of the vagina and cervix. Five or six bites are taken, with special
attention being paid to the stitches behind the cervix. These are difficult to insert and must be deep. The
stitch is pulled tight enough to close the internal os, the knot being made in front of the cervix and the
end left long enough to facilitate subsequent division. Some centers will tie a second more superficial
knot to facilitate identification of the threads at subsequent removal. If a more superficial knot is tied,
the physician removing the suture must be aware of the second knot in order to prevent cutting the suture
between the two knots and leaving the suture in place.
Many operators have modified the McDonald technique in order to only take three or four bites. This
modification makes the technique easier and is probably as effective as the original McDonald technique.
Shirodkar Cerclage
The Shirodkar technique involves dissection of the vaginal mucosa and retraction of the bladder and
rectum to expose the cervix at the level of the internal os. In the original technique, a strip of fascia lata
removed from the outer side of the thigh was used as the suture material. Today silk or braided tape
are used as in McDonald’s technique. In Shirodkar’s suture, it is possible to tie the knot anteriorly as in
McDonald’s technique, or posteriorly in the posterior fornix. The anterior knot needs to be to be exposed
in the vagina, whereas if a posterior knot is tied, the knot can be buried under the vaginal mucosa.
The possible advantage of burying the knot and thread preserves sterility and prevents infection from
spreading from the nonsterile vagina to the sterile cervical tissue. After completion of the cerclage, the
anterior and posterior incisions need to be closed.
Caspi et al. [13] described a modification of Shirodkar’s technique, using a single transverse incision in
the anterior fornix. A suture is passed on each side, under the mucosa at the level of the internal os, from
the anterior incision to exit through the mucosa of the posterior cervix, and is then tied. The modified
procedure has been compared with the original technique of Shirodkar in a randomized trial in 90 subjects
who lost their pregnancies despite having undergone McDonald’s procedure or with cervical anatomy felt
to be unfavorable for McDonald cerclage placement. Similar pregnancy outcomes were reported. The
investigators believed that the modified Shirodkar technique has the advantages of simplicity, ease of
removal, and lower incidence of severe vaginal discharge. Using the modified Shirodkar technique allows
the suture to be placed 2–3 cm above the level of the McDonald suture.
Recently, a modification of Shirodkar’s technique has been used in which the cardinal ligaments are
isolated after the anterior and posterior fornix incisions, as in vaginal hysterectomy. The stitch can then
be placed above the cardinal ligaments in the relatively avascular space just below the insertion of the
uterine arteries. However, care must be taken to place the suture immediately lateral to the uterus in
order to avoid injury to the uterine arteries and ureters. This technique can reach a height equivalent to
abdominal cerclage.
Abdominal Cerclage
There are circumstances in which the cervix has become so torn and scarred from previous trauma,
including failed vaginal cerclage, that a vaginal approach is technically impossible. There may be cervical
tears of which the apex of a tear cannot be identified, or previous trauma may have amputated the entire
intravaginal portion of the cervix. In these cases, an abdominal approach may be required. There are a
number of small series in the literature on abdominal cerclage. However, there are no evidence-based
trials. An abdominal approach was originally described using laparotomy. However, today with the
increased experience and proficiency, a laparoscopic approach can be used.
There are two main techniques. In Anthony et al.’s [14] technique, the uterine arteries are identified
and tunnels created medial to the uterine arteries. A 5 mm Mersilene tape is then passed through the
tunnels and the knot tied anteriorly. There is a problem in that if the suture requires removal, laparotomy
or laparoscopy are required.
In Topping and Farquharson’s [15] technique, the suture is passed through the muscle or uterus medial
to vessels at the height of isthmus above cardinal ligaments, and the stitch tied anteriorly.
Evidence-Based Criteria for Cerclage Placement

History-Indicated Cerclage
A minority of recurrent second trimester losses/births are primarily, and perhaps exclusively, caused by
congenital or acquired structural weakness of the cervix and can be treated effectively with support by a
“history-indicated” cerclage. The largest randomized trial for history-indicated cerclage was published in
1993 by the Medical Research Council/Royal College of Obstetricians and Gynaecologists (MRC RCOG)
[16]. There were 1292 pregnant women with a history of early delivery or cervical surgery randomized to
cervical cerclage or withholding the operation unless it was considered to be clearly indicated. There were
fewer deliveries before 33 weeks in the cerclage group (13% vs. 17%, p = 0.03). There was a corresponding
difference in very low birth weight deliveries (10% vs. 13%, p = 0.05). The authors concluded that the
number needed to treat to prevent early preterm birth was in 1 in 25 cases. The authors recommended
that cerclage should be offered to women with a history of three or more pregnancies ending before 37
weeks’ gestation. It is now suggested to insert a history-indicated cerclage at 12–14 weeks for women who
meet all of the following criteria: (i) Two or more consecutive prior second trimester pregnancy losses
or three or more early (<34 weeks) preterm births, (ii) presence of risk factors for cervical insufficiency,
including a history of cervical trauma and/or short labors or progressively earlier deliveries in successive
pregnancies, and (iii) other causes of preterm birth (e.g., infection, placental bleeding, multiple gestation)
have been excluded.
However, a Cochrane database meta-analysis [17] analyzed 15 studies of women considered at sufficient
risk to justify cerclage who were randomized to cerclage, alternative treatments (e.g., progesterone), or
no treatment. Although cerclage was associated with a statistically significant effect on reducing preterm
birth rates, there was no significant impact on perinatal morbidity and mortality. Furthermore, cerclage
was associated with increased maternal morbidity and cesarean section rates (the latter perhaps also
accounting for a nonsignificant increase in respiratory morbidity among infants born to women with a
cerclage).
Ultrasound-Indicated Cerclage
The majority of women with suspected cervical insufficiency do not meet the above criteria for history-
indicated cerclage. In women with singleton gestation, a prior spontaneous preterm birth, and short
cervical length <25 mm before 24 weeks, a meta-analysis [18] of controlled studies showed that preterm
birth prior to 35 weeks occurred in 28.4% (71/250) of patients after cerclage compared to 41.3% (105/254)
of women without cerclage (RR 0.70; CI 0.55–0.89). Cerclage also significantly reduced preterm
birth before 37, 32, 28, and 24 weeks of gestation. Composite perinatal mortality and morbidity were
significantly reduced (15.6% after cerclage compared to 24.8% without cerclage; RR 0.64; CI 0.45–0.91).
Hence, placement of cerclage upon identification of a short cervix (“ultrasound-indicated cerclage”)
is effective in reducing preterm births, results in pregnancy outcomes comparable to those with history-
indicated cerclage, and avoids cerclage in about 60% of patients with a suggestive history.
In an updated meta-analysis of randomized trials of women with singleton gestations and no prior
preterm births, the same team [19] modified their criteria for ultrasound-indicated cerclage. They reported
that with cervical length of <25 mm in the second trimester, cerclage did not seem to prevent preterm
delivery or improve neonatal outcome. However, cerclage seemed to be efficacious at lower cervical
lengths (CLs), such as <10 mm, and when tocolytics or antibiotics are used as additional therapy.
Figure 16.1a shows the sonogram of a normal cervix on ultrasound. As shortening of cervical length
seems to be a continuous process, ultrasound can detect dilatation of the internal os before the external
os is affected. Figure 16.1b shows shortening of the cervical canal. Figure 16.2 shows funneling of the
internal os and shortening of the cervical canal. However, transcervical ultrasonography has a number
of drawbacks. Figure 16.3 shows an apparently normal looking cervix. However, the application of light
fundal pressure allows the insufficiency to become apparent, and grand multipara can have open cervices
without insufficiency. Hence transcervical ultrasound is not always selective.
FIGURE 16.1 Ultrasound of cervical length. (a) Normal cervix of 35 mm length. (b) Shortened cervix of 14 mm length.
These sonograms show normal cervices. The cervix in (a) is completely closed, with a length of 35 mm (as seen between
the calipers). The cervix in (b) is 14 mm in length but can still be competent.
FIGURE 16.2 Sonogram of cervical incompetence: funneling of the cervix with a dilation of the internal os. The remaining
cervical canal from the funneling to the external os is extremely shortened.
FIGURE 16.3 A dynamic cervix. (a) Cervix with no fundal pressure. (b) Cervix with fundal pressure. The cervix was
shortened from 28 to 0 mm during the examination by light fundal pressure.
The incidence of cervical incompetence [20], midtrimester loss, and preterm labor are higher after
recurrent pregnancy loss [20,21]. It is debatable whether this higher incidence is sufficient to justify
screening the entire population on a regular basis. In patients who are screened, it is advisable to start
cervical length screening at 16 weeks, especially in women with early second trimester losses, recurrent
second trimester losses, or prior large cold knife conization. Ultrasound examination is generally repeated
every 2 weeks until 24 weeks as long as the cervical length is ≥30 mm, and increased to weekly if the
cervical length is 25–29 mm, with the expectation that preterm cervical changes will precede overt
preterm labor or membrane rupture symptoms by 3−6 weeks.
Progestogen administration has been reported to prevent preterm birth, either by 17 alpha hydroxy-
progesterone caproate, vaginal micronized progesterone, or dydrogesterone. No randomized controlled
trial has directly compared progestogen administration to cervical cerclage for the prevention of preterm
birth in women with a sonographic short cervix in the midtrimester, singleton gestation, and previous
preterm birth. An indirect comparison meta-analysis concluded that vaginal micronized progesterone
and cerclage were equally efficacious in the prevention of preterm birth in this population [22]. Based on
evidence from the direct comparisons in the randomized trials already discussed, we treat women with
prior preterm birth with intramuscular 17-alpha-hydroxyprogesterone caproate and then perform cerclage
if the cervical length shortens to less than 25 mm. We perform a single transvaginal ultrasound (TVU)
cervical length measurement at 18–24 weeks in women with risk factors for cervical insufficiency and
no prior delivery and treat those with a short cervix (≤20 mm) with vaginal micronized progesterone
supplementation. In a meta-analysis of five trials, administration of vaginal progesterone to women with
a short cervix reduced the rate of spontaneous preterm birth and composite neonatal morbidity and
mortality.
If the patient delivers preterm or has another midtrimester loss, subsequent pregnancies are managed as
previously described. If the patient delivers at term, we again perform a single cervical length measurement
at 18–24 weeks and administer vaginal micronized progesterone if the cervix is short.
Physical Examination−Indicated Cerclage

A patient may present in the midtrimester with minimal or no symptoms, and physical examination
reveals a dilated cervix. Occasionally, such findings may occur after diagnosis of a very short cervical
length (e.g., <5 mm) on TVU. The management of these patients is governed primarily by whether the
condition requires prompt delivery, e.g., if there is overt infection, ruptured membranes, or significant
hemorrhage. In the absence of indications for delivery, the gestational age and degree of cervical dilation
are the next considerations. The goal of management is to both prolong the pregnancy and improve
neonatal outcome in the likely event of preterm birth [23–25].
Data from several studies suggest that a grossly dilated cervix with visible membranes up until 27 weeks’
gestation may be an appropriate criterion for placement of a “rescue cerclage” in some cases (also called
“heroic cerclage” or “emergency cerclage”). Placement of a rescue cerclage when a dilated cervix and
visible membranes are detected on digital examination at <27 weeks appeared to prolong pregnancy and
improve pregnancy outcome compared to expectant management in a small randomized trial, a prospective
study, and retrospective cohort studies. Due to differences in patient populations, actual outcomes varied
among these studies. Physical examination−indicated cerclage in women with visible bulging membranes
should only be considered in the absence of infection, labor, and vaginal bleeding (abruption).
In women without clinical signs of infection, amniocentesis should be considered to exclude subclinical
infection.
Prior Successful Outcome after Cerclage

Prolongation of pregnancy after cerclage does not confirm the diagnosis of cervical insufficiency because
many pregnancies with premature cervical effacement have good outcomes in the absence of surgical
intervention. As discussed, in randomized trials and controlled studies, about 60% of women with a
history of early preterm birth or recurrent late miscarriage maintain cervical length above 25 mm and
have low rates of recurrent preterm birth/loss without placement of a cerclage. Therefore, repeat cerclage
in subsequent pregnancies is not mandatory.
In women who received a cerclage in a prior pregnancy without an appropriate indication, especially
those who, after removal of cerclage at 36–37 weeks, did not go into labor in the subsequent 2 weeks, the
risk of preterm birth in a subsequent pregnancy probably does not warrant a history-indicated cerclage;
instead we suggest TVU cervical length screening.
Prior Unsuccessful Outcome after Cerclage

Transabdominal cerclage may be successful in women who deliver very preterm despite placement of a
transvaginal cerclage.
Twin Pregnancy
A recent meta-analysis of cerclage in twin pregnancies comes from Saccone et al. [26]. In a meta-
analysis of three randomized trials of cerclage for a cervical length of <25 mm in twins, the results
did not favor cerclage. The gestational age at delivery was earlier in the cerclage group (30.33 vs.
34.20 weeks, p = 0.007), and preterm birth <34 weeks was worse in the cerclage group (62.5% vs.
24.0%; OR 1.17; 95% CI 0.23–3.79). Hence in twin pregnancies, cerclage was not only not indicated,
but even contraindicated. In fact, even before the Saccone [26] meta-analysis, the American College of
Obstetricians and Gynecologists [27] stated that cerclage may increase the risk of PTB in twin pregnancy
and an ultrasound-detected cervical length <25 mm and is not recommended. The same contraindication
also applies to history-indicated cerclages. Elective cerclage at 13 weeks is not indicated.
However, the question arises as to whether cerclage may be beneficial and therefore indicated in
certain circumstances. Houlihan et al. [28] carried out a retrospective cohort study of 40 biamniotic and
bichorionic twin gestations. In patients with an ultrasonic cervical length of 1–24 mm at 16–24 weeks, the
incidence of preterm birth at <32 weeks was significantly less frequent (RR = 0.40; 95% CI 0.20–0.80).
Abassi [29] reported on 27 rescue cerclages at 21.5 ± 2.6 weeks. The gestational age at delivery was
more advanced after rescue cerclage (28.9 ± 6.1 vs. 24.2 ± 2.6 weeks, respectively; p = 0.03). Preterm
birth was less likely after cerclage at <34 and <28 weeks, p = 0.02. A recent meta-analysis [30] included a
total of 16 studies with 1211 women. The outcomes indicated that cerclage placement for twin pregnancies
with a cervical length of <15 mm was associated with significant prolongation of pregnancy by a mean
difference of 3.89 weeks of gestation (95% CI 2.19–5.59) and a reduction of preterm birth at <37 weeks
of gestation (RR 0.86; 95% CI 0.74–0.99), <34 weeks of gestation (RR 0.57; 95% CI 0.43–0.75), and
<32 weeks of gestation (RR 0.61; 95% CI 0.41–0.90), compared to pregnancies in the control group. For
women with a dilated cervix of >10 mm, cerclage placement was associated with significant prolongation
of pregnancy by a mean difference of 6.78 weeks of gestation (95% CI 5.32–8.24), a reduction of preterm
birth at <34 weeks of gestation (RR 0.56; 95% CI 0.45–0.69), <32 weeks of gestation (RR 0.50; 95% CI
0.38–0.65), <28 weeks of gestation (RR 0.41; 95% CI 0.20–0.85), and <24 weeks gestation (RR 0.35;
95% CI 0.18–0.67), and improvement of perinatal outcomes compared with those in the control group.
However, for twin pregnancies with a normal cervical length (e.g., cerclage for an indication for women
with a history of preterm birth or twins alone), the efficacy of cerclage placement was less certain because
of the limited data.
The question therefore arises as to whether cerclage may be occasionally indicated. Obviously good
clinical judgment is essential.
Cervical Pessary
Another technique that has come into use for encircling the cervix is the cervical pessary. The Arabin
pessary is the most commonly used such device. However, the idea of using a pessary is not new. In 1959,
Cross described the use of a ring pessary in patients with cervical incompetence, lacerations, or uterine
malformations [31]. Since then, other devices have been used, including the Hodge pessary and donut
pessary (Figure 16.1 shows sonograms of the Hodge pessary in situ). The pessary has been described to
act by pressing the internal os closed from behind, and by changing the inclination of the cervical canal.
This change of position may prevent direct pressure on the membranes at the internal os and on the cervix
itself. The weight of the uterus may therefore be directed toward the lower anterior uterine segment rather
than the cervix. The pessary has been reported to protect the cervical mucus plug by compressing the
attachment of the remaining cervical tissue. The cervical mucus plug may protect the intrauterine cavity
from ascending infection and subsequent miscarriage or preterm labor [32,33]. Cervical elongation after
pessary insertion has also been shown by TVU [34].
The most commonly used pessary was designed by Arabin. It is a round cone-shaped flexible silicone
pessary. The dome shape resembles the vaginal fornices, and hence it attempts to encircle the cervix
close to the internal os. It comes in different sizes and has perforations in the silicone to drain the vaginal
discharge of the vaginal fornices.
Advantages of the Pessary

The pessary has a number of advantages over cerclage. The pessary can be fitted without anesthetic; it is
not invasive, as cerclageis. There is no foreign body inside the tissue of the cervix, which reduces the risk
of infection. There is no fenestration from tearing of the cervical tissue, either by contractions or pressure
necrosis of the tissue under the suture. As with other pessaries, the Arabin pessary changes the uterocervical
angle [35,36], making the angle more acute, thus moving the weight of the uterus to the anterior segment.
This change of angle is thought to prevent direct pressure on the membranes at the internal cervical os.
The pessary also protects the cervical mucus plug by pushing the internal os closed. The cervical mucus
plug may prevent ascending infection [35,36]. Cerclage, on the other hand, introduces a foreign body close
to the mucus plug and may enhance infection. If there is rupture of the membranes, suture cerclage should
preferably be removed in order to prevent infection. The pessary can, however, be left in situ if the patient
is managed conservatively [37]. In addition, removal is relatively easy. In some cases of cerclage, the suture
may become embedded, making removal extremely difficult. One of the authors (HC) has seen amputation
of the cervix due to excess pressure on the cervix in a patient with five previous first trimester miscarriages,
who conceived twins. A pessary was inserted after premature rupture of the membranes at 21 weeks.
Correct Placement
If the Arabin pessary is used, the pessary should be lubricated, squeezed between thumb and fingers,
and introduced into the introitus. Inside the vagina, the pessary is unfolded so that the smaller inner
ring faces toward the cervix. The dome is pushed toward the fornices until the cervix is completely
surrounded. Once in place, the pessary should not be felt by the patient. Subsequently, digital examination
or ultrasound can be performed to confirm that the cervix protrudes through the inner ring. Arabin and
Alfirevic [38] have published a table recommending different sizes to be used in different indications.
The pessary should be removed if delivery is imminent or if contractions are effective. However, in
normal circumstances, the pessary, as a suture, is removed at approximately 37 weeks. If there is cervical
edema, removal may be painful. In any case, the cervix should be pushed back through the inner ring of
the pessary dome.
Results
Arabin published the results of a study on 46 women with a short cervical length <25 mm before 24
weeks [35]. Twenty-three had a pessary inserted, and the results were compared to 23 women treated
expectantly. The mean gestational age at delivery was 35+6 weeks in the pessary group and 33+2 weeks
in the control group (p = 0.02).
There have been two randomized control trials of the pessary in patients with a short cervix (below
25 mm); however, the results are conflicting. In Goya et al.’s [37] trial, the use of the pessary was associated
with a statistically significant decrease in the incidence of preterm birth prior to 37 weeks compared with
the 193 women in the expectant management group (RR = 0.36; CI 0.27–0.49). There were also fewer
births before 34 weeks (RR = 0.24; CI 0.13–0.43) and before 28 weeks (RR 0.25; CI 0.09–0.73). In addition,
women in the pessary group required less tocolytics (RR 0.63; 95% CI 0.50–0.81) and corticosteroids (RR
0.66; 95% CI 0.54–0.81) than the expectant group. However, Hui et al.’s [40] trial also assessed the pessary
on women with a singleton pregnancy who were selected for a short cervical length at routine second
trimester ultrasound. The mean gestational age at delivery was 38.1 weeks in the pessary group compared
with 37.8 weeks in the expectant management group. There was also no significant difference in the rates
of delivery before 28, 34, or 37 weeks. However, in Hui et al.’s [40] study, some of the women who would
be expected to benefit were excluded; e.g., women with a cerclage in a previous pregnancy, the presence
of cervical dilatation, or a history of cervical incompetence were excluded.
The pessary has been tested in twin pregnancy [40,41]. Again, the results differ. In Hui et al.’s [40]
trial, prophylactic use of the pessary did not reduce poor perinatal outcome. However, in the subgroup
of women with a cervical length below 38 mm at 20 weeks, the incidence of poor neonatal outcomes
was 12% (9/78) for the pessary group and 29% (16/55) for the expectantly managed group. The major
effect was due to significantly fewer deliveries before 32 weeks (RR 0.49; CI 0.24–0.97). However, in
Merced et al.’s [41] trial, significant differences were observed in the preterm birth rate before 34 weeks.
PTB occurred in 11/67 (16.4%) of patients in the pessary group and 21/65 (32.3%) in the control group
(RR = 0.51; CI 0.27–0.97). No significant differences were observed in the preterm birth rate <28 weeks
or <37 weeks. The pessary group also required readmissions for new episodes of threatened preterm
labor less frequently (RR = 0.28; CI 0.10–0.80). There was also a significant reduction in the number of
infant neonates <2500 g (RR = 0.25; CI 0.15–0.43).
Comparison of Treatment Modalities

When pessary treatment is compared to other treatment modalities, there seems to be little difference in
the results. Alfirevic et al. [42] compared three cohorts of women with previous preterm births and a short
cervix. Cerclage was performed on 142 women. Fifty-nine women received vaginal progesterone, and 42
were treated by pessary. There were no significant differences in terms of perinatal loss, neonatal morbidity,
or preterm birth except for a higher rate of births before 34 weeks in the vaginal progesterone group
compared to the pessary group. Dang et al. [39] compared the pessary to vaginal micronized progesterone
in a randomized trial in 300 women with twin pregnancies. There were similar rates of preterm birth at less
than 34 weeks of gestation in women with twin pregnancies and cervical length less than 38 mm. However,
in women with cervical length of <28 mm, the pessary significantly reduced the preterm birth rate at less
than 34 weeks of gestation from 46% (16/35) to 21% (10/47) (RR 0.47; CI 0.24–0.90).
Currently there is insufficient evidence to recommend either pessary or cerclage for the routine
management of women at risk of preterm birth, both singleton and twins. Many factors lead to the common
pathway of cervical shortening and preterm birth, and the naïve thought that one treatment modality can
benefit all women is far from reality. Personal judgment and tailored treatment are required for each patient.
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17
What Genetic Screening Is Appropriate
in Recurrent Pregnancy Loss?
Howard Cuckle
Background
Antenatal screening for aneuploidy has steadily evolved over several decades from the initial concept
of a single second trimester maternal serum marker to complex protocols involving the combination
of multiple serum and fetal ultrasound markers, determined within trimester or sequentially across
trimesters. Test results were interpreted by estimating the risk of an individual pregnancy being affected
and those with high enough risk referred for counseling on invasive prenatal diagnosis.
Two recent developments have changed the situation. First, there is the discovery that a single-
marker maternal plasma cell free (cf)DNA has a vastly superior performance to any of the current
protocols. A second, and in some ways competing, development is the use of the prenatal chromosomal
microarray (CMA) to facilitate the detection of clinically significant subchromosomal microdeletions and
microduplications that are not seen on nonmolecular cytogenetic karyotyping. This considerably enriches
the diagnostic potential of fetal material obtained by invasive prenatal diagnosis.
Couples with recurrent pregnancy loss (RPL) are at increased risk of fetal chromosomal abnormalities
in subsequent pregnancies. Moreover, they are naturally averse to chorionic villus sampling (CVS) or
amniocentesis, since invasive prenatal diagnosis is associated with increased risk of miscarriage. It might
be argued that such couples may require different screening options than other couples.
In this chapter all current screening options are considered with a view to determining those most
appropriate for such couples. Screening is a public health activity, and as such, policies must be judged
in general population terms. This does not necessarily provide the best testing option for an individual
who is willing to pay for their own care. Both perspectives are discussed here.
Purpose of Routine Screening

Screening for chromosomal abnormalities has the simple aim of identifying pregnancies at sufficiently
high risk of an affected birth to warrant the hazards and costs of invasive testing. Local policy and national
convention on what counts as sufficiently high risk to warrant invasive testing has generally emerged from
the push and pull of health care providers, reimbursement tariffs, and professional bodies. Chromosomal
abnormalities are relatively rare at birth with a prevalence of about 0.6%, excluding mosaics, when 70,000
consecutive newborns were karyotyped [1]. Hence from the earliest days, universal unselective invasive
testing was not considered an option; rather there was selection based on advanced maternal age and
family history of chromosomal abnormality.
The maternal age-specific birth prevalence of Down syndrome (trisomy 21) increases to 0.11%, 0.26%,
0.98%, and 3.5% by ages 30, 35, 40, and 45 respectively [2]. The estimated risk at term for all common
autosomal trisomies—Down, Edwards (trisomy 18), or Patau (trisomy 13) syndrome—is 0.48% and
1.6% at ages 35–39 and 40–44, and for all chromosomal abnormalities 0.81% and 2.4%, respectively
[3]. A family history of chromosomal abnormality confers a much higher risk than this when a maternal
balanced translocation is found [4], whilst for paternal carriers and for non-carrier couples there is only
164
What Genetic Screening Is Appropriate in Recurrent Pregnancy Loss? 165
a modest excess over their maternal age-specific risk. With a Down’s syndrome proband and non-carrier
parents the excess at midtrimester is 0.54% for the same disorder and 0.24% for other aneuploidies [5].
Testing these two high-risk groups can have little impact on birth prevalence, as most chromosomal
abnormalities occur in young women, and they are sporadic. This consideration has led to the development
of newer methods of selection for invasive testing.
Conventional Screening Modalities

Beginning in the mid 1980s, a series of maternal serum markers of aneuploidy was discovered: human
chorionic gonadotrophin (hCG), the free-β subunit of hCG (β-hCG), α-fetoprotein (AFP), unconjugated
estriol (uE3), inhibin A, and pregnancy-associated plasma protein (PAPP)-A. Meanwhile, even more
discriminatory first trimester ultrasound markers were found: nuchal translucency (NT), nasal bone (NB),
tricuspid regurgitation (TR), and ductus venosus (DV).
Various marker combinations, determined concurrently, formed the basis for the first effective screening
protocols. The efficacy of a given policy is generally measured by applying a statistical model to calculate
the expected detection rate, proportion of affected pregnancies selected for invasive testing, and the false-
positive rate, proportion of unaffected pregnancies selected.
Quad Test
This is the best early second trimester maternal serum combination: AFP, free β-hCG, uE3, and inhibin.
When applied to all women using a 1 in 250 term risk cutoff—the norm in the United Kingdom—the
model-predicted Down syndrome detection rate and false-positive rate are 68% and 4.2% [6]. In the
United States, where a 1 in 270 midtrimester risk cutoff is favored, equivalent to about 1 in 350 at term,
the corresponding rates are 73% and 5.9%.
Combined Test
This is the most widely used first trimester combination: PAPP-A and free β-hCG, ideally at 10 weeks,
and NT at 11 weeks. The model-predicted Down syndrome detection rate and false-positive rate are 82%
and 2.4%, respectively [6]. The predicted United States rates are 84% and 3.2%, respectively.
The same markers can also detect a large proportion of Edwards syndrome cases; in the second trimester
this requires a separate risk cutoff, but in the first trimester most are detected because of increased Down
syndrome risks. Many of the remaining severe but nonlethal chromosomal abnormalities are also detected
incidentally because of high Down syndrome risk [7]. Although even more are associated with extreme
marker levels, particularly NT [8], it is not routine practice to calculate risks for these other disorders.
Sequential protocols have also been developed that considerably increase the detection rate for Down
syndrome and other common trisomies. Attendance for screening is required on two occasions.
Contingent Test
This is the most efficient sequential protocol. It starts with the first trimester combined test markers
determined at 11 weeks’ gestation but adopts an extremely high cutoff risk, selecting a small number for
immediate CVS. The remainder then have the second trimester quad test markers, and all seven first and
second trimester marker levels are incorporated into the calculation of risk. The model-predicted Down
syndrome detection rate and false-positive rate with a 1 in 250 term risk cutoff are 88% and 1.6% (1 in
270 midtrimester 89% and 2.0%) [6].
Integrated Test
The integrated test is based on using the best markers at each trimester: PAPP-A and NT in the first and
the quad markers in the second. Unlike the contingent test, all women have both determinations and the
results are not reported until the test is completed. This nondisclosure raises both ethical and practical
problems. Moreover, modeling predicts results similar to the contingent test: Down syndrome detection
rate and false-positive rate with a 1 in 250 term risk cutoff are 87% and 1.6% (1 in 270 midtrimester 89%
and 2.1%) [6]. It is not in widespread use.
Incorporating the newer first trimester ultrasound markers—NB, TR, and DV—substantially enhances
detection both of the common trisomies and other chromosomal abnormalities. For example, routinely
adding NB to the combined test would improve the above detection rates with a 1 in 250 term risk cutoff
to 90% and 1.4% (1 in 270 midtrimester 91% and 1.8%) [6]. When NB is added to the contingent test, the
rates for a 1 in 250 term risk become 91% and 0.8% (1 in 270 midtrimester 92% and 0.9%) [6].
It is also possible to incorporate second trimester ultrasound markers. One option is to determine
three “facial profile” markers that can be measured in the same plane as the biparietal diameter: nuchal
skinfold (NF), nasal bone length (NBL), and prenasal thickness (PT). Modeling predicts that combining
these with the quad test would yield results comparable with a standard first trimester combined test. The
model-predicted Down syndrome detection rate and false-positive rate with a 1 in 250 term risk cutoff
are 87% and 1.8% (1 in 270 midtrimester 89% and 2.4%) [6].
Furthermore, so-called “soft” markers determined by the late second trimester anomaly scan,
or genetic sonogram, could be used to modify the risk. These are not very discriminatory markers
of aneuploidy, and it has been estimated that routine screening with them would only have a Down
syndrome detection rate of 69% for a false-positive rate of 5% [9]. However, some clinicians do use the
scan ad hoc in women with “borderline” risks from first or second trimester screening tests. This is
often done simplistically, whereby the presence of one or more marker is taken to be sufficient to tip the
balance in favor of invasive testing, and the absence of any markers is sufficient to contraindicate testing.
This is no longer acceptable; instead, the prior risk needs to be modified by a series of likelihood ratios
derived from each soft marker [10].
cfDNA Screening
Maternal plasma cfDNA testing is more effective in screening for Down, Edwards, and Patau syndromes
compared to conventional methods; it can also be applied to sex chromosome abnormalities (SCAs).
However, both clinicians and patients need to be aware of the limitations of the new technology. In
particular, it is misleading to consider cfDNA testing as prenatal diagnosis that could replace current
invasive testing. Indeed, when a cfDNA screening test is “positive,” CVS or amniocentesis is required
to confirm the result [11].
The most recent meta-analysis of cfDNA results includes data from 47 studies [12]. These studies
are retrospective in high-risk women with complete outcome information known—plasma samples are
mostly drawn prior to invasive prenatal diagnosis—or prospective on samples drawn in a conventional
screening program. Retrospective studies can be assumed to be substantially unbiased, but prospective
studies may overestimate the detection rate because of incomplete follow-up and “viability” bias resulting
from the inclusion of detected nonviable cases.
While there are practical issues that currently limit widespread application—economics, uninterpretable
tests (“no-calls”), patient choice, and implications for other services—different strategies are in principle
possible.
Primary cfDNA Screening

This envisions offering the test to all women. Including only retrospective studies, possibly providing a
conservative estimate of performance, the estimated Down syndrome detection rate is 99.3% and false-
positive rate 0.11%.
On the basis of the same studies, the primary cfDNA Edwards and Patau syndrome detection rates
were 97% and 90%, respectively, comparable to the incidental detection rates obtained through the
conventional combined test using only a cutoff for Down syndrome. For Turner syndrome and other
SCAs, the detection rates are 93% and 94%, respectively, much higher than those for the combined test.
However, most of the studies in the meta-analysis exclude mosaic cases, which is a particular a problem
for Turner syndrome since mosaicism is common in viable cases. Consequently, it is likely that a large
proportion of the cases studied were pregnancies destined to spontaneously abort and do not fully reflect
the more clinically important surviving cases [13].
Primary cfDNA screening for all aneuploidies together will have a false-positive rate approaching
0.8%, and this is substantially lower than the 5% rate for the combined test. The positive predictive value
(the chance of being affected given a positive result) for Down syndrome at birth is about 1 in 2, which
is much higher than 1 in 50 for the combined test.
In conventional screening, twins discordant for aneuploidy have biochemical marker levels intermediate
between concordant and unaffected twins, so there is a reduced detection rate. A similar effect is seen in
cfDNA testing, but performance is much higher than the combined test. Meta-analysis of 11 published
studies [12,14–18] yields a Down syndrome detection rate of 97%, Edwards syndrome 90%, Patau
syndrome 100%, and false-positive rate 0.06%. For the combined test, in one study, the Down syndrome
detection rate was 90% and false-positive rate 5.9% [19].
A substantial proportion of cfDNA tests fail and require a redraw. About 2% of results are no-calls
mainly due to low or borderline fetal fraction (FF), the proportion of cfDNA derived from the fetus or
placenta. The failure rate is higher in twins, in samples drawn at very early gestations, and in obese
women. A repeat sample taken a week or more later may provide an interpretable result, but in about
one-third of samples this too will be a no-call.
The increased detection rate for Down syndrome at a considerably lower false-positive rate suggests
that primary cfDNA screening should replace conventional modalities. One of the limitations, though,
is the very high unit cost of a cfDNA test. From a public health perspective, the most important financial
consideration is the “marginal” cost of avoiding a Down syndrome birth where the pregnancy would have
been missed by conventional screening. Several studies have evaluated this and found that the marginal
cost will be several times higher than the lifetime costs associated with Down syndrome unless the unit
cost falls substantially; this is already beginning to happen [20].
Secondary cfDNA Screening

In this option, cfDNA is limited to women with positive conventional screening results. Since cfDNA
tests are cheaper than invasive prenatal diagnosis, it would be at least cost-neutral. However, while the
false-positive rate will be very low, the detection rate will be lower than for conventional screening.
Seven studies have reported the uptake of cfDNA in those with positive conventional screening results
[21–27]. This rate ranged markedly between studies, but only in three did substantially more women
choose cfDNA than invasive testing.
Contingent cfDNA Screening

This is like a conventional contingent test except that the quad markers are replaced by cfDNA. The
model-predicted Down syndrome detection rate and false-positive rate are 94% and 0.02% when 20%
with the highest risk are selected for cfDNA. Even at the current unit cost of a cfDNA test, this option
is cost-beneficial [20]. Contingent cfDNA screening can be enhanced by the use of additional markers
at the time of the first trimester combined test. Adding two maternal serum markers, placental growth
factor (PlGF) and AFP, would increase the Down syndrome detection rate to 96%.
Routine CVS or Amniocentesis

The American College of Obstetricians and Gynecologists recommends that all pregnant women are
offered “screening of diagnostic testing” regardless of whether they have a specific indication [28]. While
the cost of this policy, assuming a high uptake, would be prohibitive for any public health system, it is not
unreasonable as detection of chromosomal abnormalities would be maximized. However, the hazards of
CVS and amniocentesis also need to be taken into account.
There has only been one randomized clinical trial of amniocentesis, and the difference in fetal losses
between the active and control arms was 0.8% [29]. Randomized trials comparing CVS with amniocentesis
showed a small excess in fetal losses for CVS. However, when women with positive combined tests were
randomized to either invasive procedure or cfDNA, the number of fetal losses was the same in both arms
[30]. Moreover, a recent systematic review that also included nonrandomized comparisons concluded that
both procedures have a similar fetal loss rate of 0.4% [31].
One of the problems in assessing nonrandomized data is that those having invasive prenatal diagnosis
are at different a priori risk of a fetal loss compared to those not tested. Recent studies have considered
these differences. In one study involving more than 30,000 women having first trimester Down syndrome
screening, logistic regression was used to calculate risk of fetal loss [32]. There was no statistically
significant excess of losses among 2396 women having CVS. In a national study of almost 150,000 women
in Denmark, a stratified comparison was carried out on those who did or did not receive invasive testing
following a positive combined test [33]. This analysis did not find a statistically significant excess fetal
loss rate in those having CVS or amniocentesis.
The general conclusion being drawn from these recent analyses is that in experienced hands the hazards
of invasive prenatal diagnosis are much less than in the past and might even be negligible. However, it
should be noted that in some localities the proportion of women now having cfDNA testing is increasing
so rapidly that the concomitant fall in invasive procedures being carried out is likely to substantially
reduce the number of operators with sufficient experience of CVS and amniocentesis.
Prenatal Chromosomal Microarray

The limit of sensitivity of invasive prenatal diagnosis in detecting chromosomal abnormalities is 5–10 Mb
using conventional karyotyping and 3 Mb with comparative genomic hybridization. In contrast, molecular
karyotyping using a chromosomal microarray enables the detection of submicroscopic changes or
copy-number variants (CNVs) except where there is a balanced rearrangement. In the last two decades,
considerable experience has accumulated using this technique to identify CNVs in adults and children
presenting with intellectual impairment or dysmorphic features. The same approach can now be used for
CVS and amniocentesis samples.
In a back-to-back study of a CMA and standard karyotyping in 4282 samples, CMA detected all found
by karyotyping except, as expected, balanced rearrangements and triploidies [34].
In addition, among euploid pregnancies CMA detected 2.5% with either a known pathologic phenotype
or potential for clinical significance, and 3.4% had variants of uncertain significance; with experience
over time, about half are likely to be proved benign.
The choice between cfDNA and invasive testing may be different in centers where the latter includes
CMA, which can identify known or potential clinically significant microdeletion/duplication syndromes
that are not detectable by karyotyping. Since the phenotype of many such syndromes includes physical
malformations, the CMA yield is highest for those having invasive tests for an ultrasound abnormality
[34] or an isolated increased NT above 3.5 mm [35], but it is also appreciable even when the indication is
a positive conventional Down syndrome screening test. On the other hand, CMA might reveal a variant
of unknown significance or mild phenotypes resulting in considerable anxiety and potential termination
of essentially normal pregnancies.
Extended cfDNA Tests

The main commercial providers have extended cfDNA screening to include some CNVs. All of them
now include 22q11.2 deletion syndrome. Many also include some less common but severe syndromes:
for example, Williams-Beuren (7q11), Prader-Willi and Angelman (15q11-12), Miller-Dieker (17p13),
Smith-Magenis (17p11), Wolf-Hirschhorn (4p16), cri-du-chat (5p15), Langer-Giedon (8q23-24), 1p36, and
Jacobsen (11q24.1). The ability to detect microdeletions is limited by the minimum size of the identifiable
deletion and the depth of sequencing [36] or for one method the number of informative SNPs.
Unlike for the common aneuploidies, performance is difficult to quantify. For example, the detection
rate for 22q11.2 is likely to be overestimated in retrospective studies, because of biased ascertainment of
large deletions and obvious phenotype, or in prospective studies, because some phenotypically mild cases
will not surface for years. Moreover, the retrospective estimates are based on small numbers of cases
often supplemented by synthesized samples. False-positive rates are more readily estimated, but these
can only be meaningfully compared between laboratories when the studies simultaneously estimate the
detection rates. Direct estimates of positive predictive value are subject to bias due to laboratory referral
patterns. Nevertheless, taking all available data into consideration, the literature suggests that the addition
of microdeletion syndromes will not substantially increase the false-positive rate, and the predictive value
for positive results will be comparable with that of cfDNA screening for the common aneuploidies.
Public Health Policies for High-Risk Groups

Aneuploidy screening was not initially applied to all women, but only those not already regarded as high
risk based on maternal age and family history. It was argued, most forcefully in the United States, that
those in the “traditional” high-risk groups expect to be provided with a diagnostic test and the offer of
a less definitive screening alternative was unfair. Eventually it was recognized that this hybrid policy is
inefficient, since many women with potentially low risks were receiving invasive testing, and screening
is uniformly offered.
In general, RPL is associated with an increased risk of a fetal chromosomal abnormality in
subsequent pregnancies, but this risk is not very great. A study of almost 47,000 women having invasive
prenatal diagnosis found a steady increasing trend in aneuploidy risk according to the number of
previous miscarriages [37]. After adjustment for age, parity, and the indication for testing, the odds
ratio compared to no miscarriages was 1.21, 1.26, and 1.51 for one, two, and three or more miscarriages,
respectively. For a woman aged 30 with recurrent miscarriages, this would barely increase the risk
to that of women aged 35 who are no longer considered automatic candidates for invasive testing.
However, particularly high-risk couples might be identified by cytogenetic analysis of one or more
aborted fetuses or the parents.
If a fetus has an unbalanced chromosome rearrangement, cytogenetic analysis of the parents is indicated
to determine whether one of them is a carrier of a balanced form of the rearrangement. Recurrence risk
will depend on the specific rearrangement identified, and for some abnormalities, the gender of the
carrier parent [38]. Even when an unbalanced translocation was not identified in fetal tissue, parental
karyotyping may reveal a structural abnormality, but such testing is not policy. In carrier couples,
subsequent pregnancies are more likely to end in fetal loss [39,40], although unbalanced translocations
neither account for the excess of miscarriages [41] nor do they contribute much to the overall chromosomal
abnormality risk [42].
If an aborted fetus has a common autosomal trisomy it might be assumed that, as with trisomic live
births, this confers an increased risk in subsequent pregnancies. However, there is no direct evidence for
such an effect. The presence of a prior pregnancy with an SCA does not appear to materially increase the
risk for trisomy in a subsequent pregnancy [43].
Choices for Individual RPL Couples

For those known to carry a balanced structural rearrangement, invasive prenatal diagnosis with CVS or
amniocentesis would seem to be indicated in subsequent pregnancies. However, two reported series in
such couples show that the chance of finding an unbalanced translocation is not high [40,44]. Invasive
prenatal diagnosis in 26 couples from Holland and 23 from Japan detected only one case (2.0%). The
chance of a balanced arrangement is high but given that the transmitting parent is unaffected it would
be assumed that transmission of the chromosome anomaly had no phenotypic effects. For those without
a known etiology, assuming sufficient private resources, the main choice would be between invasive
prenatal diagnosis and screening.
If the aim is to maximize detection of chromosomal abnormalities and provide early reassurance,
invasive testing will be the first choice. Concerns about the iatrogenic fetal losses associated with these
procedures can be ameliorated by the latest findings. It is likely that in experienced hands CVS and
amniocentesis will not substantially increase the fetal loss rate compared with that expected in RPL.
For couples who want to avoid altogether any additional chance of a fetal loss, among the various
screening options, cfDNA testing results are superior to other approaches and would appear to be the first
choice. This is particularly attractive when the test also includes a panel of microdeletion and duplication
syndromes.
However, there is another option, not usually available in public health programs. This is continuous
sequential screening using conventional markers and cfDNA when risk is high or even borderline.
Screening markers could be determined throughout the first and second trimesters to assess and
reevaluate risk and provide reassurance. Where possible, risk would be calculated and revised using all
possible markers, not just those routinely available, including (i) first trimester ultrasound NB, TR, DV,
serum PlGF, AFP; (ii) early second trimester NF, NBL, PT; and (iii) late second trimester soft markers.
In addition to Down and Edwards syndromes, risks should be calculated and revised for all types of
aneuploidy.
A sequential screening protocol designed to continuously reassure women with recurrent pregnancy
about their aneuploidy risk will necessarily lead to a higher overall false-positive rate as the positive
results accumulate. The cutoff risks used in public health screening are chosen to predict the use of
resources, although in practice there is often less than strict adherence to the cutoff, which is merely
taken to be a guide to action. In a sequential screening situation, lower cutoffs than used in population
screening might be considered. If the next step following increased risk is cfDNA testing, the need for
invasive prenatal diagnosis might still be acceptable, but if an extreme ultrasound marker such as very
high NT or significant cardiac defect is found, invasive testing with a CMA may be indicated.
Conclusions
In recent years, public health screening programs for fetal chromosome abnormalities have become
increasingly effective, although provision may vary considerably between localities. In parallel with
this development, invasive prenatal diagnosis has become both safer and more comprehensive. Couples
with RPL are at increased risk of a pregnancy affected by a chromosomal abnormality, but this is not
sufficiently great to warrant a screening protocol different from the general population. Nonetheless, given
the heightened need for reassurance, couples may prefer to choose a screening or diagnostic modality that
offers maximum detection with minimum risk. In these cases, policy of continuous sequential evaluation
could also be undertaken.
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18
Obstetric Outcomes after Recurrent Pregnancy Loss
Rakefet Yoeli-Ullman, Howard J.A. Carp, and Shali Mazaki-Tovi
Introduction
Recurrent pregnancy loss (RPL) is one of the most challenging conditions in reproductive medicine for
patients and physicians alike. The overwhelming majority of research conducted in this field explores
the etiologies, methods of prediction, and treatment for prevention of this frustrating condition. The
conventional view is that once the patient with previous RPL passes the first trimester, the risk of
major complications is not significantly increased. However, over the last decade evidence has emerged
demonstrating a link between RPL and several complications of pregnancy including preterm birth, pre-
eclampsia, fetal growth restriction, gestational diabetes mellitus, fetal anomalies, placental abruption, and
perinatal mortality. The rationale for this association hinges on several observations: (i) Both recurrent
pregnancy loss and several complication of pregnancy share similar risk factors and predisposing
conditions, (ii) observational studies have highlighted the relationship between these two conditions,
(iii) epidemiological studies have identified recurrent pregnancy loss as a significant and independent
risk factor for several complications of pregnancy, and (iv) similar treatments have been shown to be
effective for both recurrent pregnancy loss and some of the complications of pregnancy. This chapter
presents the available evidence for the association between recurrent pregnancy loss and several common
and important complications of pregnancy, to suggest a plausible explanation(s) for this association and
to critically appraise the literature.
Spontaneous Preterm Labor

Contradicting evidence exists regarding the association between spontaneous preterm birth and recurrent
pregnancy loss (Table 18.1). Several studies do not distinguish between induced and spontaneous preterm
birth. Furthermore, both RPL and several complications of pregnancy that are frequently managed with
iatrogenic preterm induction of labor (e.g., preeclampsia, fetal growth restriction, etc.) share the same risk
factors, thus adding an additional layer of complexity in the attempt to decipher the genuine relationship
between these conditions.
Hughes et al. [1] examined the obstetric outcome in 88 women with a past history of three or more
consecutive pregnancy losses and compared the results to a control group drawn from their local obstetric
population. The rate of preterm delivery (12.5%) was no different than the control group. Sheiner et al. [2]
conducted a population-based study in which all singleton pregnancies were assessed in women with and
without two or more consecutive recurrent miscarriages. The study included 154,294 singleton deliveries.
Of these deliveries, 7503 occurred in patients with recurrent miscarriage. Spontaneous preterm birth
did not differ significantly between the two groups, although the rate of preterm premature rupture of
membranes (PPROM) was significantly higher in the study group compared to controls (6.5% vs. 5.6%,
respectively; p < 0.001).
Two randomized trials of special note were conducted in which the rate of preterm birth was relatively
low compared to the general population. Schleussner et al. [3] conducted a multicenter, randomized
controlled trial including 449 women with at least two consecutive early miscarriages or one late
miscarriage. Women in the intervention group received 5000 IU of dalteparin sodium for up to 24 weeks’
172
TABLE 18.1
Association between RPL and Spontaneous Preterm Labor
Study Group (n) Control (n) PTL in Study Group (%) PTL in Controls (%) p Comments
Hughes et al. [1] 88 12,590 11/88 (12.5%) 1075/12,590 (8.5%) NS
Cozzolino [40] 53 65 6/53 (11.3%) 1/65 (1.5%) p = 0.05 OR 8.17; CI 0.95−70.1
Sheiner [2] n = 7503 n = 146,791 6.5% 5.6% p < 0.001 PPROM
Schleussner [3] Total = 449 No control Total: 38/449 (8.5%) No control
Study group (dalteparin) n = 226 Study group: 20/226 (8.8%)
Control (no treatment) n = 223 Control: 18/223 (8%)
Kaandorp [4] Total ongoing pregnancies n = 200 No control Total: 11/200 (5.5%)
Aspirin+ nadroparin n = 69 Aspirin+ nadroparin: 7/69
Aspirin n = 61, Placebo n = 70 (10.1%)
Aspirin: 1/61 (1.6%)
Placebo: 3/70 (4.3%)
Shapira [47] No control Total: 47/306 (15.3%)
Total n = 306
Primary RPL n = 123 Primary RPL: 26/123 (21.1%)
Secondary RPL n = 183 Secondary RPL: 21/182
(11.5%)
Reginald [5] n = 175 Normal population 28% p < 0.05
Tulppala [6] n = 63 No control 9.7%
Jivraj [7] n = 162 n = 24,699 22/162 (13%) 959/24,699 (3.9%) p < 0.01
Thom [8] n = 583 n = 2820 63/583 (11.1%) 220/2820 (7.8%) RR 1.5; 95% CI 1.1−2.1
Brown [16] One previous miscarriage n = 6105 n = 44,308 One: OR 1.7; CI 1.52−1.83
Two previous miscarriages n = 1813 Two: OR 2.0; CI
≥3 previous miscarriages n = 978 1.73−2.37
>3: OR 3; CI 2.47−3.70
Hammoud [17] One previous miscarriage n = 5973 n = 52,280 One: 527/5973 (8.8%) 3552/52,280 (6.8%) p < 0.001
Two miscarriages n = 908 Two: 90/908 (9.9%)
Three or more miscarriages n = 225 Three or more: 32/225
(14.2%)
173
gestation. The rate of preterm birth was 8.8% in the study group and 8% in the controls (p = 0.24), a
rather low rate by any standard. Similarly, Kaandorp et al. [4] reported the results of a randomized trial
that included 364 women who had a history of unexplained recurrent miscarriage and were attempting to
conceive or were less than 6 weeks pregnant. Participants were randomly allocated to receive 80 mg of
aspirin plus subcutaneous nadroparin daily, 80 mg of aspirin alone, or placebo. The rate of preterm birth
was 10.1%, 1.6%, and 4.3%, respectively. In a pooled analysis, the rate of preterm birth was only 5.5%, a
very low rate for the general population in the Netherlands, where the study was conducted.
In contrast, others have found an association between preterm birth and recurrent pregnancy loss.
Reginald et al. [5], in a retrospective observational cohort study, assessed the results of 175 pregnancies
in 97 recurrently miscarrying women whose subsequent pregnancy progressed beyond 28 weeks. The
results were not compared with a control group attending the same hospital, but with standard figures from
Scotland between the years 1973–1979. A significantly higher prevalence of preterm deliveries was found.
Tulppala et al. [6] conducted a prospective study of 63 women with recurrent miscarriage and presented
the results of a detailed investigative protocol, including antiphospholipid syndrome. The rate of preterm
delivery (9.7%) appeared to be increased. Unfortunately, the results were not compared to any control
population. Jivraj et al. [7] studied a cohort of 162 women with recurrent miscarriage compared to the
local control population from 1992 to 1998. Among a total of 162 pregnancies that progressed beyond 24
weeks gestation in women with a history of recurrent miscarriage, there were 22 (13%) preterm deliveries
compared with 959 (3.9%) in the 24,699 controls (p < 0.01). Thom et al. [8] analyzed Washington State
birth certificate records for 1984–1987 to examine the association between spontaneous abortion, recurrent
miscarriage, and adverse outcomes in the subsequent live birth. The results of 638 women with three or
more miscarriages were compared to those of women with no prior spontaneous abortions (n = 3099).
Women with recurrent pregnancy loss had a higher risk of delivery at less than 37 weeks’ gestation (relative
risk 1.5; 95% confidence interval [CI ] 1.1–2.1). Importantly, several epidemiological studies have identified
recurrent preterm birth as a significant risk factor for preterm birth [9–15].
Two more recent studies have supported the association between preterm birth and recurrent pregnancy
loss by demonstrating a “dose-dependent effect.” Brown et al. [16] examined live singleton births using
data from the United States Collaborative Perinatal Project. Compared with women with no history of
miscarriage, women who had one, two, or three or more previous abortions were 1.7 (95% CI 1.52–1.83),
2.0 (95% CI 1.73–2.37), and 3.0 (95% CI 2.47–3.70) times more likely to have preterm birth (defined
as delivery <37 weeks of gestation), respectively. These results remained significant after control for
obstetric and medical history and lifestyle and demographic factors. Hammoud et al. [17] analyzed data
from the perinatal database collected from the state of Schleswig-Holstein, Germany. During the years
1991–1997 there were 170,254 deliveries and 59,386 nulliparas with singleton pregnancies. Among the
59,386 (38%) nulliparous patients included, 5973 (10.1%) had a history of one miscarriage, 908 (1.5%)
had a history of two previous miscarriages, and 225 (0.4%) had a history of three or more previous
miscarriages. The risk of preterm delivery increases with the increasing number of previous spontaneous
miscarriages. Compared to women with no history of miscarriages (3552/6.8%), women who had one
(527/8.8%), two (90/9.9%), or three (32/14.2%) previous abortions were more likely to have preterm
birth (p < 0.001). A similar increase was found in the rate of PPROM: controls: 1354/2.6% versus one
192/3.2%, two 46/5.1%, and three 15/6.7% previous miscarriages (p < 0.001). Logistic regression analysis
was performed correcting for smoking status, maternal age, and obesity. Patients with a history of three
or more previous miscarriages had a risk of preterm delivery of more than twice that of women with no
such history (OR 2.46; CI = 1.68–3.60).
Although the cause of the association between recurrent pregnancy loss and preterm birth has not been
elucidated, there are several possible explanations. Uterine evacuation, by either mechanical dilatation or
osmotic dilatation of the cervix, may explain the association. Saccone et al. [18] reported the results of a
systematic review and meta-analysis of 36 studies (1,047,683 women). Women with a history of uterine
evacuation had a significantly higher risk of preterm birth (5.7% vs. 5.0%; OR 1.44; 95% CI 1.09–1.90)
than controls. The authors concluded that a prior surgical uterine evacuation is an independent risk factor
for preterm birth. Similar findings were reported by Lemmers et al. [19] from a systematic review and
meta-analysis of 21 studies including 1,853,017 women. Lemmers et al. [19] asked whether dilatation and
curettage (D&C) increases the risk of subsequent preterm birth. D&C increased the odds ratio (OR) for
Obstetric Outcomes after Recurrent Pregnancy Loss 175
preterm birth. The OR was 1.29 (95% CI 1.17–1.42) if a 37-week cutoff was used. This risk was 1.69 (95%
CI 1.20–2.38) for 32 weeks and 1.68 (95% CI 1.47–1.92) for 28 weeks of gestation. The risk remained
increased when the control group was women with medically managed miscarriage or induced abortion
(OR 1.19; 95% CI 1.10–1.28). When women with multiple D&Cs were compared to women with no D&C,
the OR for preterm birth (>37 weeks) was 1.74 (95% CI 1.10–2.76). For spontaneous preterm birth, the
OR was 1.44 (95% CI 1.22–1.69). Collectively, these data may suggest a causal relationship between
prior surgical uterine evacuation and subsequent preterm birth. Additional risk factors that have been
implicated in both RPL and preterm birth are uterine malformation [20] and infections (i.e., bacterial
vaginosis and endocervical infections) [21].
Preeclampsia and Pregnancy-Induced Hypertension

population (see Table 18.2). The rate of preeclampsia appeared to be similar in both groups (2.3% vs. 2.6%,
respectively). Similarly, Jivraj et al. [7], who studied a cohort of 162 women with recurrent miscarriage
compared to local controls, found no difference in the rate of pregnancy-induced hypertension or
preeclampsia (6.7%) between the two groups. Sheiner et al. [2] reported a similar rate of preeclampsia in
7503 pregnant women with RPL and 146,791 controls (3.5% in both groups; OR 1; 95% CI 0.9–1.2). Of
note, there was a significant difference in the rate of severe preeclampsia between the pregnant women
with and without RPL (1.6% vs. 1.1%; OR 1.5; 95% CI 1.3–1.8; p < 0.001)
Consistent with these reports, several recent multicenter randomized trials have found a low rate of
preeclampsia in patients with RPL. Schleussner et al. [3] reported that the rate of preeclampsia was 3/226
women in the intervention group (treated with 5000 IU of dalteparin sodium) and 6/223 in the controls
(1.3% vs. 2.6%; p = 0.74). Kaandorp et al. [4] reported that the rate of preeclampsia was 2.9%, 1.6%, and
1.4% in pregnant women with a history of unexplained recurrent miscarriage treated with daily 80 mg
of aspirin plus nadroparin, 80 mg of aspirin alone, or placebo, respectively. Of note, there was only one
case of HELLP syndrome out of 364 participants.
Trogstad et al. [22] conducted a cohort study based on the Norwegian Mother and Child Cohort Study,
a large population-based pregnancy cohort. The sample consisted of 20,846 singleton pregnancies in
nulliparous women between the years 1999–2005. The authors found an increased risk of preeclampsia
following three or more miscarriages but not after one or two miscarriages. The association was
statistically significant only when combined with a history of infertility treatment (OR 2.4; 95% CI
1.11–5.18). Further support for the association between RPL and preeclampsia came from Weintraub et al.
[23] who reported a case-control study designed to evaluate the prevalence of pregnancy complications in
a live birth preceding the appearance of recurrent miscarriages. Women who had at least two consecutive
spontaneous miscarriages after one live birth (n = 58) were matched with controls, women without
recurrent miscarriages (n = 232). A statistically significant higher rate of preeclampsia (mild and severe)
was found in a live birth preceding recurrent miscarriages than in the matched controls (10.3 vs. 3.9%;
p = 0.04).
The association between preeclampsia and unexplained recurrent miscarriages may be due to
endothelial dysfunction. Germain et al. [24] determined brachial arterial reactivity and factors related to
endothelial dysfunction, such as circulating cholesterol, uric acid, nitrites, L-arginine, vascular endothelial
growth factor, and soluble vascular endothelial growth factor receptor-1, in women with previous healthy
pregnancies (n = 22), patients with severe preeclampsia (n = 25), and patients with RPL (n = 29). A
significant decrease in endothelium-dependent dilatation was found in the preeclampsia (40%) and RPL
(45%) groups compared to the controls (4.5%). In addition, lower serum nitrites and higher cholesterol
were found in both study groups as compared to control subjects.
An additional putative mechanism linking preeclampsia and recurrent pregnancy loss is obesity. Obesity
has been associated with both recurrent pregnancy loss [25,26] and preeclampsia [27,28]. A two- to fourfold
higher prevalence of preeclampsia has been reported in obese patients [29]. A meta-analysis focusing on
maternal BMI and preeclampsia has shown that the risk of preeclampsia is doubled with each 5–7 kg/m2
176
TABLE 18.2
Association between RPL and Preeclampsia/Pregnancy-Induced Hypertension
Rate of PET/PIH in Study Rate of PET/PIH
Study Group (n) Control (n) Group (%) in Control (%) p Comments
Hughes et al. [1] 88 N = 15,590 2/88 (2.3%) 333/15,590 (2.1%) NS PET
Jivraj [7] 162 N = 24,699 13/162 (8%) 2643/24,699 (10.7%) NS Hypertensive related disorders
Sheiner [2] 7503 N = 146,791 Mild PET: 3.5% Mild PET 3.4% NS
Severe PET: 1.6% Severe PET 1.1% P < 0.001
Schleussner [3] Total: 449 No control Total: 9/449 (2%) PET
Dalteparin: 226 Study group: 3/226 (1.3%)
Control (no RX): 223 Control: 6/223 (2.6%)
Kaandorp [4] Ongoing pregnancies: 200 No control Total: 4/200 (2%) PET
Aspirin+ nadroparin: 69 Aspirin+ nadroparin: 2/69 (2.9%)
Aspirin: 61 Aspirin: 1/61 (1.6%)
Placebo: 70 Placebo: 1/70 (1.4%)
Shapira [47] Total: n = 306 No control Total: 20/306 (6.5%)
Primary RPL n = 123 Primary RPL: 9/123 (7.4%)
Secondary RPL n = 183 Secondary RPL: 11/183 (6%)
Trogstad [22] Total: 3159 N = 17,687 Total: 165/3159 (5.2%) 956/17687 (5.4%) NS
One miscarriage: 2556 One: 133/2556 (5.2%)
Two miscarriages: 473 Two: 21/473 (4.4%)
>3 miscarriages: 130 Three: 11/130 (8.5%)
Cozzolino [40] 53 65 PIH: 2/53 (3.8%) PIH 2/65 (3.1%) NS
PET: 1/53 (1.9%) PET 1/65 (1.5%) NS
Weintraub [23] 58 232 6/58 (10.3%) 9/232 (3.9%) P = 0.04 PET in the pregnancy preceding RPL
increase in BMI [30]. Several explanations have been proposed to explain the association between obesity
and RPL: (i) Excess adipose tissue can produce a hypoxic state by increasing the concentrations of
glycosylated hemoglobin and decreasing the affinity for oxygen. This relative hypoxemia may cause
abnormal placentation leading to miscarriage in severe cases and preeclampsia if more moderate [31].
(ii) Subclinical inflammation is a hallmark of obesity, and an exaggerated inflammatory response may
predispose women to both preeclampsia and recurrent pregnancy loss. (iii) Obese pregnant women have a
three- to tenfold higher risk of preexisting hypertension or diabetes compared to those of normal weight.
Finally, several common and important pathophysiological mechanisms and predisposing factors have
been implicated in both conditions including antiphospholipid syndrome [32], thrombophilia [33,34],
endocrine disorders [35], fetal and maternal genetic mismatch [36], and immunologic abnormalities
[37,38].
Intrauterine Growth Restriction and Small for Gestational Age

population (see Table 18.3). The prevalence of small for gestational age (SGA) infants (3.4%) was no
different than the control group. Sheiner et al. [2] conducted one of the largest population-based studies
regarding the association between RPL and intrauterine growth restriction (IUGR)/SGA. This study
included 7503 patients with recurrent miscarriage and 146,791 controls. The rate of IUGR was identical
(2%) in both groups.
As with other complications of pregnancy, there is evidence to support the association between IUGR
and RPL. Reginald et al. [5], in a retrospective observational cohort study, assessed the results of 175
pregnancies in 97 recurrently miscarrying women whose subsequent pregnancy progressed beyond 28
weeks. The results were compared with standard figures from Scotland between the years 1973–1979.
They reported a 30% incidence of intrauterine growth restriction representing a relative risk of 3 compared
to the standard Scottish population. Tulppala et al. [6] conducted a prospective study in 63 women with
recurrent miscarriage. The results were not compared to any control population. The prevalence of fetal
growth restriction (20%) appeared to be increased. Similarly, Thom et al. [8] quoted a relative risk (RR)
of 2.0 (95% CI 1.4–2.8) for IUGR.
Basso et al. [9] analyzed a Danish population-based registry consisting of 45,449 women having a
live birth preceded by one or more spontaneous miscarriages and a random sample of 9752 women
with two consecutive live births. They reported an increased risk for IUGR in patients with two or more
miscarriages (OR 1.4; 95% CI 1.2–1.6). Similarly, Jivraj et al. [7], who studied a cohort of 162 women
with recurrent miscarriage compared to local controls, found a significant difference in the rate of SGA
in patients with or without RPL (13% vs. 2.1%, respectively).
Preeclampsia SGA or IUGR may have similar causes, including failure of physiologic transformation
of the spiral arteries, an antiangiogenic state, endothelial cell dysfunction, and an increased maternal
intravascular inflammatory response. Thus, the same factors that predispose patients with RPL to
developing preeclampsia may affect fetal growth and cause IUGR. Notably, it has been proposed that the
presence of altered metabolic states including obesity, insulin resistance, and dyslipidemia predisposes
pregnant women to develop preeclampsia, while the absence of these metabolic alterations will result in
an SGA or IUGR neonate [39].
Gestational Diabetes Mellitus

Cozzolino et al. [40] performed a retrospective cohort study investigating adverse pregnancy outcomes
in women with RPL (n = 53) compared to a control group of couples attending a low-risk antenatal unit
(n = 65) (see Table 18.4). Women with previous RPL had a significantly increased risk of gestational
diabetes with 12 cases (22.6%) in the study group and 3 cases (4.6%) in the control group (OR 6.04; 95%
CI 1.60–22.76; p = 0.007). Romero et al. [41] used fructosamine as an indicator of mean blood glucose
178
TABLE 18.3
Association between RPL, IUGR, and SGA
IUGR in
Study Group (n) Control (n) IUGR in Study Group Controls p Comments
Hughes et al. [1] 88 12,590 3/88 (3.4%) 180/12,590 (1.4%) NS
Sheiner [2] 7503 146,791 2% 2% NS
Cozzolino [40] 53 65 4/53 (7.5%) 3/65 (4.6%) NS
Schleussner [3] Total: 449 No control Total: 11/449 (2.4%) IUGR or placental
Dalteparin: 226 Dalteparin: 5/226 (2.2%) insufficiency
Control (no RX): 223 Control: 6/223 (2.6%)
Kaandorp [4] Total ongoing No control Total: 18/200 (9%)
pregnancies: 200 Aspirin+ nadroparin:
Aspirin+ nadroparin: 69 6/69)8.7%(
Aspirin: 61 Aspirin: 7/61 (11.5%)
Placebo: 70 Placebo: 5/70 (7.1%)
Shapira [47] Total: 306 No control Total: 14/306 (4.5%)
Primary RPL: 123 Primary RPL: 11/123 (8.9%)
Secondary RPL: 183 Secondary RPL: 3/183 1.6%)
Reginald [5] 175 Normal obstetric 30% Prevalence of IUGR was 3
population times higher in study group
compared to controls
Tulppala [6] 63 No controls 20%
Thom [8] 631 N = 3065 60/631 (9.5%) 141/3065 (4.6) RR 2.0, CI 1.4–2.8
Basso [9] 45,449 9752 Increased risk for IUGR with
≥2 miscarriages
OR 1.4; CI 1.2–1.6
Jivraj [7] 162 24,699 21/162 (13%) 523/24,699 (2.1%) <0.001
TABLE 18.4
Association between RPL and Gestational Diabetes Mellitus
Study Group (n) Control (n) GDM in Study Group GDM in Controls p Comments
Cozzolino [40] 53 65 12/53 (22.6%) 3/65 (4.6%) P = 0.007 OR 6.04; CI 1.60−22.76
Romero [41] 117 117 3/117 (2.5%) 0/117 NS GDM defined according
to fructosamine levels
Vaquero [42] Total n = 82 No control Total 5/63 (7.9%)
Prednisone and aspirin Prednisone and aspirin 3/22 (14%)
n = 29 IVIG 2/41 (5%)

IVIG n = 53
Shapira [47] Total n = 306 No control Total 43/306 (14%)
Primary RPL n = 123 Primary RPL 24/123 (19.5%)
Secondary RPL n = 183 Secondary RPL 19/183 (10.4%)
Hughes [1] N = 88 N = 12,590 15/88 (17%) 359/12,590 (2.8%) P < 0.05
Sheiner [2] N = 7503 N = 146,791 8.6% 4.3% P < 0.001
Tulppala [6] N = 63 No control 22.8%
Jivraj [7] N = 162 N = 24,699 3/162 (1.8%) 198/24,699 (0.8%) NS
179
in a study including 117 women with unexplained RPL, and no more than one live birth, and 117 age-
matched controls with at least one full-term uncomplicated pregnancy and no more than one pregnancy
loss. The mean fructosamine concentration was higher in women with RPL (224.1 ± 28.79 µmol/
mL) compared with controls (188.9 ± 19.3 µmol/mL, P < 0.001). This difference persisted when RPL
patients and controls were stratified according to BMI. However, the proportion of women with elevated
fructosamine (>285 µmol/L) was similar in RPL patients and controls.
Vaquero et al. [42] reported the results of a prospective, two-center trial study that included 82 women
with RPL and antiphospholipid syndrome. Twenty-nine were treated with prednisone and low-dose
aspirin in one center and 53 received IVIG in the other center. In the prednisone plus low-dose aspirin-
treated patients, gestational diabetes was found significantly more often than in the IVIG-treated group
(14% vs. 5%, p < 0.05). Clearly one cannot exclude the diabetogenic effect of prednisone to account for
this finding. The association between RPL and gestational diabetes has also been reported by Hughes
et al. [1] (study group 17% vs. 2.8% in controls, p < 0.05) and Sheiner et al. [2] (study group 8.6% vs.
4.3% in controls, p < 0.001).
The molecular mechanism(s) that may account for the association between RPL and gestational
diabetes has not been fully determined. However, an interesting publication may shed new light on this
relationship. Andraweera et al. [43] investigated the association of the fat mass and the obesity associated
gene (FTO) rs9939609 single nucleotide polymorphism with recurrent miscarriage. This was a candidate
gene association study including 202 Sinhalese women with two or more first trimester miscarriages and
no living children and 202 age- and ethnicity-matched women with no history of miscarriage and having
two or more living children. The prevalence of the AA genotype and A allele of the FTO rs9939609 single
nucleotide polymorphism was increased in women with recurrent miscarriage compared with the controls
(AA: OR 3.8; 95% CI 1.8–8.0; p = 0.0002; A: OR 1.6; 95% CI 1.2–2.2; p = 0.002).
Fetal Anomalies
The data regarding the association between RPL and congenital anomalies are scarce. Thom et al.
[8] examined the Washington State birth certificate records and included 638 women with three or
more miscarriages and a control group of women with no prior miscarriages (n = 3099). Women with
RPL had a higher risk of delivering a child with congenital malformations (RR 1.8; 95% CI 1.1–3.0).
The Recurrent Miscarriage Immunotherapy Trialists Group trial [44] showed an anomaly rate of 4%,
which is higher than expected in the general population. Schoenbaum et al. [45] reviewed 5003 records
of consecutive deliveries in 1975 and 1976 at the Boston Hospital for Women and analyzed singleton
deliveries at 27 weeks’ gestation or greater. They compared women with exactly one prior proximate
induced or spontaneous abortion with women of similar gravidity or parity with no prior pregnancy
losses. The offspring of women with a proximate miscarriage had an increased incidence of congenital
malformations. Finally, there are a few case reports reporting that chromosomal aberrations lead to both
RPL and fetal anomalies [46]. Today, with the advances in ultrasonic detection of fetal malformations,
many patients elect to terminate the pregnancy. Consequently, today the incidence of anomalies at birth
may not be higher than in the general population.
Placental Abruption
Only two case-control studies have compared the incidence of placental abruption in patients with and
without RPL. Neither Thom et al. [8] nor Sheiner et al. [2] found an increased risk of placental abruption in
women with RPL. However, if both studies are pooled the common odds ratio for abruption is significant:
5.8 (CI 5.1–6.6).
In contrast to the report of Sheiner et al. [2] and the aforementioned pooled analysis, two multicenter
randomized trials have found a very low prevalence of placental abruption in patients with RPL. Schleussner
et al. [3] reported that the prevalence of placental abruption was 0/226 women in the intervention group
(treated with 5000 IU of dalteparin) and 1/223 in the controls (0% vs. 0.4%; p = 0.5). Kaandorp et al.
[4] did not find a single case of placental abruption in a study that included of 364 patients with RPL. Of
note, Kaandorp et al. [4] excluded patients with aPL from the study.
The group of Shapira et al. [47], in a retrospective cohort study of 420 patients with two or more
consecutive pregnancy losses, found no significant difference in the incidence of abruption in the
subsequent (index) pregnancy in 162 primary aborters and 258 secondary aborters (2.4% and 0.5%,
respectively; p = 0.3).
Perinatal Mortality
Only a few studies have reported on the association between prenatal death and RPL, and none has
reported an increased risk of this complication. Hughes et al. [1] reported that perinatal mortality rate
was 0/88 (0%) in women with a past history of three or more consecutive pregnancy losses and 0.46%
in the control group. Jivraj et al. [7] reported a perinatal mortality rate of 2/162 (1.2%) in women with
recurrent miscarriage compared to 247/24,699 (1.0%) in controls. Importantly, Jivraj et al.’s [7] study only
included pregnancies that progressed beyond 24 weeks’ gestation. Sheiner et al. [2] conducted the largest
population-based study addressing the association between RPL and perinatal death. The prevalence
of perinatal mortality was 1.7% among 7503 patients with recurrent miscarriage and 1.4% in 146,791
controls (p = 0.12).
Van Oppenraaij et al. [49] reported the results of several studies in which the rate of perinatal mortality
was determined in women with a single miscarriage. These studies found an increased risk of intrauterine
fetal death (OR 1.9; 95% CI 1.1–3.6) and an increased risk of neonatal death (OR 2.2; 95% CI 1.1–4.8)
[8,45,48]. As previously mentioned, this higher risk for perinatal mortality was not found in studies
including patients with two or more miscarriages.
Conclusions
Patients with RPL seem to be at increased risk for developing several complications of pregnancy.
Nevertheless, this conclusion should be interpreted with caution because (i) the syndromic nature of
RPL (i.e., multiple etiologies and numerous underling mechanisms of disease) critically hampers the
external validity of many studies, (ii) the available data are insufficient to claim cause and effect, (iii) the
literature is sparse and the findings in the literature are inconsistent, and (iv) there are no interventional
studies comparing patients with and without RPL to determine whether treatment for the prevention of
gestational complications is effective in this set of patients. Despite these limitations, we recommend
active prenatal care and not to regard these patients as “low risk.” The role of preventative treatment for
obstetric complications (e.g., low-dose aspirin for the prevention of preeclampsia, or frequent sonographic
cervical length measurements to prevent preterm labor) is debatable. Prospective and interventional,
well-designed studies are necessary in order to identify RPL patients at increased risk for obstetric
complications and provide individualized and effective treatment.
Acknowledgment
We thank Ms. Maya Mazaki-Tovi for reviewing the manuscript for grammar, style, and language.
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19
Investigation Protocol for Recurrent Pregnancy Loss
Howard J.A. Carp
Introduction
In the first and second editions of this book, three guidelines for management of recurrent pregnancy
loss (RPL) were compared and contrasted: The protocol of the Royal College of Obstetricians and
Gynaecologists (RCOG) [1], the American College of Obstetricians and Gynecologists (ACOG) [2],
and the European Society of Human Reproduction and Embryology (ESHRE) [3]. The International
Federation of Gynecology and Obstetrics (FIGO) is also about to issue a guideline that will summarize
some of the features in the preceding guidelines. In addition, there are numerous other guidelines from
various national societies. Unfortunately, the various guidelines differ in their definition of RPL, inclusion
criteria for investigation and treatment, investigations, and management recommendations. The various
protocols classify RPL as one homogeneous condition. However, treating RPL as one homogeneous
condition does not take into account individual circumstances in different patients. The prognosis is
different in different patients. The author classifies patients into those with a good, medium, or poor
prognosis (Table 19.1). Saravelos and Li [4] classify patients as Type 1, in which RPL occurs by chance
and there is no underlying pathology and a good prognosis, and Type II, which occurs due to an underlying
pathology that is currently not yet identified by routine clinical investigations and with a poorer prognosis.
We are of the opinion that there may not be just one approach to treatment. Chapter 3 shows the differences
in approach, whether all patients are taken as one homogeneous group or whether treatment is tailored to
the individual diagnosis and needs of the specific patient. In this chapter, some of the standard protocols
are discussed along with some other approaches that may be appropriate in particular patients.
The Different Guidelines

The guidelines vary, as they were written by different groups of investigators and thought to be appropriate
in their local settings. The RCOG [1] protocol has to consider the availability of treatment in the United
Kingdom, bearing in mind that the major provider is the National Health Service (NHS). If investigations
the RCOG considers unnecessary in the majority of patients are recommended, the NHS will not be able
to cope with the costs. The RCOG is also cognizant that recommended treatments in its protocol form
the basis of the subsidy provided by the NHS. Therefore the protocol recommends the investigations and
treatments considered to be most cost effective.
The American Society for Reproductive Medicine (ASRM) [2] guideline is directed toward current
practice in the United States. The United States is a very litigious society. Physicians have been sued for
not treating according to the guidelines, and the courts have assumed guidelines to be a set of instructions
rather than a guideline. The ASRM guideline is very liberal in its approach, and not as dogmatic as the
RCOG guideline. The ASRM is cognizant not to recommend or exclude features that may be used in
litigation.
The ESHRE [3] guideline was written by a group of investigators from various European nations, each
with different philosophies in health care. The guideline is more dogmatic than the ASRM guideline in
stating that various treatment modalities are not recommended. However, “not recommended” means that
184
Investigation Protocol for Recurrent Pregnancy Loss 185
TABLE 19.1
Relative Prognoses according to Clinical Features
Good Prognosis Medium Prognosis Poor Prognosis
Number of miscarriages 2 3 4 5 6 7 8 9
Age 20s 30s 40s
Embryo aneuploidy Aneuploid Euploid Euploid
1° or 2° Aborter 2° 1° or 3° 1° or 3°
Early or late losses Early Early Late
Infertility Normal fertility Infertility
NK cells Normal High
ESHRE does not consider the evidence sufficient to make a positive recommendation. Not recommended
does not mean contraindicated.
The proposed protocol of FIGO will be based on the recommendations of the preceding three protocols.
However, FIGO will have to be cognizant that its recommendations are directed toward the developing
world, where resources are much more limited. FIGO will have to set a standard for recommending
certain investigations and treatment that many nations of the developing world will have to strive to
achieve. Therefore FIGO’s recommendations may again be different from the three established protocols.
RCOG Guideline
RCOG protocol [1] attempts to be evidence based as far as possible. Evidence is classified as in Table 19.2.
Recommendations are made about investigating various causes of miscarriage, and methods of treatment
are graded according to the level of evidence available. Areas lacking evidence are called “good practice
points,” based on the clinical experience of the guideline development group. The evidence is mainly
taken from the Cochrane Register of Controlled Trials. The guideline recommends fetal karyotyping,
three-dimensional ultrasound, hydrosonography or hysteroscopy for uterine anomalies, antiphospholipid
syndrome (APS) testing and interpretation according to the updated “Sydney criteria” [5], and treatment
with heparin and aspirin. Interestingly, parental karyotyping is not recommended except when an
unbalanced chromosome abnormality is identified in the products of conception. The rationale is that
there is only a 2% yield for a balanced parental rearrangement and only a 0.8% chance of an unbalanced
translocation if detected. Hence it was considered not to be cost effective to screen. The protocol
claims that there is insufficient evidence to assess progesterone and human chorionic gonadotrophin
(hCG) supplementation and bacterial vaginosis. Assessment of thyroid function, antithyroid antibodies,
alloimmune testing and immunotherapy, and assessment of TORCH and other infective agents are not
recommended. The protocol reserves judgment on the hereditary thrombophilias, claiming that there may
be an association with second trimester miscarriage but not first trimester miscarriage. The guideline
states that a significant proportion of cases of recurrent miscarriage remain unexplained, despite detailed
investigation, and that the prognosis for a successful future pregnancy with supportive care alone is in the
TABLE 19.2
Levels of Evidence
Ia Evidence obtained from meta-analysis of randomized controlled trials
Ib Evidence obtained from at least one randomized controlled trial
IIa Evidence obtained from at least one well-designed controlled study without randomization
IIb Evidence obtained from at least one other type of well-designed quasi-experimental study
III Evidence obtained from well-designed non-experimental descriptive studies, such as comparative studies,
correlation studies, and case studies
IV Evidence obtained from expert committee reports or opinions and/or clinical experience of respected authorities
region of 75%. However, the guideline does state that the prognosis worsens with increasing maternal age
and the number of previous miscarriages.
ASRM Guideline
The ASRM guideline [2] is much less dogmatic than the RCOG guideline. Two pregnancy losses are
recognized as warranting investigation. The ASRM guideline does not base its recommendations on a
strictly evidence-based approach and does not state that new and controversial etiologies should not be
investigated or treated. Various suspected causes of RPL are either recommended, not recommended, or
claimed to be of doubtful value. Like the RCOG guideline, the ASRM guideline does not take account
of different types of patients, or different prognoses; it does state clearly that it should not be construed
as dictating an exclusive course of treatment or procedure. The guideline also states that variations in
practice may be warranted based on the needs of the individual patient, resources, and limitations unique
to the institution or type of practice. Unlike the RCOG guideline, the ASRM guideline recommends
parental karyotyping and suggests that the couple should be offered prenatal diagnosis if one parent has
a chromosomal aberration, but genetic assessment of the abortus is not recommended. The guideline
states that assessment of the uterine cavity is advised and supports resection of a septum, although
the contribution of uterine septa to first trimester loss is claimed to be controversial. The resection
of intrauterine adhesions and polyps is also said to be controversial without good evidence of effect.
Screening is recommended for antiphospholipid antibodies, and treatment with aspirin and unfractionated
heparin is recommended rather than low molecular weight heparins. Progesterone support is said to be
ineffective but may have a place in some patients. The ASRM guideline does not recommend screening
for antithyroid antibodies or infections such as chlamydia, mycoplasma, or bacterial vaginosis. Neither
alloimmune testing, paternal leucocyte immunization, nor intravenous immunoglobin (IVIg) are
recommended. hCG supplementation is not mentioned.
ESHRE Guideline
ESHRE’s 2018 [3] guideline is the most comprehensive, consisting of 154 pages. The guideline includes
60 evidence-based recommendations, of which 31 were formulated as strong recommendations, 29 as
conditional, and 17 as good practice points. The evidence supporting investigations and treatment of
couples with RPL was said to be of limited and moderate quality. The guideline writing committee
sought as wide a base as possible before publishing their recommendations. The manuscript was sent for
comments to various “stakeholders.” ESHRE received 307 comments from 23 reviewers, representing
15 countries and two national societies. However, there are many inconsistencies in the report. ESHRE
recognizes that different choices will be appropriate for individual patients and that each patient should
be helped to arrive at a management decision consistent with their values and preferences. However,
the ESHRE protocol claims that adherence to the recommendations in the guideline could be used as
a quality criterion or performance indicator. Hence ESHRE hints that their guideline could be taken
as a set of instructions rather than an advice guideline. Another inconsistency is in the use of hCG.
Although the guideline states, “Studies on human chorionic gonadotrophin (hCG) for improving the LBR
in women with RPL have been recently summarized in a Cochrane review [6]. The results demonstrated
a significant benefit in using hCG to prevent RPL.” However, the guideline further states, “There is
insufficient evidence to recommend the use of hCG to improve live birth rate in women with RPL.” In
fact, ESHRE does not recommend any intervention except low-dose aspirin and a prophylactic dose of
anticoagulants in APS and levothyroxine for overt hypothyroidism.
ESHRE is the only guideline to assess lifestyle factors such as smoking, obesity, caffeine intake, and
excessive alcohol intake. However, evidence for adjusting lifestyle factors is extremely limited. The
guideline also suggests a research agenda.
Table 19.3 contrasts the recommendations for various investigations and treatment modalities in
the three protocols. Reliance on these guidelines can leave the physician in a quandary as to which
investigations to perform and which treatment to offer.
TABLE 19.3
Comparison of Three Protocols for the Investigation and Treatment of Recurrent Pregnancy Loss
Investigation or Treatment RCOG Protocol ASRM Protocol ESHRE Protocol
Parental karyotyping Not recommended Recommended Not routinely recommended;
depends on risk
Fetal karyotyping Recommended Not recommended Not recommended except for
explanatory purposes
Uterine cavity assessment Recommended Insufficient evidence Recommended
Resection of uterine septum Insufficient evidence Should be considered Insufficient evidence
APS assessment (ACA and LA) Recommended Recommended Recommended
Treatment of APS with heparin Recommended Recommended For >3 losses
and aspirin For 2 losses Rx for research trial
Luteal phase investigation — Not recommended Not recommended
Progesterone supplementation Insufficient evidence May be beneficial Not recommended
hCG supplementation Insufficient evidence — Insufficient evidence
Bacterial vaginosis Insufficient evidence Not recommended —
Infections — Not recommended Not recommended
Hereditary thrombophilias Recommended for Not recommended Not recommended except for
second trimester losses unless at high risk research or additional risk
for thrombosis factors
Anticoagulants for hereditary Insufficient evidence Not recommended No evidence
thrombophilia unless at high risk
for thrombosis
Thyroid function — Recommended Recommended including ATA
Prolactin estimation — Recommended Not recommended, but Rx
recommended
TORCH testing Not recommended Not recommended —
Alloimmune testing Not recommended Not recommended Not recommended
Immunotherapy Not recommended Not recommended Not recommended
Tender loving care Insufficient evidence Recommended Recommended
Diet, smoking, alcohol — — Recommended
Folic acid for — — Insufficient evidence
hyperhomocysteinemia
Vitamin supplementation — — Vitamin D advised
Steroids Not recommended Not recommended Not recommended
Male factors — Not recommended Not recommended
Inclusion Criteria
Two or Three Losses
The standard protocols listed in “The Different Guidelines” differ as to who should be investigated
and the criteria for investigation. The ASRM protocol [2] recommends investigation after two or more
pregnancy losses, whereas the RCOG [1] protocol only recommends assessment after three or more losses.
ESHRE changed their definition of RPL from the previous three or more losses to two or more losses
for their 2018 guideline.
The ASRM adopted the definition of two or more losses, as several studies have indicated that the
risk of subsequent miscarriage after two successive losses is only slightly lower (24%–29%) than that of
women with three or more spontaneous abortions (31%–33%) [7,8]. ESHRE adopted the “two or more”
definition so that women with APS would not be denied treatment. Figure 19.1 gives an estimate of the
prognosis after two, three, or more than five miscarriages. After two or more pregnancy losses, there is
an approximately 80% chance of a live birth in the third pregnancy. However, these figures have been
2 miscarriages 3 miscarriages ≥5 miscarriages

10% 10% 20% 20% 32.5% 32.5%
80% 60% 35%
Live births Genetic abortions Maternal factor abortions
FIGURE 19.1 Number of previous abortions and effect of treatment for maternal factors. Patients with two miscarriages
have an 80% chance of a live birth if untreated. If 50% of subsequent miscarriages are chromosomally abnormal, any
treatment aimed at correcting a maternal cause of miscarriage can only raise the live birth rate from 80% to 90%. A mega-
trial is required in order to show a statistical significance between 80% and 90%. Hence most treatment regimens used on
patients with two miscarriages will be ineffective. Treating patients with three miscarriages can only raise the live birth rate
by 20%. However, if treatment is used on patients with a poor prognosis, the live birth rate can be raised by 32%, making
it relatively easy to show a statistically significant effect of treatment.
disputed [7,8]. Jaslow et al. [7] have reported that the frequency of abnormal findings was neither changed
nor increased in women after two losses (41% after two losses, 40% after three losses, vs. 42% after four
or more losses).
If an 80% live birth rate is assumed after two losses, the “two or more” definition is problematic, as in any
research trial of treatment effect there will be an 80% live birth rate in the control group. Therefore inclusion
of patients with two miscarriages in a trial will preclude any positive result. ASRM therefore does state
that research be limited to patients with three or more miscarriages. However, ESHRE has no such caveat.
The “three or more” definition is also problematic especially if the woman is older than 35 years of age, or
when the couple has had difficulty conceiving and cannot wait for a third loss before initiating treatment.
However, the number of previous losses is not the only prognostic factor. Maternal age, concurrent infertility,
and previous euploid losses are powerful prognostic factors (see “Factors Affecting Subsequent Prognosis”).
The 80% prognosis for a live birth may only apply to the young patient with two losses.
Biochemical Pregnancies
The guidelines differ with regard to biochemical pregnancy losses, when no pregnancy sac can be
visualized on ultrasound. All biochemical pregnancies are, by definition, of unknown location, and
some biochemical pregnancies will be ectopic gestations. In addition, a low positive hCG result may be
due to “phantom” endometrial or pituitary hCG. Hence the recent revised definitions of the ASRM [9]
define a pregnancy loss as a pregnancy documented by ultrasonography or histopathologic examination.
The author [10] has previously defined a biochemical pregnancy as a βhCG level between 10–1000
IU/L in a cycle in which no hCG was administered and no pregnancy sac demonstrated on ultrasound.
However, the author has tended to become more restrictive, and only accepts a biochemical pregnancy
as such if there are two readings that show a rising level. ESHRE defines a non-visualized pregnancy as
spontaneous pregnancy demise based on decreasing serum or urinary βhCG levels and non-localization
on ultrasound, if performed, and as a biochemical pregnancy if no ultrasound was performed [11]. ESHRE
includes biochemical pregnancies as pregnancy losses in their guideline, as Kolte et al. [12] have shown
that each additional non-visualized pregnancy loss conferred a relative risk (RR) for live birth of 0.90
(95% confidence interval [CI] 0.83–0.97), which was not statistically significantly different from the
corresponding RR of 0.87 (95% CI 0.80–0.94) conferred by each clinical miscarriage.
Upper Limit of Pregnancy Loss

Similar confusion surrounds the upper level of pregnancy loss. Traditionally, any pregnancy that has been
lost prior to viability was considered as abortion. The more recent North American definition includes
pregnancy losses up to 20 weeks as a miscarriage. ESHRE considers a pregnancy loss a miscarriage up to
24 weeks. However, there are many live individuals who were born at or prior to 24 weeks. Hence the term
“recurrent pregnancy loss” is replacing the term “recurrent miscarriage.” Discrepancy over the upper limit
for defining a miscarriage has also caused confusion. Preston et al. [13], in a leading paper on hereditary
thrombophilias, assessed “miscarriages” as up to 27 weeks. Ober et al. (Ober, personal communication),
in the paper most often quoted to show that paternal leucocyte immunization is ineffective [14], included
nonconsecutive abortions and pregnancies up to 29 weeks. Laskin [15], in an article usually quoted to
show that steroids have no place in APS, included patients with pregnancy losses up to 31 weeks. It is
difficult to believe that that research on patients with two losses at 27, 29, or 31 weeks has relevance to
patients with 5 or more losses of blighted ova.
Genetic Assessment of the Embryo

Embryonic aneuploidy is the most frequent single cause of miscarriage. Genetic testing of the abortus
shows that between 29% [16] to 60% [17] may be aneuploid when using karyotype banding techniques in
patients with three or more miscarriages. However, an incidence of 90% has been reported if molecular
techniques are used in patients with two or more losses [18]. These chromosomal abnormalities in the
miscarriage usually arise de novo in chromosomally normal parents. Hence it is not surprising that the
incidence decreases in patients with increasing numbers of miscarriages [19]. Figure 19.2 shows the
decreasing incidence of embryonic aneuploidy in Ogasawara et al.’s [19], Goldstein et al.’s [20], and the
author’s series. Hence it is not surprising that Stern et al. [17] should report a 60% incidence of embryonic
chromosomal aberrations in women with a mean of 3.5 miscarriages, compared to an incidence of 29.5%
in women with a mean of 4.7 in Carp et al.’s [16] series.
Embryonic chromosomal analysis is not recommended by the ESHRE or ASRM protocols but is
recommended in the RCOG protocol. In practice, embryonic chromosomal analysis is the standard of care
in most leading centers in the United States but not commonly performed in the United Kingdom. ESHRE
claims that genetic analysis of the abortus is for information purposes only. However, the information is
all-important, as the genetic makeup of the embryo enables an accurate diagnosis, allows a prognosis to
be given, and can direct treatment to preimplantation genetic testing for aneuploidies (PGT-A) to prevent
a recurrence.
As embryonic aneuploidy may be a chance occurrence and not recurrent, two publications have reported
the subsequent live birth rate after embryonic aneuploidy [16,19]. Figure 19.3 shows the subsequent
prognosis according to embryonic aneuploidy. Aneuploidy in the embryo confers a good prognosis. Hence
70%
15 Goldstein et al. [20]
35 62
13 Ogasawara et al. [19]
60% 27 Carp
21
18
Propn. chromosomal aberrations
Cumulative
16
50% Expon. (Cumulative)
10
84 7
40%
39
48 4
30% 10
19 14 7 13
20
20%
10% 55 37 25 23 14 18 11 31 90
45
47 10 78 36 38 21 22 75 14 59 59
0%
2 3 4 5 6 >=7
No. miscarriages
FIGURE 19.2 Decreasing incidence of embryonic aneuploidy according to number of previous miscarriages (cumulative
results including author’s series).
67%
Carp et al. [16]
70% 62%
Ogasawara et al. [19]
60%
50%
37.8% 38%
40%
30%
20%
14/37 27/71 14/21 37/60

10%
0%
Euploidy Aneuploidy
OR for a live birth after aneuploidic abortion.

3.28 (95% CI = 0.94–11.9) Carp et al.16
2.62 (95% CI = 1.21–5.67) Ogasawara et al.19
FIGURE 19.3 Outcome of subsequent pregnancy according to fetal karyotype. OR for a live birth after aneuploidic
abortion 3.28 (95% CI = 0.94–11.9) [16]; 2.62 (95% CI = 1.21–5.67) [19].
it has been reported that if aneuploidy is present, there is little need for further investigation [21]. It is
confusing that as recently as 2018, ESHRE did not support embryonic genetic testing.
However, although ESHRE does not recommend routine parental karyotyping, it indicates that the
decision should be based on individual assessment. The author has published that in repeat aneuploidy,
or aneuploidy in the presence of a parental chromosomal aberration, PGT-A is advised [22].
Factors Affecting Subsequent Prognosis

In some subgroups of RPL the recurrence rate is unknown, e.g., recurrent biochemical pregnancies,
after in vitro fertilization, APS, or in the older woman. However, there are certain factors that help to
predict the prognosis. These are described in detail in Chapter 1 and Table 19.1. They are number of
previous pregnancy losses; maternal age [23]; primary, secondary, or tertiary aborter status (the secondary
aborter has a better prognosis than the primary aborter) [24]; karyotype of previous miscarriage [16,19];
concurrent infertility [25]; immunological features including natural killer (NK) cells (see Chapter 11);
and early or late pregnancy losses, as the patient with late losses tends to have a worse prognosis [26].. The
most important predictive factor is the number of previous miscarriages. Figure 1.1 in Chapter 1 shows
the decreasing live birth rate with the increasing number of miscarriages.
Figure 19.1 shows the effect of assessing treatment on patients with two miscarriages. If there is a
subsequent 80% live birth rate and 50% of subsequent miscarriages are chromosomally abnormal, any
treatment aimed at correcting a maternal cause of miscarriage can raise the live birth rate only from
80% to 90%. To show a statistical significance between 80% and 90% will require a mega-trial. Hence
any trial that includes patients with two miscarriages will show any treatment to be ineffective. Even the
ASRM guideline, which recognizes two or more miscarriages as the basis for investigation and treatment,
suggests that research trials should be limited to patients with three or more pregnancy losses. Table
19.1 shows a rough scale of the prognosis according to the various prognostic factors, and should give
physicians and patients a general idea as to the relative prognosis.
Good Prognosis Patients

These patients include young patients with two or possibly three first trimester miscarriages. “Good
prognosis” patients probably require very little investigation. However, they do require diagnosis and
reassurance of their prognosis, and appropriate follow-up ultrasound scans on a regular basis can reassure
the patient and their partner that the pregnancy is progressing normally. The patient should be reassured
that in the event of another miscarriage further investigations will be carried out, including complete
chromosomal analysis of the abortus and possibly embryoscopy. It is doubtful whether “good prognosis”
patients need pharmacological support on an empirical basis. A question arises regarding patients who
have undergone partial investigations; e.g., if a patient with two blighted ova is found to have a septum,
it is questionable whether the septum is the cause, or whether it should be resected. A septum has been
described over 40 years ago to cause abortions of live fetuses in the second or third trimester after a
“mini labor” [27]. Therefore, should the septum be left in situ, as there is no evidence that it is the cause
of RPL, or should it be resected, as it may cause late pregnancy loss and preterm labor? These questions
should be discussed with the patient and partner. It is important to remember that the patient’s views
are as valid as those laid down in official guidelines. In any recurrent miscarriage clinic, the majority
of patients will have a good prognosis. Their good prognosis should not influence the management of
patients with a poor prognosis.
Medium Prognosis Patients

These patients include women with three and possibly four miscarriages. The prognosis for a live birth is
approximately 60% after three miscarriages (40% after four miscarriages) (Figure 19.1). If included in a
trial, Figure 19.1 shows that treatment of maternal factors can raise the live birth rate by approximately
25%. Hence a trial of treatment for maternal factors would need large numbers to achieve the power to
show a statistically significant benefit of treatment; e.g., paternal leucocyte immunization was shown
to have a statistically significant benefit in the Recurrent Miscarriage Immunotherapy Trialists Group
(RMITG) trial of 419 patients [25], but not in Ober et al.’s [14] trial of 200 patients. We believe that
medium prognosis patients should be investigated, and treatment should be directed at the cause as far as
possible. However, even with the most rigorous testing, including embryonic genetic testing, 50% of the
RPL cases are still unexplained [7]. In these patients with unexplained losses, there may be a place for
empirical hormone support with progestogens or hCG, as there is evidence [6,28–31], although debatable
and unsupported by guidelines, that these hormones may improve the prognosis by approximately 25%.
Hormone treatment is empirical, as there is no investigation in the interval between pregnancies to
diagnose a hormonal deficiency.
A problem may arise when the clinical presentation is at variance with the laboratory investigations;
e.g., should a patient with antiphospholipid antibodies and an aneuploid abortus in a previous pregnancy
be treated by anticoagulants? As with good prognosis patients, skill and experience may be necessary to
interpret the results.
If there is a presumptive diagnosis, treatment should be prescribed accordingly. Some examples are
given as follows.
1. Opinions are divided as to whether parental chromosomal aberrations should be examined.

Testing is not recommended by ESHRE or the RCOG. However, if the fetus does inherit the
chromosomal aberration in an unbalanced form, preimplantation genetic diagnosis may be
appropriate treatment.
2. When fetal karyotypic aberrations are present, there is usually a good prognosis. However, there
are patients with repeat aneuploidy (see Figure 19.3). PGT-A may be appropriate in cases of
repeat aneuploidy.
3. Antiphospholipid antibodies are generally accepted as a cause of pregnancy loss. At present,
treatment seems to be indicated and supported by all three guidelines. However, there is no
placebo control trial on anticoagulant and aspirin treatment in APS. In addition, there is no
evidence that aspirin has a therapeutic effect. On the contrary, two meta-analyses of five trials
of aspirin failed to find any therapeutic effect [32,33].
4. Hereditary thrombophilias are controversial as to their role in pregnancy loss. They seem to
be associated with late losses rather than early losses [13]. We investigate and treat patients
with hereditary thrombophilias with anticoagulants, usually the low molecular weight heparin
enoxaparin. The rationale for treatment is shown in Figure 9.2 in Chapter 9, as there seems to
be an increase of 25% in the live birth rate.
5. There is also a dearth of trials to determine the place of uterine malformations. Hysteroscopy
or three-dimensional ultrasound have tended to replace hysterosalpingography, as they are
associated with much less discomfort. However, hysteroscopy cannot distinguish between a
septate and bicornuate uterus. Ultrasound is probably the best procedure for distinguishing
between a septate and bicornuate uterus. This distinction is essential if hysteroscopic septotomy
is considered. There is only one comparative control trial of septotomy [34], which showed a
non-significant trend to a 20% improvement in the live birth rate. This trial is not considered
sufficient by the three guidelines to recommend uterine surgery.
Poor Prognosis Patients

The author defines these patients as those with five or more consecutive miscarriages. Saravelos and Li [4]
classify these patients as Type 2 RPL. They have been poorly described in the literature and have formed
the subjects of few trials. These patients constitute approximately 30% of the patients in our service.
However, their proportion will be less in patients in centers using the ASRM or ESHRE definition of
RPL as two or more miscarriages. Poor prognosis patients have usually had all the investigations and
empirical treatments available. Hormone supplements, anticoagulants, hysteroscopic surgery, and often
in vitro fertilization have been tried. In addition, there may be APS patients who have failed treatment
and patients who continue miscarrying after surgery for uterine anomalies. However, most of these
patients have not had fetal genetic analysis performed. After five or more miscarriages, the chance of fetal
chromosomal aberrations is less than after three miscarriages. In poor prognosis patients, it is possible
to retrieve histological specimens of previous miscarriages; either fixed slides or paraffin blocks can be
used for comparative genetic hybridization (CGH) or next-generation screening (NGS) [35,36]. If one of
the embryos is aneuploid, PGT-A should be considered. If, however, the embryo is euploid, PGT-A will
not lead to a live birth. Our approach in these patients is to perform controversial testing and treatment
such as immune testing.
In poor prognosis patients in whom other forms of treatment have failed, immunotherapy with IVIg
seems to confer a greater benefit than after two or three miscarriages [37,38]. The randomized trials and
meta-analyses of paternal leucocyte immunization and IVIg are not appropriate for judging the effect on
poor prognosis patients, as the results have been obscured by the good and medium prognosis patients.
As with the medium prognosis patients, we analyze the genetic makeup of the embryo. If immunotherapy
fails and the embryo is karyotypically normal, surrogacy may offer the only possibility of a live birth.
The Resistant Patient

The patient with three miscarriages has an approximately 30%–40% chance of a fourth miscarriage. The
patient with four miscarriages has a 50%–60% chance of a subsequent miscarriage. Therefore, after three
miscarriages, 15%–24% of patients will have two subsequent miscarriages. None of the guidelines listed
in “The Different Guidelines” provide any guidance for the resistant patient, only for the initial pregnancy.
The following is the author’s approach. If a subsequent miscarriage occurs in the first trimester of the next
pregnancy, the embryo should be tested genetically. Using molecular techniques, nearly 90% of abortuses
will reveal a result. If necessary, CGH or NGS can be performed on previous histological specimens.
Alternatively, embryoscopy can be performed to exclude anomalies, if embryoscopy is available. If the
embryo is aneuploid or otherwise abnormal, the anomaly may be an isolated event and the prognosis is
After initial management, further miscarriage
Consider hereditary
thrombophilia or placental
1st trimester Late losses villositis. Treat with
loss anticoagulants or steroids
accordingly
Embryonic genetic assessment

(Either on fresh products of conception or previous paraffin block)
Treatment has failed. Reconsider
original diagnosis. Consider hCG Consider if sporadic or
or dydrogesterone repeat. If sporadic, repeat
supplementation if not previously Euploid Aneuploid initial treatment
used
Immune testing for NK PGT-A

Further pregnancy Repeat aneuploidy
cells if available.
IVIg even if empirical loss
Further pregnancy Further pregnancy loss,

loss or no euploid embryos
Ovum donation
Gestational carrier
Live birth (Sperm donation if WES shows repeat
surrogacy
aneuploidy from father)
FIGURE 19.4 Flowchart for resistant patients.
better for the next pregnancy. If treatment had been administered for a maternal cause of pregnancy loss,
the diagnosis should be reviewed. If the diagnosis is still thought to be accurate, the fetal abnormality
may be a confounding factor. In this case, it is fully justified to repeat the same treatment. However, in
cases of repeat aneuploidy, PGT-A may offer the only chance of a euploid embryo.
If the embryo is euploid on genetic testing, other forms of therapy should be considered, e.g., if
progesterone support had been given, hCG or immunotherapy might need to be considered.
If a subsequent loss occurs in the second or third trimesters, hysteroscopy may need to be performed
(if not previously performed) or repeated, in order to exclude uterine anomalies. If anticoagulants had
been used for APS, an increased dose may be indicated, or steroid therapy or hydroxychloroquine should
be considered, and possibly IVIg if the treatment failure presented as pregnancy loss for late obstetric
complications. In the very resistant cases with five or more miscarriages, unconventional or nonevidence-
based treatment may be indicated, such as intravenous immunoglobulin, or surrogacy. Figure 19.4 shows
an algorithm for treatment of the resistant patient, dependent on good or poor prognosis.
Specific Forms of Pregnancy Loss

The majority of recurrent pregnancy losses are losses of blighted ova, in which no fetal heartbeat or even
a fetal shadow is detected on ultrasound. We tend to assess these patients on the basis of their prognosis,
as listed in “Factors Affecting Subsequent Prognosis,” and to treat them according to genetic findings.
Recurrent Second Trimester Fetal Death

Patients with recurrent second trimester fetal deaths have a poorer prognosis than after first trimester
losses [26]. The chance of a second trimester loss being due to chromosomal aberrations is less than in
first trimester miscarriages. However, there may be fetal structural anomalies. Hence detailed ultrasound
may assist the diagnosis. Another possibility for diagnosing fetal structural anomalies is embryoscopy.
Diabetes should be excluded, as diabetes predisposes to fetal anomalies.
Thrombotic mechanisms, either due to APS or hereditary thrombophilias, are more likely to cause
fetal demise than first trimester miscarriages [13]. If either of these is found in the presence of recurrent
second trimester fetal deaths, treatment by anticoagulants is warranted.
Another condition that has been identified is chronic histiocytic intervillositis [39]. The condition is
strongly associated with highly recurrent, severe obstetric complications including miscarriage, fetal
growth restriction, and intrauterine fetal death [40]. The etiology remains unclear, but the aberrant
recruitment of maternal immune cells to the maternal-fetal interface suggests an anomalous maternal
immunological response to fetal tissue. Immunosuppression by steroids has been reported to be useful
and superior to anticoagulants and aspirin [41].
Losses of Live Embryos

Live embryos may be lost in the first or second trimesters. The distinguishing feature of these losses is
that the uterus starts to contract, and vaginal bleeding precedes fetal demise. There may be placental
separation and retroplacental hematoma formation. Losses of live embryos are less likely to be due to
an embryonic or fetal factor, and more likely to be due to a uterine or other maternal factor. In the first
trimester, there is a typical history. Embryonic development is normal. The uterus suddenly starts to
contract, and miscarriage ensues. In this type of miscarriage, we recommend testing for uterine anomalies
and infections. In patients who are pregnant and present with a hematoma, the hematoma may become
infected. In the case of an infection, uterine contractions rapidly follow, with expulsion of the uterine
contents. Pelinescu-Onciul [42] has reported that dydrogesterone is more effective than progesterone in
preventing retroplacental hematoma progressing to miscarriage.
In the case of second trimester losses of live fetuses, uterine anomalies, infections, and possibly diabetes
(which predisposes to infections) should be investigated. In the presence of contractions in the second
trimester, tocolytic agents may be appropriate. However, appropriate trials to determine an optimal course
of management have not been performed.
Mixed Pattern of Pregnancy Losses

In many cases, each pregnancy loss may have a different clinical presentation; e.g., there may be a blighted
ova followed by miscarriage of a live fetus in the second trimester, followed by an embryonic demise
(missed abortion). These mixed patterns of pregnancy loss are relatively frequent in patients with two or
three losses but rare in patients with five or more losses. In patients with a mixed pattern of pregnancy
loss, the cause is more likely to be due to chance, and the prognosis is good. In the author’s opinion, they
probably do not require active treatment.
Conclusions
Recurrent pregnancy loss is not one homogeneous condition. Hence there is no one protocol that is
applicable. The aim of the standard protocols is entirely laudable, to advise physicians with little
experience of RPL as to the optimal methods of diagnosis and treatment. The standard protocols try
to guarantee that the patient receives effective treatment, and that ineffective treatment is not used.
However, the standard protocols listed in this chapter might have done more harm than good, as they
treat recurrent pregnancy loss as one homogeneous group, and hence their recommendations preclude
the treatment of subgroups of patients. The development of an optimal investigation protocol depends
on reaching an accurate diagnosis of cause and directing treatment to that diagnosis. Fetal genetic
assessment and embryoscopy hold out the possibility of more accurately diagnosing embryonic or fetal
causes of pregnancy loss. Treatments that have not been shown to be effective when tried on a large
cohort of patients may be found to be highly effective when used on a subgroup of patients with an
accurate diagnosis.
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20
Debate: Should Progestogens Be Used
in Recurrent Pregnancy Loss? Yes
Ashok Kumar and Simar Kaur
Introduction
The role of progestogens in supporting early pregnancy in women with unexplained recurrent pregnancy
loss (RPL) has always been controversial and always stirs up debate. To prescribe or not to prescribe
progestogens to women with recurrent miscarriage of unclear etiology is the question that confronts
every obstetrician in day-to-day practice. Several questions arise with regard to progestogen treatment for
women with RPL: Why should progesterone work in women with RPL? Is there any scientific evidence?
Does it actually work in clinical practice? Or does it often fail? Which progestogen works better? What
is the ideal time to start and the ideal route of administration? The questions are many, and controversies
surrounding them are great.
The cause of RPL remains unestablished in approximately 50% of women with RPL [1]. The controversy
lies in the optimal management of women with unexplained RPL. With a limited understanding of
the etiology, no specific treatment regimens can be offered, though several therapies with varying
degrees of success have been proposed to manage this condition. Apart from supportive measures, or
so-called “tender loving care,” the most commonly used pharmacological option is the administration
of progestogens.
Progesterone is produced by the corpus luteum following ovulation until placental function is
established. This so-called luteoplacental shift occurs around 7−11 weeks of pregnancy. During this
shift, endogenous progesterone production may be less due to limited production by the corpus luteum
or delay in initiation of placental production. This is the phase of progesterone deficiency when most
miscarriages occur. Csapo et al. [2] studied the effect of luteectomy-induced progesterone withdrawal on
the oxytocin and prostaglandin response of the first trimester pregnant human uterus. Luteectomy prior
to 7 weeks was found to cause miscarriage. Mifepristone, a progesterone receptor antagonist, blocks
the progesterone receptors, leading to fetal death and placental separation. Women with various risk
factors (such as corpus luteum insufficiency, women undergoing in vitro fertilization [IVF], a history of
recurrent miscarriages, and also pregnant women under definite stress) suffer from decreased production
of endogenous progesterone.
The basis for the predicted success of progesterone in unexplained RPL is that it has both endocrine
and immunological function [3]. Insufficient progesterone production can manifest in inappropriate
endometrial development or an inadequate immune response to fetal antigens resulting in a spontaneous
miscarriage. Progesterone induces secretory changes in the endometrial lining, thus rendering it more
receptive to the embryo. This results in successful implantation and normal pregnancy. Progesterone
also decreases the synthesis of prostaglandins, resulting in relaxation of myometrial smooth muscles.
This induces uterine quiescence and prevents uterine contractions that lead to miscarriage. Endocrine
effects of progesterone also include reduction in cervical stromal degradation, altered barrier function to
cervical ascending inflammation or infection, reduced gap junction formation, and decreased expression
of oxytocin receptors [4].
The immunomodulatory effect of progesterone is mediated through progesterone-induced blocking
factor (PIBF). PIBF is secreted by activated maternal lymphocytes in the presence of progesterone. The
197
embryo protective immunomodulation effects mediated by PIBF mainly consist of Th1/Th2 cytokine
shift, reduction in decidual natural killer (NK) cell activity, and increased production of asymmetrical
blocking antibodies against fetal antigens [5,6].
Evidence of Effect
Hussain et al. [7] conducted a cohort study on women with three or more unexplained RPLs. Serum
progesterone levels of women were checked on the day of positive urine pregnancy test and repeated
48 hours later. Women with initial serum progesterone less than 40 nmol/L or those with a rise of less
than 15% after 48 hours were supplemented with micronized progesterone vaginal pessaries 400 mg,
b:d until 12 weeks of pregnancy. Two hundred and three pregnancy cycles were studied for the efficacy
of progesterone supplementation. The live birth rate and repeat miscarriage rate after progesterone
supplementation was 63% (95% confidence interval [CI] 56%–70%) and 36% (95% CI 30%–43%),
respectively. Since there was no control group in the study, the results were compared to similar historical
data showing a miscarriage rate of 45% after three unexplained losses, suggesting a significant reduction
in miscarriage rate after administration of progesterone (36% vs. 45%) [7].
In addition to micronized progesterone, a stereoisomer of progesterone dydrogesterone has been widely
studied in various clinical trials. Dydrogesterone has 5–6 times higher bioavailability than progesterone
itself and higher receptor binding selectivity [8]. These result in a significantly lower oral therapeutic
dose, which is approximately 10–20 times less than micronized progesterone. El-Zibdeh [9] conducted
a three-arm study on 180 women that compared dydrogesterone treatment with human chorionic
gonadotropin (hCG), both in combination with standard supportive care and with standard supportive
care alone. Women recruited for the study were less than 35 years old with history of ³3 unexplained
recurrent consecutive miscarriages. All women received standard supportive care, including multivitamin
supplements and recommended bed rest, and were followed routinely in the antenatal clinic. Eighty-two
women received dydrogesterone 10 mg twice daily with standard supportive care from diagnosis of
pregnancy until 12 weeks, 50 women received hCG 5000 IU intramuscularly every 4 days with standard
supportive care from diagnosis of pregnancy until 12 weeks, and in the third arm 48 women (controls)
received standard supportive care alone. Miscarriage was significantly (p ≤ 0.05) more common in the
control group (29%; 14/48 women) than in the dydrogesterone group (13.4%; 11/82 women). There were
no significant differences between the hCG group (18%; 9/50 women) and the control group [9].
Kumar et al. [10] conducted the most recent double-blind, randomized, placebo-controlled study, with
women receiving either dydrogesterone (10 mg twice daily; n = 175) or placebo (10 mg lactose; n = 173) from
confirmation of pregnancy until 20 weeks of gestation. Women between 18–35 years with a history of ≥3 first
trimester unexplained pregnancy losses and currently in the first trimester of a live pregnancy, preferably at
4–8 weeks of gestation, were included in the study. Significantly fewer women in the dydrogesterone group
(12/175) miscarried compared with placebo (29/173); i.e., 6.9% versus 16.8%, respectively (p = 0.004). There
was a significant increase in the mean gestational age at delivery in the dydrogesterone group compared
with placebo (38.0 ± 2.0 vs. 37.2 ± 2.4 weeks, respectively; p = 0.002). There was also a trend toward
the reduction of preterm birth, cesarean delivery, and low birth weight. The study supported the use of
dydrogesterone in women with recurrent miscarriage to reduce miscarriage risk [10].
Coomarasamy et al. [11] performed a multicenter, randomized, placebo-controlled study to investigate
whether treatment with micronized progesterone would increase the rates of live births among women
with unexplained recurrent miscarriage. The live birth rate was 65.8% in the progesterone group, which
was comparable to 63.3% in the placebo group. The trial showed no significant increase in the rate of live
births with the use of vaginal micronized progesterone.
These conflicting results require explanation. Micronized progesterone has a role in making the
endometrium receptive for implantation. However, in the PROMISE trial [11], it was started much later than
the time of implantation. Micronized progesterone may have been found to have a positive effect if started
either prior to or during implantation. Dydrogesterone, however, has a more effective role in improving
subendothelial blood flow compared to progesterone and therefore is useful even if started at 4–8 weeks.
In addition, in the PROMISE trial [11], women of up to 39 years were included in the study. Women of
Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? Yes 199
advanced maternal age have a high risk of spontaneous miscarriage due to chromosomal anomalies in the
fetus. Maternal age can impact on the rate of chromosomal abnormalities in women experiencing three
miscarriages: 60.0% for women <35 years of age; 78.3% for women ≥35 years of age [12].
A recently published randomized double-blind placebo-controlled trial by Ismail et al. [13] studied the
effect of periconceptional progesterone started early in the luteal phase before confirmation of pregnancy
in preventing miscarriage in women with RPL The main difference between Ismail et al.’s [13] study
and the PROMISE trial [11] was the initiation of progesterone prior to implantation and confirmation of
pregnancy. The women with unexplained RPL were randomized into two groups; one group (n = 340)
received progesterone vaginal pessary 400 mg twice daily, while the other group (n = 335) was given
placebo pessaries. Treatment was started immediately after documentation of ovulation using ultrasound
through luteal phase until confirmation of pregnancy and continued until 28 weeks. The progesterone
group had a significantly lower number of miscarriages before 20 weeks compared to the placebo group
(12.4% vs. 23.3%, p = 0.001). The progesterone group had a significantly higher number of live births
(273 [91.6%] vs. 199 [77.4%], p = 0.001) compared with the placebo group. The study also highlighted
the immunomodulatory effects of progesterone. The baseline serum levels of IL-10, IL-2, and IFN γ
were measured preconceptionally for women in both groups and repeated in first, second, and third
trimesters. Though there was no statistically significant difference in the cytokine levels in the two groups
preconceptionally, there was a significant increase in the IL-10 levels through the first, second, and third
trimesters and a significant decrease in IL-2 and IFN γ levels through the trimesters in the progesterone
group. IL-2 and IFN γ are proinflammatory Th-1 responses that are detrimental for maintaining the
pregnancy, whereas IL-10 is an anti-inflammatory Th-2 cytokine. Thus, the study showed that progesterone
promotes the Th1/Th2 cytokine shift, which helps maintain the pregnancy.
The findings of Kumar et al. [10] were supported by a subsequent meta-analysis by Carp [14], who
concluded that dydrogesterone was favored in unexplained RPL compared with standard treatment. The
meta-analysis included thirteen reports of dydrogesterone treatment (including two randomized trials
and one nonrandomized comparative trial) with 509 women. The number of subsequent miscarriages
or continuing pregnancies per randomized woman was compared in women receiving dydrogesterone
compared to standard bed rest or placebo intervention. There was a 10.5% (29/275) miscarriage rate after
dydrogesterone administration compared to 23.5% in control women (odds ratio for miscarriage 0.29, CI
0.13–0.65, and 13% absolute reduction in the miscarriage rate) [14].
The efficacy of dydrogesterone for luteal phase support in IVF has been demonstrated in the
recently published Lotus 1 study [15]. Lotus 1 was an international Phase III randomized control trial
that compared the efficacy of oral dydrogesterone 30 mg daily with micronized vaginal progesterone
600 mg daily for luteal support in IVF. Luteal support was started on the day of oocyte retrieval and
continued until 12 weeks of gestation. The primary objective of the trial was to study the improvement
of pregnancy rate, confirmed by the presence of fetal heartbeat at 12 weeks’ gestation, determined by
transvaginal ultrasound. The Lotus 1 trial showed that dydrogesterone was not inferior to micronized
vaginal progesterone for luteal support. Pregnancy rates at 12 weeks of gestation were 37.6% and 33.1%
in the dydrogesterone and micronized vaginal progesterone groups, respectively. Lotus 1 also showed a
similar maternal and neonatal safety profile for the two drugs, suggesting that oral dydrogesterone can
replace micronized vaginal progesterone for luteal support, due to ease of administration.
In addition to the secretory effects of progesterone on the endometrium, progesterone influences
structural and functional modification of the endometrial vasculature, which further improves endometrial
receptivity during the window of implantation. Progesterone upregulates endothelial nitric oxide synthase
(eNOS) expression in uterine and spiral arteries necessary for implantation. It is well agreed upon that
nitric oxide helps in vasodilatation, decidua formation, and endometrial remodeling during trophoblast
invasion and also regulates endometrial functions such as receptivity, implantation, and menstruation [16].
A pilot study [17] conducted in India studied sub-endometrial blood flow parameters following
dydrogesterone and micronized vaginal progesterone administration in women with unexplained RPL. In
this randomized, single-blinded study, one group of women (n = 50) received oral dydrogesterone 10 mg
twice daily while the other group (n = 51) received micronized vaginal progesterone 100 mg thrice daily
until 12 weeks of gestation, after confirmation of fetal heart on ultrasound at 6−7 weeks. Uterine artery
flow Doppler assessment was performed at 7 weeks and repeated 4 weeks later. Following progesterone
supplementation, both groups showed a highly significant reduction in Doppler indices and an increase in
end diastolic velocity. Oral dydrogesterone appeared to be equally effective as micronized progesterone
in improving endometrial blood flow. However, pregnancy salvage rates were higher with dydrogesterone
(92%) compared to micronized progesterone (82.3%) [17].
Saccone et al. [18] recently conducted a systematic review and meta-analysis of 10 randomized
controlled trials, including the PROMISE trial [11] and the study by Kumar et al. [10]. The meta-analysis
included 1586 women with unexplained RPL. The role of supplementation of progesterone in the first
trimester of pregnancy to prevent miscarriage in women with unexplained RPL was studied. The pooled
data from the 10 trials showed that women with a history of unexplained recurrent miscarriage who
were randomized to receive progestogens in the first trimester and before 16 weeks had a lower risk of
subsequent miscarriage (relative risk [RR] 0.72; 95% CI 0.53–0.97) and a higher live birth rate (RR 1.07;
95% CI 1.02–1.15) than controls. The meta-analysis also concluded that synthetic progestogens, including
weekly intramuscular 17-hydroxyprogesterone caproate, were associated with a lower risk of recurrent
miscarriage, but micronized progesterone was not associated with a lower risk of recurrent miscarriage.
A recent Cochrane review on progestogens and recurrent miscarriage by Haas et al. [19] included eleven
trials involving 2359 women. The meta-analysis concluded that administration of progestogens early in
pregnancy to women with recurrent miscarriages may lower the miscarriage rate from 26.3% to 19.4%
(RR 1.11; 95% CI 1.00−1.24). However, the analysis could not draw conclusions regarding the optimal
route of administration.
The European Progestin Club guideline of 2015 for prevention and treatment of threatened and
recurrent miscarriage also recommends administration of oral dydrogesterone for women with three or
more unexplained RPLs, to reduce the rate of miscarriage [20].
In conclusion, should progestogen supplementation be used? The answer is a resounding yes. There is
a theoretical basis and lack of side effects, and numerous reports attest to the efficacy in improving the
live birth rate and lowering the number of subsequent miscarriages. In addition, dydrogesterone seems
to have a more pronounced effect than progesterone itself, as shown in numerous series.
REFERENCES
1. Practice Committee of the American Society for Reproductive Medicine. Evaluation and treatment of
recurrent pregnancy loss: A committee opinion. Fertil Steril. 2012;98(5):1103–11.
2. Csapo AI, Pulkkinen MO, Kaihola HL. The effect of luteectomy-induced progesterone-withdrawal on
the oxytocin and prostaglandin response of the first trimester pregnant human uterus. Prostaglandins.
1973;4(3):421–9.
3. Szekeres-Bartho J, Balasch J. Progestagen therapy for recurrent miscarriage. Hum Reprod Update.
2008;14(1):27–35.
4. Arck P, Hansen PJ, Mulac Jericevic B, Piccinni MP, Szekeres-Bartho J. Progesterone during pregnancy:
Endocrine-immune cross talk in mammalian species and the role of stress. Am J Reprod Immunol.
2007;58(3):268–79.
5. Blois SM, Joachim R, Kandil J et al. Depletion of CD8+ cells abolishes the pregnancy protective effect
of progesterone substitution with dydrogesterone in mice by altering the Th1/Th2 cytokine profile.
J Immunol. 2004;172:5893–9.
6. Faust Z, Laskarin G, Rukavina D, Szekeres-Bartho J. Progesterone induced blocking factor inhibits
degranulation of natural killer cells. Am J Reprod Immunol. 1999;42(2):71–5.
7. Hussain M, El-Hakim S, Cahill DJ. Progesterone supplementation in women with otherwise unexplained
recurrent miscarriages. J Hum Reprod Sci. 2012;5(3):248–51.
8. Gruber CJ, Huber JC. The role of dydrogesterone in recurrent (habitual) abortion. J Steroid Biochem Mol
Biol. 2005;97(5):426–30.
9. El-Zibdeh MY. Dydrogesterone in the reduction of recurrent spontaneous abortion. J Steroid Biochem
Mol Biol. 2005;97(5):431–4.
10. Kumar A, Begum N, Prasad S, Aggarwal S, Sharma S. Oral dydrogesterone treatment during early
pregnancy to prevent recurrent pregnancy loss and its role in modulation of cytokine production: A
double-blind, randomized, parallel, placebo-controlled trial. Fertil Steril. 2014;102(5):1357–63.
Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? Yes 201
11. Coomarasamy A, Williams H, Truchanowicz E et al. A randomized trial of progesterone in women with
recurrent miscarriages. N Engl J Med. 2015;373(22):2141–8.
12. Choi TY, Lee HM, Park WK, Jeong SY, Moon HS. Spontaneous abortion and recurrent miscarriage: A
comparison of cytogenetic diagnosis in 250 cases. Obstet Gynecol Sci. 2014;57(6):518–25.
13. Ismail AM, Abbas AM, Ali MK, Amin AF. Peri-conceptional progesterone treatment in women with
unexplained recurrent miscarriage: A randomized double-blind placebo-controlled trial. J Matern Fetal
Neonatal Med. 2018,31(3):388–94.
14. Carp H. A systematic review of dydrogesterone for the treatment of recurrent miscarriage. Gynecol
Endocrinol. 2015;31(6):422–30.
15. Tournaye H, Sukhikh GT, Kahler E, Griesinger G. A Phase III randomized controlled trial comparing
the efficacy, safety and tolerability of oral dydrogesterone versus micronized vaginal progesterone for
luteal support in in vitro fertilization. Hum Reprod. 2017;32(5):1019–27.
16. Osol G, Mandala M. Maternal uterine vascular remodeling during pregnancy. Physiology. 2009;24:58–71.
17. Ghosh S, Chattopadhyay R, Goswami S, Chaudhary K, Chakravarty B, Ganesh A. Assessment of sub-
endometrial blood flow parameters following dydrogesterone and micronized vaginal progesterone
administration in women with idiopathic recurrent miscarriage: A pilot study. J Obstet Gynecol Res.
2014;40(7):1871–6.
18. Saccone G, Schoen C, Franasiak JM, Scott RT Jr, Berghella V. Supplementation with progestogens in the
first trimester of pregnancy to prevent miscarriage in women with unexplained recurrent miscarriage: A
systematic review and meta-analysis of randomized, controlled trials. Fertil Steril. 2017;107(2):430–8e3.
19. Haas DM, Hathaway TJ, Ramsey PS. Progestogen for preventing miscarriage in women with recurrent
miscarriage of unclear etiology. Cochrane Database Syst Rev. 2018; Article ID CD003511.
20. Schindler AE, Carp H, Druckmann R et al. European Progestin Club Guidelines for prevention and
treatment of threatened or recurrent (habitual) miscarriage with progestogens. Gynecol Endocrinol.
2015;31(6):447–9.
21
Debate: Should Progestogens Be Used
in Recurrent Pregnancy Loss? No*
Roy Mashiach
Introduction
Removal of the corpus luteum before the end of the seventh week of amenorrhea leads to miscarriage. Rescue
can be achieved with progesterone therapy but not with estrogen [1]. Corpus luteum deficiency has been cited
as the underlying pathology in 35%–40% of unexplained recurrent pregnancy losses, manifesting in low
serum progesterone levels and out-of-phase endometrial biopsies [2,3]. However, women with no history of
recurrent miscarriage (RM) may exhibit endometrial histology suggestive of luteal phase deficiency in as
many as 50% of single menstrual cycles and 25% of sequential cycles [4]. A prevalence study of out-of-phase
endometrial biopsy specimens [5] failed to show any significant difference between fertile and infertile
patients and recurrent pregnancy loss, which calls the role of this intervention into question. In a series of
74 women with RM before 10 weeks of gestation, there was no difference in pregnanediol excretion curves
between those women who either miscarried or went on to have a successful pregnancy [5]. In fact, estriol
was a better prognostic indicator, showing lower values in those destined to miscarry.
Yan et al. [6] assessed midluteal serum progesterone measurements in a preconception cycle of 132
women with unexplained RM. Midluteal serum progesterone values were compared in women who had
a subsequent miscarriage and those who had a live birth. The serum progesterone concentration in the
live birth group (n = 86) and miscarriage group (n = 46) were 42.3 ± 2.4 nmol/L (mean ± SE) and
42.5 ± 3.2 nmol/L, respectively. Therefore, midluteal serum progesterone measurements did not predict
the outcome of a subsequent pregnancy. Ogasawara et al. [7] reported that a midluteal progesterone level
of <10 ng/mL (as a marker of luteal phase deficiency) did not predict a future pregnancy loss in women
with two successive unexplained first trimester miscarriages.
Progesterone may modulate the immune response required to achieve a successful pregnancy outcome.
Progesterone can upregulate the progesterone receptors on both decidual natural killer and placental
lymphocytes. Upregulated cells can synthesize progesterone-induced blocking factor (PIBF) mediating both
the immunomodulatory and anti-abortive effects of progesterone [8]. The cellular T cell system, in particular
the Th-1 cells, modulate this immune response releasing either Th-1 cytokines (such as TNFα) that induce
cytotoxic and inflammatory reactions, or Th-2 cytokines (e.g., IL 10) associated with B cell production [9].
Serum cytokine profiles demonstrate a shift toward Th-2 in normal pregnancy, whereas in those with RM,
the Th-1 response predominates [10]. It has been reported that administration of intramuscular progesterone
injections to RM patients restored levels of soluble TNF receptors to values seen in women with no such
history [11]. PIBF appears to be the main modulator of the actions of progesterone, with significantly lower
expression in RM patients compared to those with a healthy pregnancy [12]. In human pregnancy, serum
samples from patients with infertility and paid volunteers were evaluated for both PIBF and progesterone
at various times of the cycle, whether natural or involving embryo transfer after endogenous and exogenous
progesterone exposure and after various synthetic progestins. Progesterone alone without exposure to the
fetal allogeneic stimulus was able to produce a marked increase in serum PIBF [13].
* This chapter has been updated by Roy Mashiach from the original in the 2nd edition by Aisha Hameed, Shazia Malik,
and Lesley Regan.
202
Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? No 203
Clinical Data
Daya [14] presented the first meta-analysis of three controlled trials studying the efficacy of progesterone
support for pregnancy in women with a history of RM. Although none of the three trials [15–17] reached
statistical significance, the pooled odds ratio (OR) for pregnancies reaching at least 20 weeks’ gestation
was 3.09 (95% CI 1.28–7.42), indicating that progestogens had a significant effect. However, the three
trials in Daya’s [14] meta-analysis used different progestogens, implant, medroxyprogesterone acetate,
and 17-OH progesterone caproate. At that time no physiological progesterone was available for testing.
These data were again reviewed in the Cochrane meta-analysis published in 2003, which concluded that
there was a statistically significant reduction in miscarriage that favored those women in the progestogen
group (OR 0.37; 95% CI 0.17–0.91) [18].
A further analysis of the available trials drew attention to the small participant numbers and the
fact that the trials were of poor quality (the modified Jadad quality scores ranged from 0/5 to 2/5).
These authors conceded that there was a trend toward progesterone supplementation being of benefit,
with a 42%–69% reduction in the rate of miscarriage, but emphasized the wide confidence intervals
and the lack of statistically significant differences in all but one of the four studies. Furthermore,
they highlighted that no data were available for other important and clinically relevant outcomes
such as live birth [19]. The most recent Cochrane meta-analysis of four trials involving 225 women
with a history of three or more consecutive early miscarriages reported that progestogen treatment
is associated with a statistically significant decrease in the miscarriage rate compared to placebo or
no treatment (OR 0.39; 95% CI 0.21–0.72). However, once again the quality of the methodology was
considered to be poor [20].
The PROMISE Trial

In view of the poor quality of the trials, lack of homogeneity of the trials, different progestogens assessed,
and the small number of patients in each trial, it was felt necessary to have a high-quality, large, multicenter
trial to assess the effect of the physiological hormone progesterone. Hence the PROMISE trial [21] was
conceived. The trial was randomized and controlled. The primary outcome was to assess live births after
24 weeks of gestation, after vaginal micronized progesterone in a large cohort of women (836) with three
or more miscarriages compared to placebo. A large dose of progesterone was used (800 mg) in order to
avoid using a suboptimal dose and confounding the results. The trial was multicenter and multinational.
There was no benefit found to the use of micronized progesterone. In an intention-to-treat analysis, 404
women were randomly assigned to receive either progesterone or placebo (432 women). The incidence of
live births was 65.8% (262 of 398 women) in the progesterone group and 63.3% (271 of 428 women) in
the placebo group (RR 1.04; 95% CI 0.94–1.15). Hence, the authors concluded that progesterone in the
first trimester of pregnancy did not result in a significantly higher rate of live births among women with
a history of unexplained RMs.
Criticism of the PROMISE Trial

Subgroup Analysis
The PROMISE trial has been criticized as there was no subgroup analysis performed for genetic
aberrations in the embryo. As the number of patients was large (836), randomization should have allocated
equal numbers of patients of each subgroup to each treatment arm of the trial. When various personal
demographic data were analyzed, the two patient groups were found to be similar for various prognostic
factors such as maternal age, ethnic race, and parity. In addition, there was a subgroup analysis for patients
with four or more miscarriages. There was a 45.3% live birth rate for treated patients with four or more
miscarriages (183/404) compared to 44.4% (192/432) in the control group. Therefore, the question as to
whether the physician who is faced with a patient with three or more miscarriages should be prescribed
progesterone can be answered with a definite no.
Commencement of Therapy
Treatment was commenced in the PROMISE trial as soon as pregnancy was diagnosed. It has been claimed that
progestational changes occur in the luteal phase, and that treatment may have commenced too late to show a
significant benefit. Stephenson et al. [22] have shown that when micronized progesterone is commenced in the
luteal phase, the pregnancy success was higher in women prescribed vaginal micronized progesterone compared
with controls: 68% (86/126) versus 51% (19/37); (OR 2.1; 95% CI 1.0–4.4). However, it is impossible to tell
when conception will occur in women with prior pregnancy losses. Women cannot be treated with progesterone
supplementation in all cycles until conception. It may sometimes take many years for women to conceive,
particularly in the older age groups, or women miscarrying after artificial reproductive technology.
Other Progestogens
The role of the PROMISE study was to assess progesterone, and not other progestogens. Other progestogens
should be assessed, as progestogens do not have a class action, and each has different pharmacological
actions. However, other progestogens should be subject to the same scrutiny as has progesterone. There are
no other trials of the progestogens used in Daya’s [14] meta-analysis (implant, 17-hydroxyprogesterone
caproate, and medroxyprogesterone acetate). Each of these progestogens was individually found to have
no beneficial effect. The only progestogen for which there is some evidence of effect is dydrogesterone.
The debate for the use of progestogens uses Kumar et al.’s [23] double-blind randomized study as a basis
for recommending dydrogesterone. The reader is left to decide whether this paper and several smaller
studies included in meta-analyses are sufficient to warrant the use of dydrogesterone. However, it must
be borne in mind that Kumar et al. [23] commenced therapy when a fetal heart was detected, i.e., after
blighted ova (which constitute a significant number of RMs) had already been aborted.
The PRISM Trial

Similarly to the PROMISE trial, the PRISM trial [24] was a randomized control trial to assess vaginal
micronized progesterone in women with threatened miscarriage. Although the results showed micronized
progesterone to have no significant benefit, it was indeed surprising that micronized progesterone had a
significant beneficial effect in the subgroup of women who had threatened miscarriage after RM (RR 1.28;
95% CI 1.08–1.51; p = 0.007), thus partially contradicting the results of the PROMISE study. However,
it is conceivable that women with threatened miscarriage after RM represent a subgroup of women who
may have a true progesterone deficiency and can respond to progesterone therapy.
A recent study that used 2.5-million patient database compared the rates of congenital malformations
among babies exposed in utero during the first trimester of pregnancy to Dydrogesterone to a comparison
group not receiving this medication. There were 8508 children exposed to Dydrogesterone during the first
trimester of pregnancy (4417 males, 4091 females) out of 777,422 cases in the database. Dydrogesterone
exposure was associated with increased risk for hypospadias (OR 1.28; 95% CI 1.06–1.55), for overall
cardiovascular malformations (OR 1.18; CI 91.06–1.33), spina bifida (OR 2.29; CI 1.32–3.97), and
hydrocephalus (OR 2.04; CI 1.28–3.25). In a sensitivity analysis, including cases exposed to IVF and ART
in addition to Dydrogesterone, there were also increased risks for cryptorchidism (OR 1.37; CI 1.19–1.58)
and congenital dislocation of the hip (OR 1.58; CI 1.42–1.78).
Conclusions
The number of studies examining the efficacy of progesterone supplementation in early pregnancy are
few. Prior to the PROMISE study, the total number of participants was small and did not fulfill the criteria
required to generate meaningful results. The PROMISE study has shown that micronized progesterone
supplements are not effective. The role of other progestogens remains to be elucidated; however, there is
some evidence in favor of dydrogesterone and micronized progesterone in the subgroup of patients with
threatened miscarriage in addition to RM. Importantly, although no obvious adverse effects to mother
or fetus have been reported, there are reports of the antiandrogenic effects of progesterone leading to
hypospadias, cardiovascular malformations, spina bifida, and hydrocephalus [25,26].
Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? No 205
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a precipitous rise in the immunomodulatory protein the progesterone induced blocking factor (PIBF). J Assist Reprod
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22
Human Chorionic Gonadotropin Supplementation
in Recurrent Pregnancy Loss
Carlo Ticconi, Adalgisa Pietropolli, and C.V. Rao
Introduction
Assessing the role of human chorionic gonadotropin (hCG) in recurrent pregnancy loss (RPL) remains
a challenge. Assessment is beset with numerous problems: (a) the lack of a uniform definition of RPL (it
was only very recently that two consecutive miscarriages have been considered to be RPL) [1,2] rather
than three or more, which was the previous definition; (b) the multifactorial etiology that renders women
with RPL a non-homogeneous group in which more than one risk factor may be involved [3,4], e.g.,
previous studies have been carried out while failing to exclude women with antiphospholipid antibodies
or embryonic aneuploidy; (c) different diagnostic protocols are used by various RPL clinics [5,6]; (d)
the difficulties and challenges involved in carrying out high quality studies on women with RPL [4,7];
and—perhaps even more important—(e) the extreme complexity of factors involved in successful embryo
implantation and pregnancy establishment and maintenance.
Despite the research advances on the mechanisms leading to RPL, nearly 50% of all RPLs are still
considered “unexplained” [5,8–9]. Most of the trials on hCG were performed with urinary hCG at a time
when the diagnostic criteria used today were not available. Although the administration of hCG would
appear to be a logical option for the treatment of women with RPL in which there is no evidence of genetic
or other causes, only a few well-designed clinical studies on hCG supplementation have been performed.
It is against these uncertain backgrounds that hCG supplementation must be judged.
hCG is a molecule of paramount importance for successful human pregnancy. It is involved in numerous
actions in pregnancy initiation, maintenance, and development. The function of hCG continues throughout
pregnancy up until delivery. In the present chapter, the possibilities and limitations of the therapeutic
use of hCG in women with RPL are examined in light of the physiological actions of hCG as well as the
available clinical evidence concerning its utilization in early pregnancy.
Actions of hCG in Pregnancy

The hCG Molecule(s)
hCG is a heterodimeric glycoprotein, composed of noncovalently linked α and β subunits. The α subunit
is common for TSH, FSH, hCG, and luteinizing hormone (LH) and encoded by a single gene (CGA),
located to chromosome 6qq2.1–23. The β subunit of hCG is encoded by a cluster of six different non-allelic
genes, CGB1, CGB2, CGB3, CGB5, CGB7, and CGB8, located on chromosome 19q13.32 [10–11]. The β
subunit of hCG shares 82% homology with that of LH and binds to the same plasma membrane LH/hCG
receptors, which is a seven-transmembrane G-coupled protein [12–13]. Even though LH and hCG share
common receptors, there is clear evidence that some of the downstream intracellular pathways activated
by them are different [10,14–15]. While LH is present in all species, hCG is only found in primates. hCG
seems to have developed relatively recently in evolution, arising from LH. hCG has become responsible
for multiple roles in the establishment, development, and maintenance of human pregnancy [10,15]. The
206
Human Chorionic Gonadotropin Supplementation in Recurrent Pregnancy Loss 207
Cell proliferation, migration

pERK 1/2
and invasion
DAG Cell proliferation

PLC Ca2+
IP3 Steroidogenesis
Cell proliferation
LH/hCG
hCG receptor Anti-apoptotic effect
Adenyl- Cytotrophoblasts fusion
cAMP PKA
cyclase Steroidogenesis
Relaxation of uterine arteries
Cell proliferation, survival

PI3K PIP3 AKT and growth
Cell cycle progression
Angiogenesis
FIGURE 22.1 Schematic representation of the major intracellular signaling pathways activated by hCG after binding to
its LH/hCG receptors. Pathways overlap in the mediation of some of the physiologic effects. ERK 1/2 = phosphorylated
extracellular signal-regulated kinases 1 and 2; PLC = phospholipase C; DAG = diacylglycerol; IP3 = inositol
trisphosphate; Ca = free intracellular calcium; cAMP = cyclic adenosine monophosphate; PKA = protein kinase A;
PI3 K = phosphatidylinositol 3-kinase; PIP3 = phosphatidylinositol-3,4,5-bisphosphate; AKT = protein kinase B.
major intracellular signaling pathways activated by hCG binding to the LH/hCG receptors, together with
their known cellular effects, are depicted in Figure 22.1.
hCG is a highly glycosylated molecule with about 30% of its molecular weight accounted for by
carbohydrate moieties [16]. It exists at least in five isoforms: hCG, sulfated hCG, hyperglycosylated hCG
(H-hCG), hCG free β-subunit, and free β-subunit of H-hCG. The distinct biological activities of different
isoforms may vary but are not completely understood.
The actions of hCG in fetoplacental tissues are paracrine and autocrine in nature. Its actions on target
tissues such as the corpus luteum, and various non-gonadal tissues are endocrine in nature. Recently, some
reports have suggested that regular hCG and H-hCG have a different non-overlapping cellular origin in the
placenta and have separate roles during pregnancy. Regular hCG induces the secretion of progesterone,
promotes angiogenesis, causes differentiation of trophoblast cells, and prepares the endometrium for the
implanting embryo, while H-hCG enhances the implantation by promoting the growth and invasion of
cytotrophoblast cells. In addition, H-hCG has been suggested to activate the TGFβII receptors, while
regular hCG uses the classical hCG/LH receptors [17–21]. These suggestions were based on refutable data.
Actions of hCG during Pregnancy

hCG, produced by syncytiotrophoblasts, exerts a variety of actions [22] aimed at supporting embryo
implantation, promoting pregnancy maintenance, and pregnancy continuation. Functional hCG/LH
receptors are expressed in many fetal and maternal tissues, including cells of the immune system. The
actions are summarized in Table 22.1. hCG is involved in decidualization of endometrial stromal cells,
fertilization of the gametes, endometrial implantation of the blastocyst, and continuation of pregnancy
until delivery [49–50]. Some of the hCG actions are directed toward the ovary, endometrium, placenta,
fetal membranes, myometrium, and maternal immune system, while others are directed toward multiple
other targets tissues in the mother and fetus.
Actions on the Corpus Luteum

In addition to stimulating the corpus luteum to produce progesterone, hCG stimulates the secretion of
relaxin, inhibin, and prostaglandin E2 production. Relaxin has been reported to decrease endometrial
208
TABLE 22.1
Summary of the Major Pregnancy-Promoting Actions of hCG
Action Cells/Tissue Target Type of Action Notes References
Stimulation of progesterone production Corpus luteum Endocrine This action is exerted during the first 8–10 weeks of [23]
gestation
Stimulation of progesterone production Syncytiotrophoblasts Autocrine/paracrine This action is exerted throughout pregnancy [24,25]
Stimulation of cytotrophoblast Cytotrophoblasts Paracrine This action is rapid during early pregnancy and tapers [26]
differentiation into syncytiotrophoblasts off subsequently
Stimulation of uterine vasculature Uterine arteries Paracrine This process is robust during the first half of pregnancy [27,28]
angiogenesis and blood flow and then slows down
Enhancement of umbilical circulation and Umbilical cord Placenta Paracrine and autocrine Throughout pregnancy [29]
placental growth
Inhibition of myometrial contractility Myometrium Paracrine Throughout pregnancy until the labor begins to evolve [30–34]
Enhancement of embryo implantation Endometrium Paracrine/autocrine Induction of endometrial decidualization and synchrony; [13,35–38]
reprogramming of stromal development
Immunoregulatory action at the Immune system cells Paracrine/ endocrine Stimulation of uterine NK cells [39–44]
maternal-fetal interface Generation of tolerogenic DC
Promotion of Tregs
Modulation of cytokine production and upregulation of
2,3 indoleamine dioxygenase in syncytiotrophoblasts
Anti-apoptotic and decidualization effects Endometrium Paracrine Especially Important during early pregnancy [45–47]
Enhancement of trophoblast invasiveness Extravillous trophoblasts Autocrine/paracrine H-hCG was suggested to have a dominant role; however, [11,17,18,48]
the contribution of regular hCG should not be ruled out
Abbreviations: NK, natural killer lymphocytes; DC, dendritic cells; Tregs, regulatory T lymphocytes; H-hCG, hyperglycosylated hCG.
levels of matrix metalloproteinases 1 and 3 resulting in maintenance of endometrial collagen content

[51]. hCG stimulates prostaglandin (PG) E2 production by upregulating cyclooxygenase-2 [47] and
17β-estradiol secretion through a cyclic adenosine monophosphate-mediated pathway [52].
Uterine Actions
In the 1990s there were some reports that hCG upregulates endometrial VEGF secretion [27], enhancing
the growth of new blood vessels toward the developing conceptus, and later vascular remodeling of the
spiral arteries to uteroplacental arteries. hCG is involved in the differentiation of human endometrial
stromal cells into decidua. Myometrial smooth muscle cell contractions seem to be inhibited by hCG,
possibly due to downregulation of the gap junctions.
Immune Actions
hCG may have an immunoregulatory role. There is evidence that an appropriate balance between
TH-1 and TH-2 cytokines may be necessary for the maintenance of pregnancy. TH-2 cytokines such as
interleukin (IL)-3, granulocyte macrophage colony-stimulating factor (GM-CSF), and epidermal growth
factor (EGF), which stimulate placental cell proliferation [53] in vitro, may enable the trophoblast to
secrete its hormones such as hCG and hPL [54]. Uzumcu et al. [55] assessed endometrial production of
cytokines when stimulated by hCG. Increasing doses of hCG caused a dose-dependent increase in TNFα
and IL-6 secretion. hCG has also been reported to stimulate secretion of IL-1β, and inhibit IL-2 expression
by human monocyte cells in culture [55].
Clinical Studies on hCG Use in Early Pregnancy

Based on the above pregnancy-promoting actions of hCG, various studies have been carried out to explore
the possible benefits of the therapeutic use of hCG during early pregnancy. To date, the results of these
studies show a trend toward a benefit, but they are still considered insufficient to permit firm conclusions.
However, it must be kept in mind that the studies performed to date have been used in all patients with a
clinical diagnosis of RPL, rather than to investigate clinical effectiveness in a subset of patients who are
most likely benefit from treatment.
hCG in Assisted Reproductive Technologies

Many studies have been performed to investigate the effect of hCG for luteal support in women undergoing
assisted reproductive technologies (ARTs). These studies have been reviewed in a recent Cochrane review
along with the use of progesterone [56]. The analysis concluded that luteal phase hCG supplementation
may be associated with a higher incidence of live births compared with placebo, although the evidence
was not conclusive. However, if hCG supplementation is used, there may be an increased incidence of
ovarian hyperstimulation syndrome [56]. A more recent and larger Cochrane review, which included
12 randomized controlled trials (RCTs) carried out on 4038 subjects on the intrauterine administration
of hCG in sub-fertile women undergoing ART, concluded that pregnancy outcome following a dose of
500 IU or greater is promising when embryo transfer was performed at cleavage stage [57]. However, due
to the high risk of bias found in 9 out of 12 of the selected studies and the fact that positive results were
obtained in a subgroup analysis, the authors concluded that caution should be used before recommending
large-scale hCG use. Other more recent prospective studies suggest a significant beneficial effect of the
intrauterine administration of hCG in women with recurrent implantation failure [58,59].
Threatened Miscarriage
A Cochrane analysis has reviewed three RCTs (312 participating women) on the use of hCG in the treatment
of threatened miscarriage [60]. One of the studies was considered to have used poor methodology. The
other two studies could not support the routine use of hCG [60]. Hence the literature does not support
the use of hCG for threatened miscarriage at present. It is interesting to point out that none of the above
meta-analyses reported any adverse effects of hCG.

Two meta-analyses have been performed to investigate the effectiveness of hCG in preventing subsequent
miscarriage in women with RPL [61,62]. In the year 2000, Scott and Pattison [61] selected and reviewed
four RCTs and found that the hCG use was associated with a reduced risk of miscarriage in women with
a history of RPL (OR 0.26%; 95% CI 0.14–0.52). However, they urged caution in interpreting the results
due to the poor methodological quality of two of the included studies. The overall conclusion was that
“there was not enough evidence to evaluate the use of hCG during pregnancy to prevent miscarriage in
women with a history of unexplained recurrent spontaneous miscarriage” [61].
In an updated Cochrane meta-analysis, Morley et al. initially selected 12 studies for assessment
[62]. However, five were excluded due to concurrent fertility treatment [63], patient selection bias [64],
concurrent administration of other drugs [65], lack of either a placebo or untreated control group [66],
and non-applicable study design [67]. Two additional studies were not included as they were awaiting
classification at the time of the analysis [68,69]. An additional RCT by Bailie and Sadler [70] seems to
have been missed from both Cochrane meta-analyses. The initial analysis performed on five studies
showed a statistically significant benefit of hCG treatment in reducing subsequent miscarriages in women
with RPL [71–75] (Table 22.2). However, due to statistical heterogeneity in the combined comparison
(I2 = 39%), a successive analysis was performed by excluding two of the older studies [71,72]. This
restricted analysis revealed a statistically non-significant effect of hCG with a pooled RR = 0.74 (95% CI
0.44–1.23). However, it is important to point out that all the published studies show benefit. So, the loss
of significance is more related to the removal of numbers than the removal of bias papers. The authors’
conclusion was that there is a statistically non-significant trend toward a benefit with the use of hCG to
prevent further pregnancy loss in women with a history of RPL. Hence there is still insufficient evidence
to support the use of hCG supplementation in women with RPL of unknown etiology in clinical practice
[62]. Again, no detrimental effects of hCG were observed.
In addition to the above meta-analyses, other non-randomized studies on hCG treatment in RPL suggest
a beneficial effect. In a series of 328 women, 199 of which were treated with u-hCG and 129 were
untreated controls, Carp observed a statistically significant benefit of 15% by using hCG (OR 1.88%;
95% CI 1.16–3.04) [76]. This result was less than the expected 20% benefit, based on a theoretical model
developed by the author [77]. However, when the analysis involved only the subgroup of women with five
or more miscarriages—those with a “poor” prognosis—the beneficial effect of hCG was more evident
with an absolute benefit of 34% (OR 4.33%; 95% CI 1.7–11.3) and fitted the above theoretical model [77].
TABLE 22.2
Randomized Clinical Trials Included in the Cochrane Systematic Review by Morley et al. [62] on the
Therapeutic Use of hCG to Prevent Miscarriage in the Successive Pregnancy in Women with RPL
Number of Outcome (Number
Women (hCG of Miscarriages in
Authors (Year Treated/ Type of hCG hCG-Treated/
of Publication) Controls) Notes Used Controls) Risk Ratio (95% CI) Reference
Svigos (1982) 13/15 No treatment u-hCG (Pregnyl®) 1/9 RR = 0.13 (0.02–0.51) [71]
Harrison (1985) 10/10 Placebo u-hCG (Profasi®) 0/7 RR = 0.07 (0.00–1.03) [72]
Harrison (1992) 36/39 Placebo u-hCG (Pregnyl®) 6/8 RR = 0.81 (0.31–2.11) [73]
Quenby (1994) 42/39 Placebo u-hCG (Profasi®) 6/6 RR = 0.93 (0.33–2.64) [74]
El-Zibdeh 50/48 No treatment u-hCG (Profasi®) / 9/14 RR = 0.62 (0.30–1.29) [75]
(2005) (Pregnyl®)
Total 151/151 22/44 RR = 0.51 (0.32–0.81)
Carp’s [76] study has been included, together with Sadler and Baillie’s study [70], in an updated meta-
analysis by Walker [78]. Included in Walker’s report were 671 women (hCG-treated and controls), in
which the RR was a statistically significant 0.44 (95% CI 0.31–0.63).
Another retrospective cohort study on hCG supplementation (with a single injection in the midluteal
phase) in women with unexplained RPL has been recently published by Fox et al. [79]. Ninety-eight
women with RPL, defined as two or more consecutive first trimester losses, received either hCG (r-hCG/u-
hCG; Pregnyl®/Novarel®) as midluteal phase support or no treatment in monitored cycles. The results
suggested a beneficial effect of hCG. Among all the variables considered, only the use of hCG was
associated with a statistically significant successful ongoing pregnancy rate (OR 4.65; 95% CI 1.61–11.69).
Moreover, the use of hCG resulted in an increased RR of 2.4 (95% CI 1.3–4.5) for a successful pregnancy
and a reduction of 38% of the risk of miscarriage (RR 0.38%; 95% CI 0.19–0.76) [79]. However, it must
be kept in mind that luteal phase hCG may cause an erroneous diagnosis of “biochemical pregnancy”
when no pregnancy exists.
Conclusions and Future Directions

hCG is essential for human pregnancy initiation and maintenance. The successful use of hCG in the
prevention of RPL in a subset of women presumes that hCG levels either are inadequate and/or the
quality of hCG is compromised. In either case, exogenous hCG administration is expected to compensate
for the endogenous hCG. The therapeutic use of hCG in early pregnancy complications, particularly
RPL, has been limited to studies that show a possible and/or potentially beneficial effect. The Cochrane
meta-analyses of hCG treatment have drawn equivocal conclusions about the significant benefit in RPL.
However, the current evidence is hampered by the small number of studies performed, heterogeneity
between the studies, and patient selection. Hence the most recent guideline on RPL stated “There is
insufficient evidence to recommend the use of hCG to improve live birth rate in women with RPL and
luteal phase insufficiency” [2].
In our opinion, the considerable body of evidence regarding the beneficial effect of hCG supplementation
cannot be ignored, but there is an urgent need to carry out further well-designed RCTs to confirm or refute
the clinical benefit of hCG supplementation in the prevention of miscarriages in RPL. All the previous
studies carried out so far, with the partial exception of Fox et al. [79], have used urinary hCG. As women
with RPL are a heterogeneous group, it is possible that specific subsets of women with unexplained RPL
(e.g., women with high numbers of pregnancy losses) could significantly benefit from the hCG treatment
rather than all RPL patients. Until such studies are available, clinicians should consider using hCG when
they believe that there may be a significant benefit.
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23
Antiphospholipid Syndrome: Management
of the Obstetric Patient
Ashley E. Benson and D. Ware Branch
Introduction and Background

Antiphospholipid syndrome (APS) is an autoimmune disease characterized clinically by thrombotic or
obstetric morbidities and associated with the presence of circulating antiphospholipid (aPL) antibodies.
The diagnosis is confirmed when the aPL antibodies are shown to be repeatedly positive in significant
titer as measured at least 12 weeks apart [1]. aPL antibodies comprise a group of autoantibodies that bind
to glycoprotein moieties complexed with phospholipids. Current international diagnostic consensus [1]
holds that there are three relevant aPL antibodies: lupus anticoagulant (LAC), anticardiolipin (aCL), and
anti-β2-glycoprotein-I (aβ2-GP-I) antibodies.
APS-related thrombosis may manifest in virtually any arterial or venous vascular bed. The most
common thrombotic presentation is deep venous thrombosis (DVT), especially DVT of the lower
extremities. The most common arterial thrombotic presentations are stroke and transient ischemic attack
(TIA) [2,3]. Experts have estimated that 6%–15% of DVT cases are associated with positive aPL antibody
results and thus primarily attributable to APS [3], while 9%–24% of stroke/TIA are associated with
positive aPL antibody results [3,4]. Superficial venous thrombosis is not considered a clinical criterion
for APS.
The obstetric clinical criteria for the diagnosis of APS include [1]:
• Recurrent (three or more consecutive) pre-embryonic or embryonic miscarriage <10 weeks’

gestation (recurrent early miscarriage)
• One or more otherwise unexplained fetal deaths ≥10 weeks’ gestation
• Delivery prior to 34 weeks for preeclampsia or placental insufficiency
These obstetric clinical criteria are relatively nonspecific in nature, each having numerous contributing
or etiologic factors (e.g., maternal age for recurrent early miscarriage or nulliparity for preeclampsia).
Existing literature describing the association of aPL antibodies with the clinical criteria for APS has
significant limitations. These limitations pertain to the variety and number of aPL antibody tests used,
the definition of positive results, the methods of establishing thresholds for positive results, the lack of
confirmatory testing in many studies, and the nature of the study designs [3,5,6]. Against this background,
the actual relationship between each of the obstetric clinical criteria and aPL antibodies deserves ongoing
investigation.
In the authors’ referral practice experience, recurrent early miscarriage is the most common obstetric
clinical criterion for which APS is diagnosed. Based on existing literature, a reasonable estimate is that
2%–6% of women with recurrent early miscarriage have positive aPL antibody results [3]. Some experts
[7–9], including our group at the University of Utah [10], have found that fewer than 5% of women with
recurrent miscarriage and no other obvious autoimmune or thrombotic disease features have aPL results
meeting international consensus criteria [1]. Since several percent of otherwise healthy subjects have
positive aPL antibody results [11,12], further study to determine the exact relationship between recurrent
early miscarriage and aPL antibodies would seem in order.
215
Fetal death and early delivery for severe preeclampsia and/or placental insufficiency are widely
considered more specific clinical features of APS [2]. Regarding otherwise unexplained fetal death, one
case-control study of over 100 women with fetal death after 22 weeks’ gestation and over 250 controls
found a non-significant OR of 2.0 (95% CI 0.9–4.8) for at least one positive aPL result, but the OR
was 4.3 (95% CI 1.0–18.4) for LAC [13]. The Stillbirth Collaborative Research Network’s multicenter,
population-based, case-control study of stillbirths and live births [14] found positive tests for aPL (aCL
or aβ2-GP-I antibodies) in 9.6% of fetal death cases ≥20 weeks of gestation. After excluding cases that
were otherwise explicable, positive results for IgG aCL and IgM aCL antibodies were associated with a
fivefold odds and twofold odds of stillbirth, respectively, while IgG aβ2-GP-I antibodies were associated
with threefold odds of stillbirth. Two prospective observational studies of women with well-characterized
APS noted fetal deaths in more than 10% of cases despite treatment with a heparin agent and low-dose
aspirin (LDA) [15,16].
Early studies of the association between aPL antibodies and early delivery (<34 weeks) for severe
preeclampsia suggested that approximately 8%–15% of such cases test positive [17–20]. The two
prospective, observational studies mentioned in the preceding paragraph found that 9%–10% of women
with well-characterized APS develop severe preeclampsia in their observed pregnancy in spite of treatment
with heparin or low molecular weight heparin (LMWH) and LDA [15,16]. Placental insufficiency in the
absence of preeclampsia is less well studied. A prospective case-control study of women delivered prior
to 36 weeks of gestation for severe preeclampsia or placental insufficiency found that just over 10% of
cases were positive for aPL antibodies compared to less than 2% of controls [21].
Laboratory Considerations in the Diagnosis of APS

The diagnosis of APS depends upon the detection of LAC and/or aCL and/or anti-β2GPI antibodies in
medium-to-high titer. Because transient positive test results may occur in non-autoimmune conditions, the
classification of APS requires two positive aPL results at least 12 weeks apart [1]. All three tests (LAC,
IgG and IgM aCL, and IgG and IgM anti-β2GPI antibodies) are required to assess the aPL profile and to
categorize the patients according to the presence of one or more positive test [1,22]:
• Category I: More than one laboratory criteria present (any combination)

• Category IIa: LAC present alone
• Category IIb: aCL antibody present alone
• Category IIc: Anti-β2GPI antibody present alone
The presence of LAC (i.e., either category I or IIa) is the single most important antibody risk factor for
thrombosis and for second and third trimester pregnancy complications [15,23]. Individuals meeting
international laboratory criteria for all three aPL antibodies, LAC, moderate-to-high titer aCL, and
moderate-to-high titer anti-β2GPI, are known as “triple positive.”
Management of APS with Regard to Pregnancy

Preconception assessment should include determination of aPL antibody status, keeping in mind that
the diagnosis of definite APS requires repeated positive test results meeting international criteria as
determined at least 12 weeks apart. A sense of patient risk stratification is important:
• Women with LAC or “triple” positivity for the three aPLs should be counseled that adverse
pregnancy outcomes, including fetal death and early delivery for severe preeclampsia or
placental insufficiency, occur in at least one-third of cases in spite of standard treatments
[15,24,25]. Women who are negative for LAC (and hence are not triple positive) generally have
good outcomes using standard treatments.
Antiphospholipid Syndrome: Management of the Obstetric Patient 217
• A maternal history of thrombosis or another autoimmune condition (e.g., systemic lupus) also
places the patient at increased risk of second or third trimester adverse pregnancy outcomes
[15,26], again in the setting of standard treatments.
• Women with chronic hypertension or renal insufficiency are at increased risk for adverse
pregnancy outcomes.
Optimal management of APS during pregnancy would minimize the risks of adverse maternal and
fetal/neonatal outcomes. Maternal risks include APS-associated thromboembolism, catastrophic APS,
and risks associated with gestational hypertensive disease. Fetal/neonatal risks include miscarriage, fetal
death, and risks associated with early delivery. The current treatment of choice for APS pregnancy
is heparin or LMWH and LDA. This regimen certainly provides maternal thromboprophylaxis and
may improve pregnancy outcomes. Experts recommend preconceptional LDA because of its possible
beneficial effect on early stages of implantation and that it may improve live birth rates [15]. Heparin, or
more usually LMWH, is started in the early first trimester after demonstrating either an appropriately
rising hCG or an ultrasound-proven intrauterine live embryo.
APS patients with a history of thrombosis, most of whom are maintained on long-term anticoagulation,
require transitioning from their long-term anticoagulation agent to therapeutic levels of LMWH prior to
or very early in pregnancy (Table 23.1). APS patients without a history of thrombosis are treated with
thromboprophylactic-dose LMWH and LDA.
TABLE 23.1
Treatment of APS during Pregnancy
Clinical Manifestation of APS Treatment Options Comment
APS with history of thrombosis Patient on long-term Patients with thrombotic APS are at risk
anticoagulation: Full- for recurrent thrombosis and are most
anticoagulation-dose low often managed using long-term
molecular weight heparin agent anticoagulation, e.g., with warfarin.
and low-dose aspirin. Warfarin should be discontinued prior to
Patient not on long-term 6 weeks’ gestation to avoid risk of
anticoagulation: Intermediate warfarin embryopathy.
dose or full-anticoagulation dose
low molecular weight heparin
agent and low-dose aspirin.
APS without a history of thrombosis
Recurrent early miscarriage Low-dose aspirin or prophylactic- With regard to recurrent early miscarriage,
dose low molecular weight heparin some studies show high rate of
agent and low dose aspirin. successful pregnancy on LDA alone and
others show no benefit to the addition of
a heparin agent (see text for further
discussion).
History of fetal death or history of Prophylactic-dose low molecular The evidence that a heparin agent
early delivery for severe weight heparin agent and improves pregnancy outcome in women
preeclampsia or placental low-dose aspirin. with a history of fetal death or early
insufficiency delivery for preeclampsia or placental
insufficiency is of low quality.
Women with repeatedly positive LAC
results or repeatedly positive for LAC
and moderate-to-high titers of aCL or
aβ2-GP-I antibodies are likely at
increased risk for thrombosis during
pregnancy; prophylactic-dose low
molecular weight heparin agent should
be considered.
TABLE 23.2
Selected Features of Heparin Agent and Low-Dose Aspirin (LDA) Treatment Trials
% Live Births % Live Births
Positive aCL IgG Positive CL IgM with Heparin with Heparin
Study (year) N (GPL Units) (MPL Units) LAC and LDA and LDA
Kutteh (1996) 50 ≥27 ≥27 +LAC excluded 80% 44%
Rai (1997) 90 >5 >5 RVVT 71% 42%
Farquharson (2002) 98 >9 >5 DRVVT 78% 72%
Goel (2006) 72 >17 Not done Not done 85% 62%
Laskin (2009) 42 >15 >25 DRVVT, PTT-LA, 77% 76%
DiPT, KCT
Alalaf (2012) 141 >15 >25 aPTT, KCT, 86% 72%
DRVVT, DiPT
Median (range) 79% (71%–86%) 67% (42%–76%)
Abbreviations: aPPT, activate partial thromboplastin time; DiPT, dilute prothrombin time; DRVVT, dilute Russell venom time;
KCT, Kaolin clotting time; PTT-LA, partial thromboplastin time–lupus anticoagulant sensitive; RVVT, Russell viper
venom time.
Randomized heparin or LMWH treatment trials of pregnant women with APS have involved patients
with predominantly recurrent early miscarriage [27–32]. Four of these trials found that the addition of a
heparin agent to LDA resulted in a higher live birth rate, though the range of live births in the treatment arms
of these studies varied considerably (Table 23.2). Two of these trials [28,30] proved negative, finding no
benefit to the addition of LMWH to LDA; in these studies, the live birth rates in the LDA-only patients were
quite good (70%–75%). Successful pregnancy outcomes in excess of 70% also have been reported among
APS patients predominantly with recurrent early miscarriage who were treated with LDA alone [33,34].
Two studies comparing unfractionated heparin to LMWH, each paired with LDA, found no difference in
pregnancy outcomes, again among APS patients predominantly with recurrent early miscarriage [35,36].
Experts have criticized the existing trials as highly heterogeneous with regard to clinical events (e.g.,
number of previous pregnancy losses, gestational ages of pregnancy losses) and laboratory criteria (e.g.,
different thresholds for positive test results, inclusion of patients with low titers, and lack of confirmatory
testing) [5,6], and lack of exclusion of embryonic aneuploidy as a cause of miscarriage. Moreover, several
of the trials [27–29] were completed before the publication of the current international consensus criteria
[1], and many of the subjects included in each of the published trials would not meet the current consensus
criteria for definite APS. With regard to APS diagnosed because of fetal death or previous early delivery
due to severe preeclampsia or placental insufficiency, treatment trials are simply lacking.
Against this background, a critical assessment would conclude that the efficacy of current recommended
treatment regimens to prevent adverse obstetric outcomes is somewhat uncertain. However, several clinical
points deserve consideration. First, women with APS and prior thrombosis should be treated with appropriate
anticoagulant agents during pregnancy and the postpartum period [37,38]. Second, women positive for LAC
or “triple” positive for aPL, and perhaps those repeatedly positive for medium-to-high titer aCL or aβ2-GP-I
antibodies, are at increased risk for pregnancy-associated thrombosis [39]; in these patients, clinical judgment
favors treatment with heparin or LMWH during pregnancy and the postpartum period. Finally, many women
suspected to have APS-related adverse pregnancy outcomes will choose treatment over no treatment if
the regimen is known to be relatively safe. Experience strongly suggests that thromboprophylactic-dose
anticoagulants, particularly LMWH, are very unlikely to cause untoward side effects such as osteopenia
[40,41], clinically significant bleeding, or heparin-induced thrombocytopenia [42] when properly managed.
Obstetric Care in APS Pregnancy

Obstetric morbidity in women diagnosed with APS varies depending upon patient history and laboratory
features. Otherwise healthy women who meet the criteria for APS because of recurrent early miscarriage
are at no more than modest risk for second or third trimester adverse outcome such as fetal death,
preeclampsia, and placental insufficiency. For example, in the NOH-APS observational study [16], severe
preeclampsia occurred in 5% of women diagnosed with APS because of recurrent early miscarriage
(compared to 1.6% of controls). In contrast, 14% of women with prior fetal loss had severe preeclampsia
in the study pregnancy. The prospective, observational PROMISSE study found that nearly 20% of
women with APS suffered an adverse outcome (fetal death or early delivery for preeclampsia or placental
insufficiency) despite treatment with a heparin agent and LDA, and outcomes were twofold worse for
women with LAC or prior thrombosis [15]. Given the risk profile for women with APS, experts recommend
frequent prenatal care visits, serial obstetric sonography, monitoring of maternal blood pressure, and
fetal surveillance beginning at 32 weeks, or earlier if clinical concerns arise. Periodic monitoring of the
relevant maternal laboratory results, including maternal platelet counts, is prudent.
High-Risk APS and Refractory Cases

As noted in the preceding sections, women with APS can be stratified according to adverse pregnancy
outcome risk on the basis of certain laboratory and clinical features. For example, in the PROMISSE
study, the 64 women with repeatedly positive LAC results had a 39% rate of fetal death, preterm delivery
prior to 34 weeks due to gestational hypertension or placental insufficiency, small for gestational age
infant, or neonatal death linked to early delivery [15]. The retrospective PREGNANTS study [43] of
750 women with aPL antibodies and at least one clinical feature of APS found that women who were
triple-positive for aPL antibodies had only a 30% rate of successful pregnancy in spite of treatment with
a heparin agent and LDA. In contrast, APS patients with low antibody titers [44], IgM isotype anti-
β2GPI antibodies [45], or a single-positive aCL or aβ2GPI antibody result in 77%–97% rate of successful
pregnancy [15,26,46]. With regard to clinical features, treated APS pregnancies are more likely to result
in adverse outcomes among women with a history of prior thrombosis, prior pregnancy morbidity, or
systemic lupus erythematosus [15,26,43].
Clinicians have sought and tried alternative “treatments” in high-risk obstetric APS cases and patients
refractory to treatment with heparin LMWH and LDA. These alternative treatments are nearly always
in addition to a heparin agent and LDA. Cautious interpretation of reports regarding such alternative
treatments is in order because they are anecdotal or retrospective in nature and have not included
proper comparison to patients matched for confounders known to be associated with adverse pregnancy
outcomes of interest. Investigators have reported modestly improved pregnancy outcomes adding low-
dose prednisolone (10 mg per day until 14 weeks) [47] or hydroxychloroquine (HCQ) [48] to heparin
LMWH and LDA. A more recent retrospective international multicenter study of high-risk APS
pregnancies concluded that the addition of HCQ treatment was associated with a significantly higher live
birth rate in women with a history of one or more pregnancies refractory to conventional therapy [49],
although this report did not take account of embryonic aneuploidy as a cause of miscarriage. Varying
degrees of successful pregnancy outcomes have been reported in retrospective case series of high-risk or
refractory obstetric APS using intravenous immunoglobulin (IVIG) infusions and/or apheresis [50–57].
Most recently, a retrospective multicenter study found that triple-positive APS patients with previous
thrombosis treated with additional therapies had a significantly higher live birth rate compared to those
receiving conventional therapy alone [58]. Finally, improved outcomes in APS pregnancies treated with
pravastatin has been reported by one group [59].
Study Design Limitations and Future Directions

Best intentions notwithstanding, the existing “treatment” studies of women with high-risk APS or those
“refractory” to treatment with heparin and LDA are flawed by being retrospective in nature, lacking
an appropriate control population with analysis for confounders, having included patients of different
clinical and laboratory features, having included different treatment regimens according to individual
physician preference, and using an inappropriate compilation of prior pregnancy outcomes in the same
patients for statistical comparison. Thus, we view the existing studies as virtually impossible to interpret.
However, we also recognize that properly-designed and -powered, controlled, randomized treatment
trials will not likely be done in women with high-risk or “refractory” obstetric APS. Rare diseases are
inherently difficult to study due to a small number of eligible participants, geographic dispersion, and
lack of appropriate comparators. Further, a rigorously designed study is difficult in part due to costs and
difficulties inherent in large, properly designed and implemented, multicenter efforts. Thus, understanding
what, if any, treatments are beneficial when added to a heparin agent and LDA in women with high-risk
or “refractory” obstetric APS will require a multicenter effort with the following characteristics [60]:
• Prospective in nature
• The use of core laboratories for aPL confirmation
• Meticulous definition of prior adverse pregnancy outcomes and other relevant past medical
history
• Standardized treatment and antenatal management protocols
• Identification and statistical accountability for protocol deviations
• An appropriate control group, even if retrospective in nature, with sufficient background data
to enable matching with regard to laboratory features and potential confounders, e.g., chronic
hypertension or history of thrombosis
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24
Can Recurrent Pregnancy Loss Be
Prevented by Antithrombotic Agents?
Audrey A. Merriam and Michael J. Paidas
Introduction
Antithrombotic agents encompass two classes of drugs: antiplatelet agents (i.e., aspirin) and anticoagulant
agents (i.e., heparin and low-molecular weight heparin [LMWH]). Antiplatelet agents prevent platelets
from aggregating and forming blood clots. This class of drugs has recently gained popularity in obstetrics
due to its ability to decrease the risk for preeclampsia in certain high-risk populations. Anticoagulant
medications act by preventing fibrin formation, decreasing clot formation and growth. In addition,
heparins have anti-inflammatory actions by inhibiting tumor necrosis factor (TNF)α production [1] and
increasing TNF-binding protein [2].
Heparin has also been reported to enhance trophoblast invasion in antiphospholipid syndrome [3] and
increase hCG production. Aspirin has been reported to inhibit the proinflammatory cytokines TNFα
and IL-8 in stroke. TNFα induces thrombin generation [4,5]. IL-8 causes polymorph accumulation [6].
Polymorphs react with fibrin and damaged tissues to form clots. Hence aspirin may also modify cytokine
mediated thrombosis.
These agents have been studied in the context of recurrent pregnancy loss in women with and without
a personal history of a thrombophilia. This chapter explores the evidence for use of these agents in
preventing recurrent pregnancy loss in women with and without a diagnosed thrombophilia.
Hereditary Thrombophilias
The full thrombophilia workup is described in Chapter 9. There is a problem in that some thrombophilias
should not be assessed during pregnancy (particularly protein S, the levels of which fall physiologically in
pregnancy), so it is important to perform screening for hereditary thrombophilias during preconception
counseling so patients who are found to have a thrombophilia can be appropriately managed in a
subsequent pregnancy and potentially decrease their risk for recurrent pregnancy loss.
Anticoagulants in Patients with a Thrombophilia

The presence of a thrombophilia can increase the risk of multiple adverse pregnancy outcomes. Perhaps
one of the most concerning is the increased risk of venous thromboembolism (VTE). The association
between thrombophilias and recurrent pregnancy loss (RPL) is not as strong as the risk for VTE. The
cause of RPL in the presence of a thrombophilia is not truly understood. It is theorized that women
with inherited thrombophilias may experience uteroplacental insufficiency due to thrombosis in the
uteroplacental circulation. However, until now no specific placental lesion has been found in the presence
of hereditary thrombophilia
The initial studies that found an association between RPL and inherited thrombophilias were small
case-control studies and meta-analyses. Larger, prospective trials have not proven causality between
inherited thrombophilias and RPL or first trimester single-antigen bead (SAB). The European Prospective
223
TABLE 24.1
Indications for Anticoagulation Therapy
Indication Description Antepartum Postpartum
High-risk thrombophilia History of one prior VTE Therapeutic or Therapeutic or prophylactic
• FVL homozygous prophylactic LMWH/ LMWH regimen or
• Prothrombin G20210A UFH postpartum warfarin;
mutation homozygous dosing/level to match
• FVL/prothrombin antepartum regimen
G20210A mutation No history of VTE Prophylactic LMWH/UFH Prophylactic LMWH or
double heterozygous postpartum warfarin
• Antithrombin III
deficiency
Low-risk thrombophilia History of one prior VTE Prophylactic LMWH/UFH Prophylactic LMWH/UFH
• FVL heterozygous or surveillance without or postpartum warfarin
• Prothrombin G20210A anticoagulation
mutation heterozygous No history of VTE Surveillance without Surveillance without
• Protein C deficiency anticoagulation or anticoagulation or
• Protein S deficiency prophylactic LMWH/UFH prophylactic LMWH/UFH
or postpartum warfarin if
patient has additional risk
factors
Two or more prior VTE On long-term Therapeutic LMWH/UFH Resumption of long-term
episodes (thrombophilia or anticoagulation anticoagulation therapy
no thrombophilia) Not on long-term Therapeutic or prophylactic Therapeutic or prophylactic
anticoagulation LMWH/UFH LMWH/UFH for 6 weeks
Source: Modified from American College of Obstetricians and Gynecologists. Practice Bulletin No. 197 [9].
Abbreviations: FVL, factor V Leiden; LMWH, low-molecular-weight heparin; UFH, unfractionated heparin; VTE, venous
thromboembolism.
Cohort on Thrombophilias (EPCOT) examined pregnancy outcomes comparing women with and without
a documented inherited thrombophilia and found an association with stillbirth but not first trimester
SAB [7]. These findings were also confirmed on studies in a United States population [8]. These studies
did demonstrate an association, albeit weak, between inherited thrombophilia and pregnancy loss after
10–14 weeks’ gestation.
Recommendations regarding the use of anticoagulants (heparin and LMWH) in pregnancy in women
with known inherited thrombophilias have been established by the American College of Obstetricians
and Gynecologists (ACOG) [9]. These recommendations have been established for VTE prevention but
as a result, they impact studies examining the use of anticoagulants, primarily heparin and LMWH,
and inherited thrombophilias and RPL and first trimester SAB. Tables 24.1 and 24.2 list the current
recommendations for anticoagulation during pregnancy for women with a thrombophilia, regardless of
obstetric history.
Available literature examining use of anticoagulants in recurrent pregnancy loss is limited to small
case reports, case series, and meta-analyses. These studies vary in their inclusion criteria but all focus
TABLE 24.2
Summary of Recommendations Regarding Intervention for Women with RPL with and without a History of
Thrombophilia
Condition Anticoagulant Anticoagulant plus Aspirin Aspirin
RPL without a thrombophilia No No No
RPL with a thrombophilia No No No
RPL with a thrombophilia and history of a VTE Yes No No
RPL with or without a thrombophilia and a No No Yes
history of preeclampsia
Can Recurrent Pregnancy Loss Be Prevented by Antithrombotic Agents? 225
on prophylactic dosing of anticoagulants. Carp et al. treated 37 women with RPL and an inherited
thrombophilia with a prophylactic dose of LMWH (enoxaparin, 40 mg daily) and compared them to women
with the same history who did not receive any treatment. The odds of a live birth was 3.03 (confidence
interval [CI] 159–52.48) in the group receiving treatment with LMWH but the trial was flawed in its lack
of randomization of patients [10]. A prospective study, flawed by the control group selection (the patient’s
previous poor pregnancy outcome was used as the control) examined the used of LMWH in women with
RPL and thrombophilia and found a benefit to treatment with LMWH; however, given the flawed study
design, recommendations from this trial are presented with a note of caution [11]. Another multinational
trial by Roger et al., examining the use of dalteparin in women with an inherited thrombophilia, did not
find a difference in pregnancy loss or other adverse pregnancy outcomes, compared to no dalteparin use
antepartum [12]. The lack of consensus called for a meta-analysis of trials of thromboprophylaxis in
cases of hereditary thrombophilias. Skeith et al. [13] published a meta-analysis of 8 publications and 483
women (including the dalteparin trial). Treatment with prophylactic-dose LMWH did not reduce the risk
of pregnancy loss in women with an inherited thrombophilia compared to similar women treated with
aspirin alone or no treatment [13]. However, Skeith et al.’s [13] meta-analysis was not limited to RPL.
Four of the eight trials included patients with first and second trimester losses [14], patients with one loss
[15], and two trials included patients with previous pregnancy complications [16,17]. In addition, there has
been no attempt at a dose-finding study. The LIVE-ENOX trial compared two doses of LMWH, 40 mg
daily versus 40 mg twice a day. In women with a thrombophilia, the live birth rate was not significantly
different between the treatment groups (p = 0.48) [18].
However, the question is whether anticoagulants are warranted in patients with RPL and a thrombophilia.
The editor performed a meta-analysis of the figures in Skeith et al.’s [18] meta-analysis for patients with
RPL [19–22] and a trial by Aynioglu [23]. The figures are shown in Figure 9.2 of Chapter 9. There was a
statistically significant increase in the live birth rate (odds ratio [OR] 4.88; CI 2.82–8.47). However, the
OR is dependent on Aynioglu’s [23] trial, which is at variance with the other three trials. Hence the need
for the use of treatment with prophylactic anticoagulation, either heparin or LMWH, in women with a
history of thrombophilia and RPL or second trimester fetal demise, is an open question. It is inappropriate
to recommend treatment on the basis of one randomized controlled trial [23], as benefit, defined as
successful pregnancy, has not been consistently demonstrated. In addition, anticoagulant medication
does carry risk to the patient, even at prophylactic doses. Heparin-induced thrombocytopenia and major
bleeding episodes are rare but can occur. More commonly, aversion to daily injections, cost, and potential
injection site reactions can be seen. These potential serious and non-serious side effects question the use
of anticoagulant medications solely for improved pregnancy outcomes if benefit is not consistent.
Additional randomized controlled trials are needed to further examine if treatment with prophylactic
anticoagulation would result in a successful pregnancy in women with a history of inherited thrombophilia
and RPL or second trimester fetal demise. One important note is that women with a thrombophilia
and prior VTE may require prophylactic or treatment dose anticoagulation during pregnancy according
to ACOG guidelines. These recommendations will likely make future research examining successful
pregnancy outcomes in this patient population difficult.
Treatment with Anticoagulants and Antiplatelet Agents

In obstetrics, aspirin is the primary antiplatelet agent considered safe in pregnancy. Aspirin has become
increasingly popular lately given its use in prevention of preeclampsia in certain high-risk populations.
Despite the potential benefits of aspirin in certain populations, its use alone in patients with a history of
an inherited thrombophilia with RPL or second trimester fetal demise in the absence of preeclampsia
(≥20 weeks’ gestation) is not currently recommended [24]. Results of randomized control trials, including
a Cochrane review and meta-analyses looking at aspirin use to prevent intrauterine fetal demise, are
inconclusive and do not show the same strong benefit that previous retrospective cohort studies showed
[25–27].
Trials looking at the use of aspirin in combination with anticoagulants (heparin or LMWH) are equally
inconclusive. There are no randomized control trials examining successful pregnancy outcomes in women
with an inherited thrombophilia and RPL. A small study with 52 women (29 treatment and 23 control)
examined low-dose aspirin and LMWH in women with an inherited thrombophilia and RPL and did find
a lower miscarriage rate in the treatment groups; however, the methodology was flawed in that the study
was not randomized [28]. In one study, 153 women diagnosed with an inherited thrombophilia and at
least two prior early pregnancy losses were randomly assigned to no treatment or treatment with aspirin
80 mg/d and LMWH 100 IU/kg. Those in the treatment group had a lower occurrence of intrauterine fetal
demise (n = 14, 33.3%) versus the control group (n = 31, 56.4%) and anembryonic pregnancies (n = 6,
14.3%) versus the control group (n = 17, 30.9%). The authors did not mention if there were first trimester
SABs not due to anembryonic pregnancies [23]. This study is flawed by its inclusion of anembryonic
pregnancies and the dose of LMWH used is higher than in other studies. There are subgroup analyses of
women with inherited thrombophilias in larger trials looking at treatment for RPL where the treatment
group received aspirin plus anticoagulation, but these subgroups were too small, and the trials were not
powered to detect a difference in this subset of women with inherited thrombophilias [19–21]. The only
study to show a benefit was Aynioglu et al. [23] where there was a clear benefit of effect for combined
treatment. However, we feel that additional trials are required to confirm or refute Aynioglu et al.’s figures.
A Cochrane meta-analysis of 9 studies with 1228 women included women with and without a history of
inherited thrombophilia. The authors concluded that anticoagulation either with or without aspirin did
not improve the live birth rate in women with a history of RPL [29]. However, the Cochrane review was
written in 2014 and may need updating. Therefore, it is clear that randomized control trials are needed
to further address this question. At this time, aspirin plus an anticoagulant is not recommended for the
purpose of successful pregnancy outcome in women with a history of inherited thrombophilia. Women
with an inherited thrombophilia, especially those with a history of VTE, may require anticoagulation
with heparin or LMWH, and women with a history of preeclampsia may require low-dose aspirin for pre-
eclampsia prevention in a subsequent pregnancy, but neither of these medications alone or in combination
are recommended for the purpose of preventing RPL or second trimester fetal demise in women with an
inherited thrombophilia.
Patients without a Thrombophilia

The majority of patients with RPL will not be diagnosed with a thrombophilia, which can make
recommendations regarding potential treatment difficult. Patients with negative workup for the various
causes of RPL may be tempted to try treatments such as LMWH and aspirin, as they are thought to be
low-risk interventions. However, the data do not support their use in achieving a successful pregnancy
outcome, and particularly in the case of anticoagulation agents, such as heparin and LMWH, there are
potential risks with taking these medications.
Treatment with Anticoagulants

Multiple prospective studies exist looking at the effect of anticoagulants on successful pregnancy outcome in
women with RPL and no inherited thrombophilia. Table 24.3 summarizes the results. There are two positive
studies, both from Egypt. An additional study of 80 women without an inherited thrombophilia which
found that treatment with LMWH showed improvement in the number of live births [30] was excluded, as
the comparison was the patient’s previous poor pregnancy outcome, which is not the correct control group.
A prospective study by Fawzy et al. showed a 33% improvement in the enoxaparin-treated arm
compared to the control arm [31]. A more recent randomized trial by Shaaban et al. with 300 women
showed a significantly improved pregnancy continuation rate and live birth rate in women treated with
tinzaparin 4500 IU/d. This study was well designed, and all participants had complete follow-up to
pregnancy loss or delivery [32].
All other studies are negative. Badawy et al. studied 340 women (170 per treatment group) treated with
either enoxaparin 20 mg/d and folic acid supplementation or folic acid supplementation alone. The odds
of early spontaneous miscarriage (OR 1.41%; 95% CI 0.16–1.2) and late pregnancy loss (OR 1.21%; 95%
CI 0.06–3.18) were improved in the treatment group but analysis showed the 95% CI crossed 1 [30,33].
There is one multicenter, randomized, double-blind placebo-controlled trial to examine this topic that
TABLE 24.3
Heparins in Unexplained RPL
Heparin Controls RR (CI)
Positive Trials
Fawzy et al. [31] (enoxaparin 20 mg vs. aspirin 75 mg) 46/57 (81%) 24/50 (48%) 1.68 (1.22–2.34)
Shaaban et al. [32] (tinzaparin and folic acid vs. folic acid) 110/150 (73.3%) 72/150 (48%) 1.52 (1.26–1.85)
Negative Trials
Badawy et al. [33] (enoxaparin 20 mg and folic acid vs. 161/170 (94.7%) 151/170 (88.8%) 1.07 (1.00–1.14)
folic acid alone)
Dolitzky et al. [35] (enoxaparin vs. aspirin) 44/54 (81.5%) 42/50 (84.0%) 0.92 (0.58–1.46)
Clark et al. [20] (heparin and aspirin vs. surveillance alone) 111/143 (77.6%) 111/140 (79.3%) 0.95 (0.73–1.25)
Kaandorp et al. [19] (nandoparin and aspirin vs. placebo) 45/92 (48.9%) 47/81 (58.0%) 0.84 (0.64–1.11)
Visser et al. [21] (enoxaparin and placebo vs. aspirin) 35/51 (68.2%) 34/57 (59.6%) 1.24 (0.79–1.92)
Schleussner et al. [22] (dalteparin vs. placebo) 185/215 (86%) 183/211 (86.7%) 0.99 (0.86–1.44)
Shaaban et al. [32] (tinzaparin and folic acid vs. folic acid) 110/150 (73.3%) 72/150 (48%) 1.52 (1.26–1.85)
Pasquier et al. [34] (enoxaparin vs. saline) 92/138 (66.6%) 86/118 (72.9%) 0.91 (0.78–1.07)
Note: Proportion of live births are shown in parentheses.
found contrary results to the Shaaban et al. paper. In women with RPL the chance of a live birth did not
improve with use of enoxaparin during pregnancy [36,34]. This paper is perhaps the best designed study to
date and does not show a benefit with use of anticoagulants to prevent RPL. There are three other placebo
control studies which did not find a difference in ongoing pregnancy or live births in women treated with
LMWH and a history of RPL in the intervention group [20–22].
There are two studies comparing heparin to aspirin. In the HABENOX study, enoxaparin was not found
to improve the live birth rate in women with RPL [21], which is similar to their findings in women with
an inherited thrombophilia. Dolitzky et al. [35] also found enoxaparin to have similar results to aspirin.
Given the heterogeneity in the trial design as well as the findings, it is difficult to compare the studies. One
meta-analysis of the Badawy et al., Fawzy et al., and Shaaban et al. studies did not find improvement in the
live birth rate in women with RPL and no history of inherited thrombophilia [36]. Similarly, the Cochrane
review, which included nine studies by de Jong et al., did not find an improvement in pregnancy outcomes
in women with RPL and no history of thrombophilia when anticoagulants were used during pregnancy
[29]. Given these findings, the use of anticoagulants during pregnancy in women with RPL but without a
history of a thrombophilia is not recommended, and multiple societies have come out against this practice,
including ACOG, the Royal College of Obstetricians and Gynecologists, and the American College of Chest
Physicians [37–39]. Regarding the results of anticoagulants compared to aspirin, similar to the conclusions
drawn in women with RPL treated with anticoagulants alone, the combination of antiplatelet treatment with
aspirin and anticoagulation does not increase the live birth rate or successful pregnancy outcome.
There is one trial looking at women presenting with a threatened miscarriage (vaginal bleeding with a
confirmed intrauterine pregnancy in the first trimester) being treated with LMWH which found that the
live birth rate was higher in women who discontinued LMWH after vaginal bleeding in the first trimester.
The authors concluded that stopping LMWH in this very specific population resulted in improved
pregnancy outcomes (i.e., a higher live birth rate) and called into question the practice of using LMWH
to improve outcomes in women with RPL and no history of inherited thrombophilia [40].
Treatment with Antiplatelet Agents

Many trials have been performed examining the combination of antiplatelet agent aspirin in women with
RPL and without a history of inherited thrombophilia. Table 24.4 summarizes the results of aspirin in
unexplained RPL. None of the studies shows any benefit; however, the trial designs are inconsistent with
various control groups.
TABLE 24.4
Aspirin in Unexplained RPL
Aspirin Control RR (CI)
Tulppala et al. [41] 22/27 (81.5%) 22/27 (81.5%) 1.0 (0.78–1.29)
Rai et al. [42] 373/556 (67.1%) 308/449 (61.7%) 1.26 (0.92–1.64)
Kaandorp et al. [19] 42/82 (51.2%) 47/81 (58.0%) 0.90 (0.66–1.22)
Visser et al. [21] (aspirin and enoxaparin 32/48 (66.7%) 35/51 (68.3%) 0.96 (0.62–1.46)
vs. enoxaparin and placebo)
Note: Proportion of live births are shown in parentheses.
Tulppala et al. originally looked at aspirin versus placebo for women with RPL and no history of
thrombophilia (27 women in each treatment arm) and found no difference with aspirin treatment in
live births between the groups [41]. Rai et al. also looked at aspirin alone in women with RPL and in
women with late pregnancy loss. There was no difference in the rates of early miscarriage but there
was a suggestion of improvement in late pregnancy loss with the use of aspirin alone. The confidence
interval neared 1.0 and the authors concluded more studies were needed before aspirin alone should
be used to prevent later pregnancy loss [42]. ACOG currently does not recommend the use of aspirin
alone for prevention of early miscarriage in women with RPL or for prevention of later pregnancy fetal
demise unless the latter occurs with a diagnosis of preeclampsia [24]. The HABENOX trial, in addition
to assessing enoxaparin, also had an arm for enoxaparin and aspirin compared to aspirin alone in women
with RPL and found no difference in live birth rate in the combination treatment group, which included
63 women compared to control patients [21]. The ALIFE study contained three treatment arms (aspirin
only, aspirin plus nadroparin, or placebo) and found no difference in live birth rates among women with
RPL [19]
Aspirin may be considered in women with a prior poor pregnancy outcome related to preeclampsia
but should not be used for RPL or poor pregnancy outcome in a prior pregnancy not attributed to pre-
eclampsia [24,43].
Conclusions
• Screening for inherited thrombophilias is not recommended for women with a history of RPL
or second trimester fetal demise.
• MTHFR mutations are not considered to be an inherited thrombophilia and patients should not
be treated with anticoagulants or antiplatelet agents during pregnancy for VTE prevention or
for promotion of a successful pregnancy outcome.
• Anticoagulation (heparin or LMWH) is not recommended by professional organizations in
women with an inherited thrombophilia and a history of RPL or second trimester fetal demise
solely to improve pregnancy outcome. However, more work is required to clarify the issue.
• Aspirin alone is not recommended in women with an inherited thrombophilia and a history of
RPL or intrauterine fetal demise (in the absence of preeclampsia or risk factors for preeclampsia).
• Aspirin plus anticoagulation (heparin or LMWH) is not recommended in women with an
inherited thrombophilia and a history of RPL or second trimester fetal demise solely to improve
pregnancy outcomes. However, more work is required to clarify the issue.
• Anticoagulants (heparins) are not recommended in women with RPL or second trimester fetal
demise and no history of inherited thrombophilia to improve pregnancy outcomes.
• Aspirin plus anticoagulation (heparin or LMWH) is not recommended in women with RPL or
second trimester fetal demise and no history of thrombophilia to improve pregnancy outcomes.
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loss treated by enoxaparin. Thromb Haemost. 2000;83:693–7.
12. Rodger MA, Hague WM, Kingdom J et al. Antepartum dalteparin versus no antepartum dalteparin for the prevention
of pregnancy complications in pregnant women with thrombophilia (TIPPS): A multinational open-label randomised
trial. Lancet. 2014;384:1673–83.
13. Skeith L, Carrier M, Kaaja R et al. A meta-analysis of low-molecular-weight heparin to prevent pregnancy loss in
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15. Gris JC, Mercier E, Quéré I et al. Low-molecular-weight heparin versus low-dose aspirin in women with one fetal
loss and a constitutional thrombophilic disorder. Blood. 2004;103:3695–9
16. Martinelli I, Ruggenenti P, Cetin I et al. Heparin in pregnant women with previous placenta-mediated pregnancy
complications: A prospective, randomized, multicenter, controlled clinical trial. Blood. 2012;119:3269–75.
17. Rodger MA, Hague WM, Kingdom J et al. Antepartum dalteparin versus no antepartum dalteparin for the prevention
of pregnancy complications in pregnant women with thrombophilia (TIPPS): A multinational open-label randomised
trial. Lancet. 2014;384:1673–83.
18. Brenner B, Hoffman R, Carp H et al. Efficacy and safety of two doses of enoxaparin in women with thrombophilia
and recurrent pregnancy loss: The LIVE-ENOX study. J Thromb Haemost. 2005;3:227–9
19. Kaandorp SP, Goddijn M, van der Post JA et al. Aspirin plus heparin of aspirin alone in women with recurrent
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consecutive recurrent miscarriages. Fetil Steril. 2006;86:362–6.
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2013;19:656–73.
25
Empirical In Vitro Fertilization for Recurrent
Pregnancy Loss: Is It a Valid Concept?
Michal Kirshenbaum and Raoul Orvieto
Introduction
In recurrent pregnancy loss (RPL), when no underlying cause can be identified, empirical treatments are
offered to couples with RPL, including assisted reproductive technique (ART). Although most patients
with RPL do not have fertility problems, RPL patients are often offered a selection of adjunct treatments
or “add-ons” aiming to improve their chance of a live birth. Other so-called benefits are quoted to the
patient such as lessening the time to conceive, improving embryo quality, improvement of implantation,
and improved synchrony between endometrium and embryo. The latter is even more important in the light
of recent work on the role of the endometrium in allowing the implantation of abnormal asynchronized
embryos [1] (and Chapter 5). In this chapter, we present the clinical evidence and assess the benefit or
lack of benefit of the various options offered in ART in RPL patients.
Subsequent Live Birth Rate

In unexplained RPL, the subsequent live birth rate is known to be dependent on certain factors described
in Chapter 1. Briefly, these include the number of previous live births, maternal age, genetic aberrations
in the previous pregnancy, and whether the losses are early or late. Therefore if in vitro fertilization
(IVF) is used, the subsequent live birth rate should be matched for all these prognostic parameters.
This type of analysis has not been performed. Chapters 1 and 19 give the figures for subsequent live
births according to the number of previous losses. A thorough literature search has not revealed any
publications on subsequent live birth rates with IVF in patients with RPL according to the number of
previous miscarriages or any other factor that affects the prognosis in RPL.
Another problem in assessing the live birth rate after IVF in RPL is how to quote the live birth rate.
In RPL, the live birth rate is quoted as the number of live births per pregnancy. In IVF, the clinical
pregnancy rate is often used as a measure of the success of treatment. In RPL, the clinical pregnancy
rate is meaningless, as RPL patients generally conceive easily. Even if the rate is quoted as live births
per cycle, the time lost in preparing for IVF will not be taken into account. If IVF is not used, the patient
may well conceive in the interval while planning for IVF.
Perfetto et al. [2] has quoted the subsequent live birth rate in 98 patients who conceived spontaneously
compared to 68 patients undergoing infertility treatment, including ovulation induction, intrauterine
insemination, or IVF, and a third group undergoing pregestational testing for aneuploidy (PGT-A). Eighty-
eight percent of patients conceived within 6 months in the spontaneous pregnancy group compared to 84%
in the fertility treatment group and 70% in the PGT-A group. The live birth rates were similar in both
groups 77% and 73% and 70%, respectively (p = 1.0). The clinical miscarriage rate and the biochemical
pregnancy rate were also similar between the three groups: 18% and 6% in the spontaneous conception
group, 16% and 11% in the IUI/IVF group, and 13% and 9% in the preimplantation genetic screening
(PGS) group, respectively.
231
However, it must be borne in mind that up to 33% of patients with RPL do have periods when they
fail to conceive [3]. Some of these patients will require IVF, but the IVF will be for failure to conceive
rather than RPL. Hence there are no data to support using empirical fertility treatment, including IVF,
to improve the live birth rate in RPL.
Time to Conceive
Previous studies have reported a longer mean interval to subsequent conception after a pregnancy loss
compared to the time to conceive before a pregnancy loss [4,5]. The emotional impact of RPL and
the strong desire to conceive as early as possible may lead patients and physicians to consider fertility
treatment, aiming at reducing the time interval to the next pregnancy. Kaandorp et al. [6] assessed the
time to conception in 251 women with unexplained RPL. In their study, time to conception was calculated
from the diagnosis of unexplained RPL until the first day of the menstrual cycle in which conception
occurred. The mean patients’ age was 34 ± 5 years, the median number of preceding miscarriages was
three (range 2–15) with a median gestational age of 8 weeks (range 6–17). Thirteen percent of the study
patients conceived with ART, although no separate analysis was performed for this group. The cumulative
incidence of conception was 56% after 6 months, 74% after 12 months, and 86% after 24 months, of
which 65% resulted in a live birth. The median time to subsequent conception was 21 weeks (interquartile
range of 8–55). According to the literature, cycle fecundity in normal fertile couples is 20%–30% and the
cumulative fecundity is 85% and 93% after 1 or 2 years, respectively [7,8]. Given that the mean patients’
age in the study by Kaandorp et al. [6] was 34 years, the cumulative pregnancy rate observed in this study
is similar to that reported for the general population.
As quoted above, Perffeto et al. compared the time to pregnancy as well as the miscarriage rate and
subsequent live birth in fertile patients with RPL who attempted to conceive spontaneously and those that
opted to undergo fertility treatment [2]. In their study, 190 patients with two or more clinical miscarriages
were followed for a subsequent pregnancy for a minimum 6 months, beginning after a complete workup
investigation of RPL. Among the 98 patients who conceived spontaneously, the median time to pregnancy
was 2 months (range 1–10) and 88% conceived within 6 months. The median time to pregnancy among
the 68 women who conceived with fertility treatment was significantly longer: 3 months (range 1–9) for
controlled ovarian stimulation with intrauterine insemination (IUI), 4 months (range 1–12) with IVF, and
5 months (range 2–10) for PGT-A. In patients achieving pregnancy with fertility treatment, excluding
PGT-A, 84% conceived within 6 months. For patients conceiving with PGT-A, the time to conceive was
significantly longer. Only 70% conceived within 6 months. The authors concluded that in young, fertile
patients with RPL, there does not appear to be a clinical benefit to using fertility treatment to reduce
the time to subsequent pregnancy. Two differences between the study groups might influence the study
results and conclusions. The patients who attempted to conceive spontaneously were slightly younger
than the patients undergoing fertility treatment (34.5 vs. 35.6) and the subset of women who used PGS
were even older, with a mean age of 36.7 years. Although this difference was not statistically significant
(p = 0.12), it might have affected the time to pregnancy, as the conception rate declines with advanced
maternal age [9,10]. Moreover, women in the fertility treatment group had a significantly longer median
time to conceive in prior pregnancies (3 vs. 2 months). While time to pregnancy seems to be similar across
successive pregnancy attempts [11], it is possible that the difference in time to pregnancy between the
groups was due to a different fertility potential.
Murugappan et al. retrospectively compared outcomes among patients with RPL intending to pursue
PGT-A and patients who were managed expectantly and attempted spontaneous conception for an interval
of 6 months [12]. All cycles of PGS were included, including cancelled cycles and those that did not lead
to embryo transfer. The median time to pregnancy was longer in the PGS group (6.5 months) than that
of the spontaneous conception group (3 months). Murugappan et al. concluded that PGT-A should not be
offered for patients who feel an urgency to conceive.
In fertile couples with RPL, it seems that there is no benefit in fertility treatment, including IVF, to
shorten the time to the next pregnancy.
Empirical In Vitro Fertilization for Recurrent Pregnancy Loss: Is It a Valid Concept? 233
Sperm Selection Techniques to Improve Sperm Quality

Until recently, the assessment and treatment of couples with RPL has been directed exclusively to
investigation of the female rather than the male partner. Chapter 13 gives a comprehensive account of
the sperm factor in RPL. This present section will be limited to the evidence that sperm selection can
improve the prognosis when empirical IVF is used for RPL.
It has been reported that the sperm of men with RPL have significantly reduced viability, with an increased
proportion of DNA damage when compared with fertile controls [13–15]. Sperm function parameters
such as hypo-osmotic swelling, acrosomal status, and nuclear chromatin decondensation have also been
found to be reduced in the male partners of RPL couples compared to fertile males [16]. In regular IVF,
various sperm preparation techniques have been used to try to improve fertility potential. These techniques
include semen washing, density gradient centrifugation, swim-up technique, and electrophoretic sperm
selection, followed by IVF. Another method for sperm improvement selects non-apoptotic sperm, based
on the presence of phosphatidylserine on the external surface of the sperm membrane in the early stages
of apoptosis. Magnetic activated cell sorting and glass wool separation columns utilize the magnetic
properties of phosphatidylserine to separate apoptotic sperm from non-apoptotic sperm. However, none of
these techniques have been reported to improve the live birth rate in RPL
Physiological intracytoplasmic sperm injection (PICSI) utilizes the presence of hyaluronic acid (HA)
binding sites on the sperm plasma membrane. HA binding sites indicate sperm maturity and ability to
attach the extracellular matrix of the cumulus oophorus. A recent Cochrane review aiming to evaluate
the impact of advanced sperm selection techniques on ART outcomes could not find sufficient evidence
to allow the review authors to determine whether sperm selected by surface charge, sperm apoptosis, or
HA binding have any additive value over conventional selection [17]. No difference was found between
the methods in terms of live births, clinical pregnancy, or miscarriage rates. However, this meta-analysis
was not restricted to RPL patients.
Intracytoplasmic morphologically selected sperm injection (IMSI) is a technique to select sperm for
injection to the egg by examining the organelle morphology, such as the acrosome, postascrosomal lamina,
neck, mitochondria, tail, and nucleus (motile sperm organelle morphology examination [MSOME])
using ultra-high magnification (≥6000×) microscopy. Although initial reports have shown that IMSI is
associated with a higher pregnancy rate and lower miscarriage rate [18,19], both the effectiveness and
safety of IMSI in clinical practice remain unclear. A Cochrane review has found an increased clinical
pregnancy rate using IMSI compared to intracytoplasmic sperm injection (ICSI), although there was no
difference regarding the live birth rate or the miscarriage rate [20].
In conclusion, although sperm selection techniques might improve sperm quality and overcome
potential male subfertility in RPL couples, the evidence is insufficient to recommend use in RPL.
Improved Embryo Quality

Embryo Morphology
Embryo morphology is thought to be highly indicative of pregnancy outcome and therefore morphological
grading of the embryo may allow the selection of “the best” embryos for transfer. Over the past decade,
with the development of sequential culture media, there has been a steady shift in practice to transfer
day 5 or 6 embryos, at the blastocyst stage. The argument for blastocyst transfer is that the blastocyst has
undergone a self-selection process in which only the most viable embryos have survived and developed
into blastocysts. A Cochrane review demonstrated a higher clinical pregnancy and live birth rates in fresh
blastocyst transfer compared to fresh cleavage stage embryo transfer [21]. Again, the meta-analysis was
performed for infertile rather than RPL patients.
A large proportion of morphologically normal day 3 embryos are chromosomally abnormal. Aneuploid
embryos on day 3 often fail to reach day 5 [22,23]. Several studies have investigated the relationship
between morphology, euploidy, and the implantation rate of cleavage stage and blastocyst stage embryos.
Majumdar et al., in a retrospective analysis, demonstrated that blastocyst morphology and the rate of
development were significantly associated with euploidy, whereas cleavage stage morphology was not.
Nonetheless, implantation rates were similar for all transferred euploid blastocysts irrespective of their
morphology or their rate of development [23]. Similarly, Capalbo et al. found a correlation between
blastocyst morphology and euploidy, although the implantation potential of euploid embryos was
similar despite different morphologies and development rates [24]. The association between blastocyst
morphology and aneuploidy explains the higher implantation potential of good quality embryos reported
during conventional IVF cycles. However, the commonly used parameters of blastocyst evaluation are
not good indicators to improve the selection of euploid embryos.
In conclusion, when IVF treatment is used for selecting high-quality embryos, blastocyst morphology
can be used to slightly reduce the risk of transferring aneuploid embryos. Nonetheless, in the absence of
studies evaluating this potential advantage in women with RPL, we cannot recommend its use.
Time-Lapse System Embryoscopy

Traditionally, embryo assessment has involved removing embryos from a conventional incubator for
quality assessment by an embryologist, under a light microscope. Recently, time-lapse systems (TLS)
have been developed that can take digital images of embryos at frequent time intervals. Hence, the quality
of the embryos can be assessed without physical removal from the incubator. The potential advantages of
TLS include maintenance of a stable culture environment, limiting the exposure of embryos to changes
in gas composition, temperature, and movement. TLS has the potential advantage of improving embryo
selection for ART treatment by utilizing additional information gained through continuously monitoring
embryo development. Although the clinical value of TLS has been validated in some studies [25,26],
literature reviews have provided controversial data, leading to ongoing debate. A Cochrane review and a
recent meta-analysis studied the advantage of TLS embryonal assessment versus conventional embryonal
incubation and assessment [27,28]. No difference was found between the two interventions in terms of
live birth rate or miscarriage rate. Another review evaluated the association between morphokinetic
parameters and embryo ploidy to evaluate if TLS can replace PGT-A [29]. No single or combined
morphokinetic parameter was consistently identified as predictive of embryonic euploidy.
Currently there is insufficient evidence that TLS is superior to conventional methods for human
embryo incubation and selection or predictive of embryonic euploidy. Although maximizing embryonal
quality might improve pregnancy outcome in couples with RPL, TLS during IVF cannot currently be
recommended.
Preimplantation Genetic Testing–Aneuploidy

Preimplantation genetic testing–aneuploidy (PGT-A), utilizing trophectoderm biopsy and next-generation
sequencing (NGS) for embryonic aneuploidy, was predicated on an apparently improved ability to
accurately diagnose embryonic aneuploidies without compromising the embryo’s implantation potential.
While several retrospective studies and supposedly prospective trials have, indeed, alleged improved
clinical outcomes following PGT-A, the value of PGT-A as a screening test for in IVF patients or to
improve live birth in RPL patients has yet to be determined [30,31], and is discussed in Chapters 26 and 27.
Improving Implantation
Assisted Hatching
Assisted hatching (AH) is a manipulation of the zona pellucida in order to facilitate implantation. AH
involves thinning the coat surrounding a fertilized egg or making a hole in the zona pellucida. A variety of
techniques have been employed to assist embryo hatching, including partial mechanical zona dissection,
zona drilling and zona thinning, making use of acid tyrodes, proteinases, piezon vibrator manipulators,
and lasers [32]. Harper et al. reviewed the literature evaluating the effect of AH on IVF treatment and
concluded that it increases clinical pregnancy and multiple pregnancy rates but not live birth rate [26].
Since no single study has been able to demonstrate sufficient evidence of a benefit in the live birth rate of
AH in RPL, we cannot recommend its use.
Biologic Glue
In an attempt to increase the success rate of IVF, various compounds have been added to the embryo
transfer medium to improve adherence and subsequent implantation and pregnancy rates. HA forms a
viscous solution that might enhance the embryo transfer process and prohibit expulsion or may facilitate
diffusion and integration of the embryos in the viscous solution that characterizes intrauterine secreted
fluid [33]. The contribution of HA to implantation may also be receptor mediated, as the primary receptor
for HA is CD44, which is expressed both on the preimplantation embryo and on the endometrial stroma
[34]. A Cochrane review of 17 randomized control trials (RCTs), aiming to evaluate the supplementation of
HA to embryo transfer medium, demonstrated an improvement in clinical pregnancy and live birth rates,
with an associated increase in the multiple pregnancy rate [35]. A more recent RCT found no significant
difference in clinical pregnancy, implantation, or delivery rate between the HA group and the control group
[36]. The use of HA in RPL couples might potentially improve implantation and ongoing pregnancy rate.
However, before conclusions can be drawn, RCTs are needed to evaluate efficacy in RPL patients.
Immunological “Add-Ons”
The general IVF patients, as well as patients with recurrent miscarriage, are routinely offered a selection
of “add-ons,” aiming to improve outcome. Various adjuvant immunotherapy regimens have been used
to correct an immunological imbalance. A recently published report by the Practice Committee of the
American Society for Reproductive Medicine [37] has evaluated the role of immunomodulating therapy
in ART. It was concluded that immunotherapies have largely proven to be ineffective or have been
insufficiently investigated to make definitive recommendations for their use in improving live birth in IVF
treatment. Other chapters in this book have described that treatment may need to be personalized rather than
extrapolating the results of large trials to individual patients with special circumstances, and Chapter 11
discusses which immunological testing may be appropriate. However, before offering immunotherapy to
the general ART population or to RPL patients, further trials are necessary in appropriate patients.
The Window of Implantation—Improving Synchronization

Timing of conception in relation to ovulation may affect the spontaneous miscarriage rate. Previous
studies have suggested that prolonged exposure of the gametes to the female reproductive tract may have
a devastating effect on the ongoing pregnancy rate. Furthermore, aging of both spermatozoa and ova
before fertilization is accompanied by a higher probability of miscarriage [38,39]. Gray et al. assessed the
effect of timing of conception on the risk of miscarriage in women conceiving naturally [40]. Conception
on the day of ovulation or the day preceding ovulation was considered optimal. Among patients who
had miscarried in a prior pregnancy, the incidence of miscarriage was significantly higher in the index
pregnancy with nonoptimally timed conceptions (22.6%), as compared with optimally timed conceptions
(7.3%). This association was not observed among women with no history of pregnancy loss. Likewise,
studies that assessed the optimal time of conception among women with no history of miscarriage
reported no increased risk of miscarriage following conception remote from the day of ovulation [41,42].
The authors postulated that some couples are predisposed to genetic abnormalities in the gametes if
fertilization does not occur at the optimal time of the cycle.
In addition to the potential aging and chromosomal abnormality of the gametes, if conception occurred
on post-ovulation days, the conceptus could be chromosomally normal but unable to successfully implant
due to the endometrium being out of synchrony with the expected ovulation date. Synchronization
between embryonic development and endometrial decidualization is essential for adequate implantation.
The window of implantation (WOI) is a temporally restricted phase that is multifactorial, during which
changes occur at the molecular, cellular, and tissue levels. It is assumed that the endometrial WOI begins
on cycle days 19 or 20 of an idealized 28-day cycle and lasts for 4−5 days [43]. Wilcox et al. studied
the relation between the time of implantation and the outcome of pregnancy in couples with no history
of fertility problem trying to naturally conceive [44]. Daily urine hormone assays were used to identify
ovulation and implantation. They found that in most successful human pregnancies, the conceptus
implanted 8−10 days after ovulation and later implantation, i.e. beyond the normal period of endometrial
implantation, is strongly associated with increased early pregnancy loss.
Noyes et al. first assessed the uterine receptivity timeline and defined a series of morphological
criteria to date the endometrium [45]. RPL has been reported to be associated with retarded endometrial
development in the peri-implantation period, known as the luteal phase defect (LPD). In LPD, there is
inadequate synchronization between the embryo and the endometrium. Identification of LPD is usually
based on the morphological study of a precisely timed luteal phase endometrial biopsy, according to
the classic method of Noyes. A maturation delay has been described in 17%–28% of patients with RPL
[46,47]. RPL has also been associated with abnormal endometrial expression of various mediators and
metabolic factors in the secretory and peri-implantation phases [48,49]. Moreover, a comparison of
genetic microarray profiling of secretory phase endometrium of women with RPL compared with fertile
women has indicated abnormal regulation of the genes related to cell adhesion, cell differentiation, and
angiogenesis in patients with RPL [50].
ART might be justified in patients with RPL in order to avoid non-optimal timing of intercourse or
conception. In natural conception, timing of ovulation might be determined by noninvasive methods
such as basal body temperature charts, observation of cervical mucus, urine or plasma hormone levels,
or serial ovarian ultrasound [51]. However, these methods are often inaccurate and extremely variable
[52]. IVF has the potential advantage of determining precise synchronization between the embryo and
endometrium. Moreover, assessing the endometrium in the cycle prior to embryo transfer might enhance
synchronization and evaluate the quality of endometrial receptivity.
In the past, Noyes et al.’s histologic criteria have been the gold standard for evaluating endometrial
development and receptivity. However, histologic dating is prone to intra- and inter-observer variability
and tissue fixation artifacts. Consequently, histological endometrial dating is not accurate or precise
enough to diagnose LPD with accuracy, or to guide the clinical management of women with reproductive
failure [53,54]. In a search for accurate methods to evaluate endometrial receptivity, many structural
characteristics and molecules have been studied including ultrasonographic measurement of endometrial
thickness, structural examination by electron microscopy, immunological markers, steroid hormones and
receptors, and protein expression profiles [43,55]. A new approach to assessing endometrial function is
the endometrial receptivity array (ERA) test, based on analysis of expression of 238 genes that are found
to be involved in the receptivity of the endometrium [56]. The value of the ERA test is controversial, with
some studies supporting the utility and accuracy of the ERA test and some not [57,58,59]. However, no
report has assessed patients with RPL.
Conclusions
Although subfertility is not a problem in most couples with RPL, ART is often advised in RPL couples.
However, scientific evidence is lacking. Patients might be interested in IVF in order to shorten time to
conceive, but to date, IVF has not shown any benefit regarding the time to conceive.
Embryo quality has a significant role in the success of an ART cycle. ART includes methods to improve
gametes and embryo quality, such as sperm selection, PGT-A, and morphologic examination. Although
maximizing embryonal quality might improve the pregnancy outcome in couples with RPL, further
adequately powered studies are needed to assess the results.
An abnormal endometrial microenvironment and changes in the functional expression of endometrial
genes and protein might contribute to an abnormal embryonal-maternal interaction, resulting in pregnancy
failure. Endometrial sampling for assessing endometrial receptivity and accurately timed embryonal
transfer might improve this embryonal-maternal interaction. Nonetheless, due to the lack of studies
investigating these methods in RPL patients, IVF cannot be recommended for this purpose.
Several “add-ons” to IVF treatment, including assisted hatching, biologic glue, and immunologic
therapy have also been suggested to improve implantation and live birth rates. Since their efficacy is
controversial, these cannot be currently recommended.
In conclusion, ART without secondary subfertility cannot be supported as a treatment intervention for
couples with unexplained RPL, because of the lack of adequate clinical studies.
REFERENCES
1. Teklenburg G, Salker M, Heijnen C, Macklon NS, Brosens JJ. The molecular basis of recurrent pregnancy loss:
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26
Debate: Should PGT-A Still Be Performed
in Recurrent Pregnancy Loss? Yes
Carmen M. García-Pascual, Pilar López, Nasser Al-Asmar, Pere Mir,

Lorena Rodrigo, Carlos Simon, and Carmen Rubio
Incidence of Aneuploidy in Products of Conception

Pregnancy loss is a common occurrence in humans, which may be attributable to several factors,
either maternal or fetal. The most common contributor to spontaneous miscarriage is a chromosomal
abnormality, or aneuploidy, in the embryo/fetus. Indeed, aneuploidies account for about 50% of all
pregnancy losses before the 12th week of gestation [1]. When the products of conception (POC) are
analyzed, the incidence of aneuploidies has been reported to be as high as 62.7% [2,3]. Thus, the potential
for aneuploidy is a key consideration for reproductive success.
Up to 5% of couples of reproductive age experience recurrent pregnancy loss (RPL), defined as the loss
of two or more pregnancies from the same partners [4]. In addition to aneuploidies, pregnancy loss may be
due to single gene disorders and telomeric deletions [5]. Despite improvements in our understanding of the
etiology of RPL, about 50% of cases remain unexplained, or idiopathic. Among patients with idiopathic
RPL, approximately 45%, but possibly up to 90% [6] of the lost pregnancies may be due to embryo
aneuploidy. Hence preimplantation genetic testing for aneuploidies (PGT-A) is proposed as an approach
to avoid miscarriages due to chromosomal abnormality by selecting euploid embryos for transfer.
PGT-A: General Considerations

PGT-A analyzes the chromosomal status of the embryo before transfer, therefore only chromosomally
normal embryos are replaced into the uterus. By enabling the selection and transfer of euploid embryos,
PGT-A can be used not only to increase implantation rates and pregnancy rates in infertile patients,
but also to reduce the number of miscarriages and minimize the risk of having aneuploid offspring.
Analyzing embryos before transfer also decreases the number of transfers needed and the time to achieve
a pregnancy. Therefore, with lower number of transfers and miscarriages, PGT-A represents a more cost-
effective approach than standard blastocyst transfer, at least in patients ≥38 years [7].
Notably, although the risk of having a live birth with a chromosomal aberration is low following PGT-A,
the risk is not zero. PGT-A does carry the possibility of a false-positive result; studies using more dated
platforms suggest that this occurs in up to 2%–4% of cases [8,9]. In contrast, the percentage of false
negatives is almost zero [10]. Another critical aspect of PGT-A is the percentage of non-informative results
in each lab. With experienced embryologists and molecular biology labs, this rate should be low (below
2% of the biopsies analyzed), and rebiopsy can be offered if embryos are not arrested [11,12]. While
misdiagnosis of embryos remains unlikely, a noninvasive prenatal test (NIPT) may also be recommended
[13]. Because it uses only maternal blood samples, NIPT does not affect the trajectory of pregnancy,
unlike invasive methods such as amniocentesis. Such invasive testing would be required only if a positive
result is observed in the NIPT test.
In recent years, the value of PGT-A has come under debate, as none of the randomized studies assessing
PGT-A showed a clear benefit [14]. However, the negative results may be at least partially attributable
239
to the technical limitations of the cytogenetic technique employed in past studies: fluorescence in situ
hybridization (FISH). FISH allows analysis of only a limited number of chromosomes, and analysis
is dependent on the quality of nuclear spreading. Past studies also used less effective embryo biopsy
techniques, and culture conditions could explain the suboptimal results [15–19].
Technological advances have since resulted in the ability to assess all 24 chromosomes in embryo
biopsies through array comparative genome hybridization (aCGH) [20], single-nucleotide polymorphism
(SNP) microarray [21,22], and quantitative polymerase chain reaction (qPCR) [23]. Randomized controlled
trials using these technologies for different indications show a benefit of PGT-A in terms of improved
live birth rates, reduced miscarriage rates, and fewer multiple pregnancies [9,7,24]. More recently,
next-generation sequencing (NGS) techniques have been extended to PGT-A. NGS offers important
advantages as a versatile platform that can be used for the detection of whole chromosome and segmental
aneuploidies (Del/dup ≥10Mb) and different levels of mosaicism. When compared to aCGH, NGS has
a higher resolution and broader dynamic range, which facilitates diagnosis [25]. Thus as laboratory
techniques improve, PGT-A becomes more efficient and reliable.
PGT-A for RPL

Increasing evidence supports the use of PGT-A in idiopathic RPL patients. In a study published by
Bianco et al. [26], in which prenatal diagnosis was performed in 46,939 women, an increased risk of
karyotypic abnormalities was confirmed in the POCs in idiopathic RPL patients. The first evidence
proving that couples with RPL have an increased number of chromosomally abnormal embryos (ranging
from 50%–80%) was published by our group in 1998 [27], and these results were later confirmed by
other studies [28–35].
Using FISH for chromosomes 13, 15, 16, 18, 21, 22, X, and Y to select chromosomally normal embryos
for transfer demonstrated that reproductive outcome was improved by PGT-A [29]. This study showed that
after PGT-A, even when not all the chromosomes were analyzed, significantly higher implantation rates
were obtained in couples that previously experienced aneuploid miscarriages. We concluded that PGT-A
should be recommended when RPL is associated with chromosomal aberrations in up to five previous
miscarriages, and when there is a high incidence of chromosomal abnormalities in sperm [29]. In fact, a
systematic review of the evidence for the efficacy of PGT-A in patients with idiopathic RPL versus controls
not undergoing PGT-A suggested that the miscarriage rate may be lower after undergoing PGT-A [35].
A retrospective study in RPL patients comparing PGT-A using FISH analysis for 9 chromosomes
against PGT-A using aCGH for 24 chromosomes showed a significant increase in the pregnancy rates
per transfer and pregnancy rates per initiated cycle in the aCGH group compared to the FISH group [36].
On the contrary, Murugappan et al. [37] have published data from a control trial rebutting the benefits
of PGT-A in RPL patients compared to patients not undergoing PGT-A. However, the Murugappan et al.
[37] study has had its validity discussed by Rienzi and colleagues [38]. No randomization was performed,
and women of the expected control group were, on average, 2 years younger; which could have biased the
results [39]. In conclusion, the study of Murugappan et al. [37] does not give strong evidence for avoiding
PGT-A in RPL patients.
Conclusions
The chromosomal analysis of embryos before transfer in couples with either idiopathic RPL or RPL due to
previous aneuploid embryos should be considered in order to improve pregnancy rates and live birth rates
per pregnancy, and decrease the number of miscarriages, particularly if the miscarriages result from IVF.
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27
Debate: Should PGT-A Still Be Performed
in Recurrent Pregnancy Loss? No
Raoul Orvieto and Norbert Gleicher
Introduction
A large majority of early pregnancy losses are the consequence of chromosomal abnormalities of the
conceptus. If performed correctly, genetic analysis of products of conception therefore offers important
information about potential causes of pregnancy loss and assists in the planning of appropriate
investigations and treatment. Even following comprehensive evaluation, more than half of all women
with RPL will have no identifiable cause, and most will do well in the next pregnancy.
Empirical treatment, including assisted reproductive technology (ART), is often offered, even if no
underlying causes can be identified. Though most patients with RPL do not have fertility problems, under
claims of improving the chance of a live birth, they are frequently offered adjunct treatments, so-called
“add-ons,” such as preimplantation genetic testing for aneuploidy (PGT-A).
PGT-A (previously called preimplantation genetic screening [PGS] and preimplantation diagnosis of
aneuploidy [PGD-A]) currently utilizes trophectoderm biopsy and next-generation sequencing (NGS) in
an attempt to detect embryonic aneuploidy in a trophectoderm biopsy obtained at the blastocyst stage.
The current version of PGT-A is claimed to have significantly improved our ability to accurately diagnose
embryonic aneuploidies without compromising the embryo’s implantation potential.
Within this context, the European Society of Human Reproduction and Embryology (ESHRE) recently
published a somewhat surprising new guideline on RPL [1], in which PGT for monogenic/single gene
defects (PGT-M) or chromosomal structural rearrangements (PGT-SR) were described as established
alternatives to invasive prenatal diagnosis and might avoid pregnancy termination in couples with a high
risk of transmitting genetic disorders. ESHRE offered this recommendation despite extremely limited and
very low-quality supportive evidence, and clearly, no established benefit of any form of PGT on outcomes
in couples with RPL. Importantly, the ESHRE guideline made no recommendation for any form of PGT
in couples with unexplained RPL without known chromosomal abnormalities. PGT-A is therefore not
indicated in couples with unexplained RPL according to the ESHRE guideline.
A Brief History of PGS/PGT-A

Verlinsky and Kuliev [2] initially proposed what was previously called PGS by polar body biopsy under
the hypothesis that removing aneuploid embryos prior to embryo transfer would improve implantation,
pregnancy, and live birth rates for the remaining embryos [3]. Their hypothesis of PGS was widely
embraced, but quickly advanced to the technically simpler cleavage stage biopsies (day 3) [4]. Yet, after
more than two decades of clinical practice, PGS, now renamed PGT-A, still has not fulfilled its promise
of improving IVF outcomes and reducing miscarriage rates [5–13].
One of the milestones in the history of PGS was the 2007 clinical trial by Mastenbroek et al. [14]. Their
study demonstrated, in addition to a lack of efficacy in improving IVF outcomes in older women (i.e.,
poor prognosis patients), that PGS was harmful in terms of lower pregnancy rates. Until then, the basic
hypothesis of PGS was largely undisputed, and proponents of the procedure attributed the procedure’s
243
failure largely to technical aspects, ignoring the questions raised about the basic underpinnings of the PGS
hypothesis [5]. The commercial purveyors of PGS services argued that better techniques and technologies
would lead to expected outcome improvements and validate the PGS hypothesis [15].
New diagnostic platforms did clearly improve the accuracy of chromosomal assessment and allowed
the investigation of complete chromosome complements instead of the prior limited chromosome panels
used for in situ fluorescence hybridization (FISH). By moving embryo biopsies from the single (or double)
blastomere biopsies at the cleavage stage (day 3) to trophectoderm (TE) biopsy (TEB) at the blastocyst
stage (days 5/6) first proposed in 1990 [16], more genetic material could be obtained, presumably
improving the accuracy of PGS (referred to as second-generation PGS) [17]. Utilization of first-generation
PGS quickly declined in favor of second-generation PGS, again without prior validation studies defining
the efficacy of this new testing procedure. Second-generation PGS saw a remarkable increase in clinical
utilization in most regions of the world.
However, as more investigators started raising questions about the basic biological veracity of the PGS
hypothesis, TE mosaicism became a substantial issue of contention, with skeptics considering mosaicism
a profound problem [18], while proponents of PGS generally described mosaicism as a non-issue [15,19–
21]. Further research established that TE mosaicism was much more common than had been suggested
by supporters of PGS/PGT-A and that skeptics had indeed been correct in considering TE mosaicism as
a major reason for questioning the PGS hypothesis on biological grounds.
Clinical Outcomes of Second-Generation PGS

Several studies claimed improved clinical IVF outcomes following second-generation PGS, summarized
in a meta-analysis [22]. The authors concluded that only in patients with normal ovarian reserve (i.e.,
good prognosis patients) PGS significantly improved clinical and sustained pregnancy rates. Those study
results have to be questioned on statistical grounds. The studies included in the meta-analysis favored
second generation PGS and were severely biased, as they uniformly reported IVF outcomes only with
regard to embryo transfers in first fresh IVF cycles. Statistically correct outcome analyses, however,
should be based in intention-to-treat analyses and should include total reproductive potential for each
initiated IVF cycle. Analyses should therefore include fresh plus subsequent frozen/thawed transfers. Any
analysis starting with embryo transfer excludes poor prognosis patients who fail to reach transfer [23].
Kang et al. reported on second-generation PGS on IVF results in women above 37 years of age [24].
With reference to embryo transfer, significant improvements in clinical pregnancy and live birth rates
(LBR) were found. With reference to cycles initiated, however, the results differed remarkably and both
clinical pregnancy and LBR (21.5% and 19.9%) were significantly lower than in non-PGS patients (49.5%
and 39.8%). Similar results were also reported by Kushnir et al. after reanalyzing U.S. national PGS
outcome data, initially erroneously reported to demonstrate an advantage after PGS [25]. Mastenbroek
et al. had previously reported similar findings in their study that utilized first-generation PGS [14].
Due to the lack of properly conducted prospective clinical trials, a theoretical model was published
for second-generation PGS. The theoretical model relied on evidence- based data in the literature on
blastulation and aneuploidy rates, the rate of mosaicism, technical errors, and implantation/live birth
rates of PGS and non-PGS cycles at cleavage and blastocyst stage [26]. The model clearly revealed the
highest LBRs in patients not undergoing PGS on day 3 embryo transfers (21.4%–50%), followed by non-
PGS blastocyst cycles (18.2%–22.2%). Patients undergoing PGS blastocysts transfers achieved the lowest
LBR (7.6%–12.6%).
Accuracy and Precision of PGS

Starting in 2015, the clinical utility of second-generation PGS faced increasing scrutiny. In addition to the
above-noted corrected re-analyses of published studies [23], the literature started reporting cases where
patients experienced spontaneous miscarriages after PGS, in which chromosomal reassessment was
found to be aneuploid, raising the specter of false-negative TEBs [27]. At the same time, concerns about
Debate: Should PGT-A Still Be Performed in Recurrent Pregnancy Loss? No 245
false-positive TEBs arose in relatively good prognosis patients who repeatedly underwent IVF cycles
without ever reaching embryo transfers because all embryos were reported as aneuploid. Suspicion that
false-positive embryos were erroneously labeled as aneuploid led to the transfer of such embryos, resulting
in a surprisingly high number of normal live births and surprisingly low miscarriage rates [28–30].
The rate of TE mosaicism in human embryos, however, remained controversial. Though initially
claimed to be in low single digits, it has since been reported to be as high as 70% and 90% in cleavage
and blastocyst-stage embryos, respectively [31], and is increasingly believed to represent a normal
physiological phenomenon [32]. Mitotic clonal, rather than meiotic universal errors, appear to represent
the majority [33]. While Liu et al. reported that 69% of abnormal blastocysts from women of advanced age
are mosaic for ICM and TE [34], Johnson et al. demonstrated that in younger women 20% of blastocysts
are aneuploid, with a majority of the abnormal blastocysts presenting with only one or two structural
chromosome abnormalities [35]. Even young women, therefore, still show a critical level of mosaicism
at the blastocyst stage [31]. A recent investigation into the cytogenetic constitution of blastocysts using
high-resolution next-generation sequencing revealed only 43% of blastocysts to be supposedly euploid
[36]. The obviously high prevalence of mosaicism at the blastocyst stage questions the basic argument
in favor of switching from first to second generation of PGS, i.e., reduction in false-negative and false-
positive embryo biopsies and lower mosaicism risk in TEBs than cleavage-stage biopsies [15]. Indeed,
the opposite appears to be the case.
Further evidence for inaccurate diagnoses in cases of TE mosaicism came from studies of multiple TEB
biopsies in the same embryos, demonstrating up to 50% divergence between biopsies of the same embryos
in the same laboratories, and up to approximately 80% divergence between multiple biopsies in different
laboratories [28,29,37]. A recently published study evaluated eight embryos. There was concordance of
multiple TEBs regarding TE and ICM biopsies in four embryos, and discordant results (i.e., mosaicism)
three out of eight embryos [38].
These studies suggest presence of TE mosaicism in at least half of all embryos biopsied, but the
prevalence can be expected to increase in parallel with growing numbers of biopsies. In addition,
laboratory platforms used in assessing TEBs offer different diagnostic sensitivities and specificities in
detecting chromosomally abnormal cell lines, as was recently acknowledged by the Preimplantation
Genetic Diagnosis International Society (PGDIS) when exclusively recommending NGS platforms [39].
Can We Improve PGS Accuracy and Precision?

The aforementioned observations are not surprising, since both the TE and the ICM are products of
different cell lineages [40], with the ICM giving rise to the fetus, while the TE becomes placenta. Even in
normal euploid offspring, the placenta has frequently been known to be seeded with islands of aneuploid
cells [41]. This observation alone should therefore have led to caution about how TEBs are interpreted.
Recent mouse data has also demonstrated more mosaicism in the TE than the ICM, and more efficient
self-correction in the ICM that eliminates aneuploid cell lineages. The same mouse study also demonstrated
considerable self-correction of even significant degrees of aneuploidy in the ICM, downstream from the
blastocyst stage, resulting in 100% chromosomally normal pups with up to half of ICM cells being
aneuploid at the blastocyst stage. Even with two-thirds of ICM cells aneuploid, a significant minority of
pups were chromosomally normal at birth [32]. If abnormal embryos at the blastocyst stage still have the
ability to self-correct, and assuming that human embryos have similar abilities to the mouse, any rationale
for blastocyst stage TEBs disappears.
There is also the question of whether a single TEB can reliably define ploidy of the whole TE.
Mathematical models, assuming a 6-cell TEB (the average reported cell number of a TEB) and an
approximately 300-cell total TE, demonstrated that the likelihood of false-negative and false-positive
diagnoses was too high to permit determination as to whether an embryo could be transferred or should
be discarded [42]. A larger biopsy involving more TE cells might therefore be suggested as a possible
solution. Gleicher et al. mathematically demonstrated that such a biopsy, under the best of circumstances,
assuming an even distribution of aneuploid cells, would require 28 cells [42]. A recent study by Neal et al.
[43], however, invalidated this suggestion. Neal et al. clearly demonstrated that the lowest live birth rates
after single-embryo transfer were associated with TEBs with the highest relative DNA content (i.e., with
biopsies with the highest cell numbers). This observation supports Paulsen’s argument of TEBs causing
significant damage to an embryo’s implantation potential [44]. While a higher cell number in a TEB may,
at least theoretically, improve the precision of PGS 2.0, higher cell numbers are likely highly detrimental
to blastocyst implantation.
PGT-A for RPL Patients

In a systematic review on PGS for unexplained RPL patients, Musters et al. [7] concluded that there is
no improvement in the live birth rate with PGS. Of note, the included studies were of small sample sizes,
with different endpoints, and used FISH.
In a recently published study comparing PGT-A and expectant management (EM), Murugappan
et al. [45] reported similar pregnancy, live birth, and clinical miscarriage rates at PGT-A or expectant
management with a shorter time to pregnancy (3.0 vs. 6.5 months) in the patients managed expectantly.
Moreover, Murugappan et al. [45] did not find PGT-A a cost-effective strategy for increasing live births
[46].
Based on the above observations, the previously referred-to new ESHRE guideline [1] made no
recommendation with regard to the use of PGT-A in patients with unexplained RPL. The ESHRE
guideline mentioned the study by Shahine et al. [47], reporting that in couples with unexplained RPL with
diminished ovarian reserve there was a higher percentage of aneuploidy in blastocysts and more initiated
IVF cycles with no embryo transfers. Hence Shahine et al.’s [47] study clearly contradicts the use of PGT-A
in unexplained RPL. Furthermore, the recently published combined American Society for Reproductive
Medicine (ASRM) and Society for Assisted Reproductive Technology (SART) committee opinion on the
use of PGT-A [48] concluded that the extremely challenging questions of false-positive testing, embryonic
damage, and loss of euploid embryos between day 3 and blastulation remain unanswered. The routine use
of blastocyst biopsy with aneuploidy testing in all infertile patients is therefore, according to this opinion,
at present not recommended. To date, not a single study in the literature has suggested improved live birth
rates in RPL patients after PGT-A.
Conclusions
Although in most couples with RPL subfertility is not a problem, ART with PGT-A is often advised
despite the absence of any supportive evidence. Patients might be interested in PGT-A to shorten the
time to conceive, improve reproductive outcome, and reduce the miscarriage rate, but to date PGT-A
has not shown any benefit in any of these parameters. Properly randomized controlled trials, which
evaluate the cumulative live birth rates following a single oocyte retrieval, utilizing all fresh and frozen
embryos in couples with unexplained RPL and no known chromosomal abnormality may be helpful in
further clarifying the potential benefits of PGT-A. However, it appears increasingly obvious that the basic
biology of the preimplantation human embryo simply does not support the PGS-hypothesis. It is therefore
becoming increasingly difficult to expect any benefit from PGT-A.
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28
Third Party Reproduction in
Gautam Nand Allahbadia, Rubina Merchant, Akanksha Allahbadia Gupta, and A.H. Maham
Introduction
A higher frequency of spontaneous miscarriage has been reported among infertile couples, as well as a
higher prevalence of infertility among patients with recurrent spontaneous miscarriages, compared with
the general population [1,2]. Recently, assisted reproductive techniques (ARTs) have been used to prevent
further miscarriages in women with recurrent miscarriage using either (i) screening or diagnosis of
embryonic chromosomes prior to embryo replacement by preimplantation genetic testing for aneuploidy
(PGT-A) [3–6] or (ii) surrogacy. While PGT-A assumes that the embryo is chromosomally abnormal and
that the mother should receive a chromosomally normal embryo, surrogacy assumes that the embryo
is normal and that the maternal environment needs to be substituted [7]. Both of these methods are
described in other chapters in this book. However, there is a group of patients with RPL in whom third-
party reproduction (TPR) needs to be used. TPR involves the use of donor gametes (sperm or oocytes),
embryos, or surrogates by couples who may not be able to conceive with their own gametes or gestate a
fetus, respectively [8]. TPR may be classified as:
1.
Sperm donation. The third party is a sperm donor who provides sperm that can be used for
insemination of the future mother or to fertilize an oocyte IVF with the transfer of the resulting
embryo into the mother or a surrogate mother.
2.
Oocyte donation. The third party is an oocyte donor who donates oocytes for IVF with the
transfer of the resulting embryo into the mother or a surrogate mother.
3.
Embryo donation. The third party is an embryo donor, donating surplus embryos for use by a
couple in need or a commissioned surrogate after the woman for whom they were originally
created has successfully carried one or more pregnancies to term, or embryos specifically
created for donation using donor eggs and donor sperm.
4.
Surrogacy. The third party is a surrogate woman used to carry a baby through pregnancy to
term for a woman incapable of doing so [8].
This chapter highlights the role of TPR as a treatment option for RPL.
Causes of RPL
Embryonic Causes
Aneuploidy is the most common embryonic cause of recurrent miscarriage, with the overall incidence
being quoted as 40% [9] when using the older banding karyotype techniques, but higher incidences have
been reported using molecular techniques [10]. However, aging gametes is another cause. Aging gametes
in the female genital tract before fertilization, maternal age, and the number of previous miscarriages are
independent risk factors for a further miscarriage. A higher incidence of small amniotic sac syndrome and
249
euploid miscarriages has been reported in infertility patients older than 35 years, the risk of miscarriage
being highest among couples where the woman is ≥35 years of age and the man ≥40 years of age
[1]. Patients >40 years undergoing in vitro fertilization (IVF) have also been shown to have a 29%
spontaneous miscarriage rate after ultrasound evidence of a fetal heartbeat [11]. Aneuploidy is the
most significant single factor affecting early pregnancy failure and miscarriage. The risk of aneuploidy
increases significantly with increasing maternal age. There has been a tremendous advance in technology
that has made preimplantation genetic testing (PGT-A) readily accessible. However, in the older age
groups, there may be an insufficient number of oocytes to make PGT-A a viable option. In addition, all
the embryos may be aneuploid. In these cases, there may be a need for third party reproduction involving
ovum donation. The embryo is taken from a younger donor and is therefore assumed to be euploid.
However, there is a question whether PGT-A should be performed on an embryo from oocyte donation in
order to prevent aneuploidy and subsequent miscarriage of an aneuploidy embryo.
Parental Causes
Regarding the male partner, standard semen parameters are poor predictors of fertility potential. Owing
to the role of sperm factors in early embryonic development, evaluation of sperm DNA integrity in
idiopathic RPL is a useful diagnostic and prognostic marker with clinical implications [12]. The sperm
from men with a history of idiopathic RPL have a higher percentage of DNA damage with a sperm DNA
fragmentation index (DFI) of approximately 26% in male partners of couples experiencing idiopathic
RPL. Men with a high DFI are infertile, whereas the sperm of men with a lower DFI (26%) fertilize and
allow conception but there may be subsequent RPL [12]. Environmental factors, such as occupational
and chemical exposure, stress, alcohol, and radiation have also been reported to be associated with an
increased risk of recurrent miscarriages [13]. Hence there may be a need for sperm donation.
The maternal causes of recurrent pregnancy loss are described in other chapters in this book. However,
evaluation of defects in endometrial receptivity with native techniques based on endocrine parameters and
newer techniques based on microRNAs, proteomics, and epigenetics may help to elucidate other maternal
causes of RPL [14]. Pregnancies obtained after IVF and embryo transfer (IVF-ET) are at increased
risk for an adverse outcome compared with natural pregnancies. Special investigations in ART include
evaluation for inhibin-A, day 11 total beta-hCG, CA-125, PGT-A/preimplantation pregnancy diagnosis
(PGD), and aneuploidy testing [15].
Auto- and cellular immune responses seem to be associated with RPL. Vitamin D (VD) has been
shown to play a role in the modulation of the immune system. Inappropriate immune modulation,
possibly involving VD deficiency (VDD) in pregnancy, has been associated with sporadic spontaneous
miscarriage (SA), preeclampsia, gestational diabetes, fetal growth restriction, and preterm labor. A high
prevalence of VDD has been reported in women with RPL, which may be associated with immunological
dysregulation and consequently with RPL [16]. For a pregnancy to proceed to term, early modulation of
the immunologic response is required to induce tolerance to the semi-allogenic fetus.
However, there are resistant patients who continue miscarrying despite immunomodulation. There
are patients who continue to lose euploid embryos after the immunomodulation described in Chapter 29.
Failure of immunotherapy in patients with large numbers of miscarriages may be an indication for
surrogacy. Resistance to anticoagulants and aspirin or other treatment modalities in the antiphospholipid
syndrome may also indicate the need for surrogacy. In addition, side effects of the antiphospholipid
syndrome condition itself, such as thromboembolism, and so on, may make another pregnancy too
dangerous. In these circumstances, again, surrogacy may be indicated.
Anti-Müllerian hormone (AMH) levels are a marker of biological ovarian age and embryonic aneuploidy
risk in RPL. Jiang et al. [6] investigated 422 IVF cycles in 394 unexplained RPL patients undergoing
PGT-A. There was a significant difference in embryonic aneuploid rate according to the levels of AMH.
The incidence of embryonic aneuploidy was significantly higher in the low AMH group, compared to the
normal AMH group (p = 0.002) and patients with a high AMH (p = 0.015). Even after age stratification,
the embryonic aneuploidy rate was still significantly different among AMH groups, with a similar trend in
women ≥35 years old but not in younger women. Hence, maternal diminished ovarian reserve along with
oocyte aging may contribute to impaired chromosomal competence of the embryo [6]. Murugappan et al.
Third Party Reproduction in Recurrent Pregnancy Loss 251
[17] also confirmed that AMH < 1 ng/mL is associated with decreased likelihood of live birth among
RPL patients pursuing expectant management. Hence, low AMH levels in older women may indicate a
need for ovum donation.
Sperm Donation for Recurrent Pregnancy Loss

Indications
Sperm donation may be indicated in severe male factor infertility due to: (i) A high risk of fertilization
failure or two previous fertilization failures with conventional IVF. (ii) Semen parameters below the
threshold required for standard IVF treatment, for example, oligoasthenoteratozoospermia (OAT), severely
oligozoospermic and teratozoospermic men (using strict normal sperm morphology ≤5%) with a very
high (>70%) frequency of defective sperm-zona pellucida (ZP) interaction and hence a high risk of a low
or zero fertilization rate in IVF. Severely impaired spermatogenesis (nonobstructive azoospermia), severe
oligozoospermia, and OAT often have a genetic origin that necessitates sperm donation [18]. (iii) The
absence of acrosomes or the presence of immotile spermatozoa. (iv) Genetic disorders such as Klinefelter
syndrome 47, XXY. (v) Sperm autoimmunity (high titers of antisperm antibodies/sperm-bound antibodies
that interfere with gamete interaction) [18]. (vi) When PGD is indicated in pregnancies that are at high risk
of aneuploidy because of genetic factors associated with azoospermia, to avoid contamination by extraneous
DNA in the case of PCR-based testing and increase the number of embryos available for testing [18].
Role of Sperm Donation

Patients with the above criteria usually present as infertility rather than RPL. However, if patients with
RPL present with the above criteria, sperm donation may be indicated. In addition, if DNA analyses
from men with a history of RPL have a high percentage of DNA damage with a sperm DFI of 26% or
above, sperm donation may be indicated. The question arises as to whether sperm donation is indicated
in idiopathic RPL. In couples with a good prognosis, sperm donation is not indicated. The literature
differs with regard to sperm aneuploidy. No increased incidence of aneuploidy or structural anomalies
was found in the sperm of men whose partners had RPL above the level seen in normally fertile men [19].
In addition, Carp et al. [20] have reported 99 parental chromosomal aberrations in recurrent miscarriage
(RM), 55 maternal, 43 paternal, and one in both partners. However, Rubio et al. [21] analyzed 12 sperm
samples from IVF couples with two or more miscarriages. Diploidy and disomy were assessed for
chromosomes 13, 18, 21, X, and Y using fluorescence in situ hybridization. Sex chromosome disomy
from RM significantly increased compared to controls (0.84% vs. 0.37%). In addition, increases in disomy
have been related to increased aneuploidy in the offspring [22]. The editor has described that in the Tel
Hashomer registry, there were 62 cases of a change in the male partners of 1925 patients, 22 had three
partners, and one had five partners. In these cases, a change of the male partner had not alleviated the
problem of RPL. Therefore, sperm donation should probably be limited to the indications above. In
addition, there is no series in literature on sperm donation in RPL.
Oocyte Donation
Indications
Oocyte donation may be indicated in: (i) carriers of genetic disorders, for example, 46, XY pure gonadal
dysgenesis, Turner syndrome (45, XO), (ii) repeated IVF failure with autologous oocytes, (iii) advanced
maternal age, and (iv) contraindications for spontaneous or induced ovulation, such as those with von
Willebrand disease or other major bleeding disorders [23]. Of these, advanced maternal age is the major
indication in RPL. In addition, women of advanced maternal age with low AMH levels as described above
may benefit from oocyte donation. There is also an indication in women who repeatedly lose aneuploid
embryos and in whom PGT-A fails to find euploid embryos for replacement.
Experience with Oocyte Donation for RPL

Simón et al. [24] reported that in 92 cycles of ovum donation, there were 64 implantations, 30 (32.6%) viable
pregnancies, and 34 (37.0%) miscarriages. In Remohi et al.’s series [25], ovum donation was performed
in eight RPL couples, in which the woman was a low responder to gonadotropins. Twelve cycles were
performed. There was a 75% pregnancy rate and a delivery rate of 66.6%. The miscarriage rate was 11.1%
per cycle. The authors suggested that the oocyte may be the origin of infertility in women with idiopathic
recurrent miscarriages [25]. In Remohi et al.’s [25] study, patients were downregulated with gonadotropin-
releasing hormone analogs and supplemented with estradiol valerate for a minimum of 15 days until
fertilized embryos from donor oocytes were transferred in IVF. Progesterone was then administered until
day 100 of pregnancy. The results of oocyte donation compared favorably with low responders without
a history of RPL undergoing oocyte donation during the study period. However, the live birth rates need
to be compared to the spontaneous live birth rates for the patient’s age and number of miscarriages [25].
Issues with Oocyte Donation

Although oocyte donation is a successful option for achieving conception in appropriate patients, there
is a high risk of complications in pregnancies thus obtained, especially in women with genetic disorders
and advanced maternal age. Hence the criteria for the selection of such patients should be strict, and
rigid protocols for the medical management of such pregnancies is an absolute requirement [8]. For
instance, although patients with Turner syndrome may achieve high pregnancy rates, comparable to
those observed in patients with other indications for oocyte donation, high miscarriage rates, potentially
severe cardiovascular complications during pregnancy, and early implantation failure often ensue. These
complications may possibly be associated with a deficiency of X-linked genes regulating endometrial
receptivity. In addition, a subsequent high rate of cesarean section is commonly observed [8].
Oocyte donation is a long and labor-intensive process with a significant amount of emotional, financial,
and physical involvement from all parties [26]. In order to ensure safety and success of the procedure, all
the participants should be extensively screened medically and psychologically, and all parties involved
should understand all aspects of the procedure [27]. Written informed consent should always be obtained
from donors and recipients prior to commencing the program.
Embryo Donation
Indications
Embryo donation may be medically indicated in couples where both sperm and oocyte donation are
mandatory to achieve a normal conception, as in unexplained genetic disease and failure of ART due
to poor fertilization or poor embryo quality. The embryos may be obtained from couples consenting to
donate surplus embryos following self-use or specifically created by using a chosen sperm and oocyte
donor [8]. Embryo donation may be offered as a viable treatment option in the event that all embryos
are chromosomally abnormal following PGT-A. If pregnancies are miscarried despite the transfer of
genetically normal embryos following PGT-A, there may be a role for embryo donation, but embryo
donation should only be advised if all the therapies for maternal causes of RPL have been exhausted.
Issues with Embryo Donation

Creation of embryos for donation might be fully justified; however, wastage of surplus embryos not
intentionally created for future use, donation, or research triggers ethical, legal, and moral issues that
boil down to the moral status of the embryo. The main ethical issues concern the effect on offspring,
consent and counseling of donors and recipients, avoidance of mixing embryos or gametes from different
sources, and payment of the donor’s expenses. The main legal issues concern whether embryo donation is
viewed as gamete donation or adoption, the rearing rights and duties of donors and recipients in resulting
offspring, liability, compensation issues, and the legality of monetary compensation for donors [28].
Surrogacy
Surrogacy is a reproductive technology involving one woman (surrogate mother) carrying a child for
another person(s) (commissioning person/couple), based on a mutual agreement requiring the child to
be legally relinquished to the intended parent(s) or the commissioning couple/person following birth
[27]. IVF allows the creation of embryos from the gametes of the commissioning couple and subsequent
transfer of these embryos to the uterus of a surrogate host. Clinical pregnancy rates achieved in large
series are up to 40% per transfer and series have reported live births in 60% of hosts [29].
Indications
Apart from its indications in patients with congenital (Mayer-Rokitansky-Kuster-Hauser syndrome) or
surgical absence of the uterus (hysterectomy) and various gynecological cancers, surrogacy may be offered
as a treatment option in women with repeated IVF failure of euploid embryos, high-order unexplained
recurrent miscarriages with a maternal cause, severe medical conditions, such as severe heart or renal
disease in which pregnancy is contraindicated or life-threatening, or following treatment for numerous
oncological and non-oncological conditions that result in uterine damage and poor reproductive outcomes
[27], or patients with the antiphospholipid syndrome or other hereditary thrombophilias that have caused
severe thrombotic episodes in the past which make pregnancy undesirable or life threatening. Other
maternal causes of RPL that may benefit from surrogacy include autoimmune causes, anatomical uterine
defects or Müllerian fusion defects following failed surgical correction and/or repeated miscarriage, and
endocrine disorders that fail medical treatment. Oncological treatment for gynecologic cancers results in
a reduction in the size of the uterus or possible damage to the uterine vasculature leading to decreased
feto-placental blood flow. Decreased feto-placental blood flow may increase the risk for pregnancy-
related complications, including later pregnancy losses, preterm labor and delivery, low birth weight, and
placental abnormalities. In these indications, surrogacy may be the only option [30]. Surrogacy assumes
that the embryo is normal and that the maternal environment needs to be substituted [7].
Types of Surrogacy
Surrogacy may be of two types: (i) traditional surrogacy, where the surrogate or birth mother is also the
oocyte donor, and hence the genetic mother. The intended father is the genetic father; pregnancy may be
achieved by artificially inseminating the surrogate with the intended father’s sperm for IVF. (ii) Gestational
carrier surrogacy involving IVF, where the gametes from the intended parents or commissioning couple
(the couple requesting surrogacy) are fertilized in vitro and the embryo transferred into the gestational
carrier surrogate, who only “rents” the womb. The surrogate is not genetically linked to the child. The
child is legally adopted by the commissioning couple following delivery [8]. Gestational carrier surrogacy
is the most acceptable form of surrogacy practiced today, and in contrast to traditional surrogacy, is
largely complication-free without major ethical or legal complications. The treatment results are good,
and reassuring with regard to follow-up of children, commissioning couples, and surrogates [29].
In addition to the classification of surrogacy by parental roles, surrogacy can also be classified by
financial compensation as (i) altruistic surrogacy that does not financially compensate the surrogate
for her role apart from fees and costs associated with bringing an embryo to term and (ii) commercial
surrogacy, which financially compensates a surrogate above the expenses associated with the pregnancy,
that is, the surrogate is paid for her gestational “services.” Altruistic surrogacy is the most common among
family members or close friends where the decision to be a surrogate stems from a willingness to help [31].
Issues with Surrogacy

Surrogacy is often beset with legal, social, ethical, and psychological complications. Some of the most
significant problems that could result from improper surrogacy arrangements are: (i) failure to relinquish
the baby immediately after birth, (ii) separation of a commissioning couple prior to treatment initiation, (iii)
withdrawal of a patient from treatment following initial counseling of the implications of the treatment, (iv)
poor response to follicular stimulation, particularly after Wertheim hysterectomy[32], and (v) the possibility
of the birth of a handicapped or genetically affected child and fear of rejection [33]. Hence, all the parties
(commissioning couple, surrogate, and the gamete or embryo donor and recipients when employed) involved
in a surrogacy arrangement should be bound by a surrogacy contract and thoroughly counseled on all the
medical, legal, financial, ethical, and psychological aspects and risks and implications of the treatment. The
implications of multiple pregnancy and the possibility that the surrogate host may spontaneously abort a
pregnancy should be discussed with the commissioning couple prior to commencing the program. A written
informed consent should be obtained from all third-party participants [27]. Consent built upon effective
lines of communication between clinical staff and legal counsel, assuring that parentage, relinquishment,
and recontact information in donor–recipient agreements are consistent with clinic consent documents, and
desires of both parties are mandatory in all gamete donations. All decisions must be adequately documented
and honored, and long-term counseling needs should be addressed [34]. Prior to embarking on a surrogacy
program, commissioning couples, or alternatively, gamete donors, when employed, should be screened
thoroughly to ensure that they do not transfer infection or a genetic disease to the offspring. Surrogates
should likewise be screened and deemed physically, medically, and psychologically fit to undertake the
responsibility of carrying the pregnancy to term. The British Medical Association has adequately detailed
issues for discussion with the commissioning couple and surrogate prior to signing a surrogacy contract [26].
The guidelines for surrogacy, laid down by the “Guidelines for Accreditation, Supervision and
Regulation of ART Clinics in India” include the following: (i) A child born through surrogacy must be
adopted by the genetic (biological) parents unless they can establish through genetic (DNA) fingerprinting
(of which the records will be maintained in the clinic) that the child is not theirs. (ii) Surrogacy by assisted
conception should normally be considered only for patients for whom it would be physically or medically
impossible/undesirable to carry a baby to term. (iii) Payments to surrogate mothers should cover all
genuine expenses associated with the pregnancy. Documentary evidence of the financial arrangement
for surrogacy must be available. The ART center should not be involved in this monetary aspect. (iv) A
surrogate mother should not be over 45 years of age. Before accepting a woman as a possible surrogate
for a particular couple’s child, the ART clinic must ensure (and put on record) that the woman satisfies
all the testable criteria to go through a successful full-term pregnancy. (v) A relative, a known person,
as well as a person unknown to the couple may act as a surrogate mother for the couple. In the case of a
relative acting as a surrogate, the relative should belong to the same generation as the woman desiring
the surrogate. (vi) A prospective surrogate mother must be tested for HIV and shown to be seronegative
for this virus just before embryo transfer. She must also provide a written certificate that (a) she has
not had a drug intravenously administered into her through a shared syringe, (b) she has not undergone
blood transfusion, and (c) she and her husband (to the best of her/his knowledge) has had no extramarital
relationship in the last 6 months. (This is to ensure that the person would not develop symptoms of HIV
infection during the period of surrogacy.) The prospective surrogate mother must also declare that she
will not use drugs intravenously and not undergo blood transfusion excepting of blood obtained through
a certified blood bank. (vii) No woman may act as a surrogate more than thrice in her lifetime [35].
Different interpretations of surrogacy in various countries, based on their definition, application, social,
religious, and legal influences have complicated matters further, extending the practice across political
borders and beyond judicial limits [27], necessitating a complete appraisal of the law of the land to protect
all parties concerned, and especially the offspring to be.
Experience with Surrogacy for RPL

In 8 years’ experience of an IVF surrogate gestational program, Raziel et al. [36] reported 33% and
12% pregnancy rates per patient and per transfer, respectively, in patients with IVF implantation failure,
recurrent miscarriages, and deteriorating maternal diseases, compared to 70% and 20%, respectively,
in patients with Rokitansky syndrome and post-hysterectomy. The authors concluded that the existence
or absence of the uterus in the commissioning mothers is irrelevant for their IVF performance and
conception rates. In patients who conceived after more than three IVF cycles, an additional “oocyte factor”
might be present [36]. Raziel et al. [37] reported a normal live birth in a patient with 24 prior pregnancy
losses. The editor has advised surrogacy (unpublished) in a secondary aborter with 12 miscarriages, one
primary aborter with 6 miscarriages and triplets of 25 weeks who died from prematurity, and a primary
aborter with 8 missed abortions including 2 euploid miscarriages, who continued miscarrying despite
immunoglobulin therapy. In all three cases, the surrogate carrier delivered normal twins. The logic of
surrogacy in patients with large numbers of miscarriages is due to the poor prognosis and low incidence
of chromosomal aberrations.
Conclusions
Recurrent pregnancy loss is a frustrating and debilitating experience that leaves patients despairing and
emotionally drained. Third-party reproduction has a definite role to play in patients with a poor prognosis.
Repeat aneuploidy following IVF-PGD/preimplantation genetic screening (PGS) may be an indication
for TPR with embryo donation if the maternal environment is supportive, or alternatively, surrogacy in
patients with a maternal cause for recurrent miscarriage, such as a severe autoimmune disorder, which
is resistant to treatment, and carrying a pregnancy is contraindicated. Oocyte donation may be offered
as a treatment option in patients with exclusively maternal X-linked disorders without a related sperm
chromosomal abnormality, advanced maternal age, and repeated failure with autologous oocytes, bearing
in mind the future prognosis of the pregnancy, while sperm donation may be an option in couples where
the male partner has a sperm chromosomal abnormality. In balanced parental chromosome aberrations,
it is uncertain which treatment mode is indicated [7]. However, ART with PGD/PGT-A or surrogacy may
have a place only in those patients with a poor prognosis in whom ART will be shown to improve the
subsequent live birth rate above the spontaneous rate [7].
Individualizing the recurrence risk and building on an evidence-based approach in management and
counseling should be the recommended clinical practice [38]. However, to date, there are no evidence-
based trials. The patients who are selected for TPR techniques are usually highly selected, and small in
number. Hence it has been impossible to devise a controlled trial of treatment.
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29
Leucocyte Immunotherapy for Recurrent Miscarriage
Salim Daya
Introduction
The prevalence of recurrent spontaneous miscarriage (i.e., three or more miscarriages) has been reported
to be as high as 4.6% [1]. In many women the cause of the miscarriages remains unexplained, leading a
number of researchers in this field to propose that there is an immunologic etiology. Normal pregnancy
involves maternal immunological recognition of the trophoblast [2]. An absence or attenuation of the
maternal recognition response to paternal antigens expressed by the conceptus allows miscarriage to
occur as a maternal rejection phenomenon. Repeated rejection of the feto-trophoblast unit resulting from
alloimmune recognition failure might provide a reason why recurrent miscarriages occur in some women.
Thus, attempts have been made to stimulate protective immune responses by immunizing the female with
allogeneic leucocytes. The efficacy of this approach is explored in this chapter.
The Mowbray Trial

The approach of using leucocyte immunization to treat unexplained recurrent miscarriage began several
decades ago with either third-party leucocytes or paternal leucocytes. The option of using third-party
leucocytes was based on the observation that renal allograft rejection could be delayed by third-party
blood transfusions [3]. In contrast, the option of using paternal leucocytes was based on the belief that
antibodies generated in the female against paternal antigens were necessary to block an immune rejection
response against the developing pregnancy [4]. Over the years, and especially after concerns about the
development of AIDS, the latter approach has become more favorable.
The first clinical trial of treatment efficacy was published by Mowbray et al. in 1985 [5]. This was
a randomized paired sequential trial. Patients were paired on entering the trial, placing randomly one
member of each pair on the experimental treatment arm and the other on the control treatment arm
[6]. Each pair was monitored to determine whether a successful pregnancy or miscarriage occurred in
one or both subjects in the pair. The results can be plotted graphically as shown in Figure 29.1, which
summarizes the results of the Mowbray trial. The x-axis shows the number of pairs so far recorded. In
the y-axis, one plots the difference between the results for husband’s cells or wife’s cells. There was a
significant benefit from immunization with paternal lymphocytes. In the experimental group, out of 22
women who had received paternal lymphocyte immunization, 17 had a successful pregnancy (77.3%). In
contrast, out of 27 women who had received autologous lymphocyte immunization, 10 had a successful
pregnancy (37%) (OR 5.78; 95% CI 1.4−25.4). The absolute treatment effect was 40.3%, resulting in a
number need to treat of 2.5. Thus, for every five women with recurrent miscarriage treated with paternal
lymphocyte immunization prior to the next pregnancy, two additional women would have a successful
pregnancy compared to immunization using the woman’s own lymphocytes.
It is important to note that in the Mowbray trial, the subjects were healthy women with no collagen
disease, at least two miscarriages, no more than one live child, no family history of recurrent miscarriage,
and no miscarriages in previous pregnancies of either partner. Immunization was performed before the
onset of the next pregnancy. Patients and obstetricians were unaware of the treatment allocation until the
end of the next pregnancy.
257
Husband’s cells cance

5% signifi
10
8
6
Excess preferences
4
2 No significant
difference
between groups
2
4
6
8
10
5% s
ignifi
Wife’s cells canc
e
FIGURE 29.1 Randomized paired sequential trial of paternal leucocyte immunization versus control. (From Mowbray
et al. [5] with permission.)
Worldwide Collaborative Prospective Study of

Immunotherapy for Recurrent Miscarriage
Since the first demonstration of the efficacy of paternal lymphocyte immunization, several subsequent
trials were unable to detect significant differences between experimental and control groups [7–9]. These
conflicting observations have been partly explained on the basis of inadequate sample sizes, heterogeneity
of study samples among trials, and the effect of cointervention [10]. In addition, concern was raised that
the significance of the placebo effect in original trial using autologous lymphocytes was obscured by the
fact that these women did not develop an inflammatory reaction at the immunization site, unlike those
receiving heterologous (paternal) leucocytes [11].
Owing to the uncertainty with respect to the efficacy of immunotherapy, a collaborative observational
study was carried out using data from the different centers using immunization, which would include
defining the subjects in treatment and control populations and stratifying for important prognostic factors.
Data were obtained from questionnaires on each patient from contributing centers. Both published and
unpublished data were accepted. The data were separated into three categories depending on the study
design: (i) randomized controlled trials, (ii) prospective cohort studies with treated and untreated women,
and (iii) case series of treated women. Data from the first two categories were analyzed independently
by epidemiologists in two centers using the data forms that had been submitted from each participating
center [12]. The data were combined using the principles of meta-analysis to generate overall estimates
of the effect of immunotherapy. The data analyses were undertaken by two independent groups to permit
more reliable conclusions. The primary question was whether immunization with leucocytes is more
efficacious in improving the live birth rate in women with recurrent miscarriage.
Results of the Worldwide Collaborative Prospective Study

of Immunotherapy for Recurrent Miscarriage
Fifteen clinical trial centers met the criteria for inclusion in the collaborative study; in seven centers the
trial design was double-blind and randomized, in two centers the trial design was randomized but not
blinded, and in six centers the trial design was comparative, in which treatment was not determined by
random allocation but by choice. Only the nine randomized trials were selected for analysis. To avoid
the perception of bias, the analysts from group #1 chose to exclude the trial data from their own center.
Thus, this group analyzed the data from eight centers. The analysts from group #2 chose to exclude the
trial data from Milan because the subjects from this center did not meet the inclusion criteria. Thus, this
group analyzed the data from eight centers.
Leucocyte Immunotherapy for Recurrent Miscarriage 259
Analysis from Group #1

In total, 430 subjects were included in the analysis; the experimental group was comprised of 180 subjects
treated with husband’s leucocytes and 51 treated with donor leucocytes, whereas the control group was
comprised of 199 subjects. The data from husband’s and donor leucocytes were combined because no
difference between them was observed. When the data from all eight centers were combined with a meta-
analysis, immunotherapy was observed to be more efficacious than control treatment (RR 1.16%, 95% CI
1.01−1.34; p = 0.031). The absolute treatment effect was 7.6% (68.4% in the experimental group vs. 60.4%
in the control group), representing 8 additional live births per 100 treated patients. The number needed
to treat was 13, implying that for every 13 women treated with immunotherapy there was one additional
live birth compared to control treatment.
Analysis from Group #2

In total, 449 subjects were included in the analysis; the experimental group was comprised of 240 subjects
treated with leucocytes (both husband’s leucocytes and donor leucocyte groups were pooled), whereas
the control group was comprised of 209 subjects. When the data from all eight centers were combined in
a meta-analysis, immunotherapy was observed to be more efficacious than control treatment (RR 1.21%,
95% CI 1.04−1.37; p = 0.024). The absolute treatment effect was 10% (61.7% in the experimental group
vs. 51.7% in the control group), representing 10 additional live births per 100 treated patients. The number
needed to treat was 10, implying that for every 10 women treated with immunotherapy there was one
additional live birth compared to control treatment.
Although the teams made different decisions on the eligibility of patients based on different data
entry quality control methods and they used different statistical methods for analysis, it is remarkable
that their estimates of a relative treatment effect of immunotherapy are almost identical and implies that
the conclusion is robust. The live birth ratios (ratio of percent live births in treatment vs. control groups)
with their 95% confidence interval were 1.16 and 1.21, respectively, and both are statistically significant.
The absolute differences in live birth rates between experimental and control groups were 8% and 10%
in the respective analyses. The treatment effect across all trials was consistent within each analysis: the
small difference between the two analyses (16% vs. 21% increase in the likelihood of live birth with
immunotherapy) is probably explained by the different criteria for eligibility for centers and subjects [13].
Subgroup Analysis of the Worldwide Collaborative Prospective

Study of Immunotherapy for Recurrent Miscarriage
The worldwide collaborative observational study and meta-analysis demonstrated that immunotherapy
significantly improved the live birth rate in women with recurrent miscarriage [14]. However, the treatment
effect was relatively small. Several possibilities can explain this observation. One possibility is the lack of
appropriate diagnostic tests to identify patients most likely to benefit from immunotherapy. This problem
is further complicated by combining the data from women primary recurrent miscarriage (i.e., women in
whom no pregnancy had advanced beyond 20 weeks’ gestation) with those from women with secondary
recurrent miscarriage (i.e., in whom recurrent miscarriages were preceded by at least one live birth or
stillbirth beyond 20 weeks’ gestation) [15].
A further complicating factor is the observation from the worldwide study that the presence of antipaternal
antibodies is a negative prognostic indicator for the efficacy of immunotherapy [14]. Heterogeneity was
addressed by performing a subgroup analysis of the data from women with unexplained primary recurrent
miscarriage in whom there was no evidence of antipaternal antibodies [16]. The data from the eight
randomized trials selected for analysis in group #1 were reanalyzed to answer the following question: “In
women with unexplained primary recurrent miscarriage and no evidence of antipaternal antibody, does
immunization with allogeneic leucocytes improve the live birth rate?” [16].
In the experimental group, because no significant difference in live birth rates was observed between
paternal and donor leucocyte treatment groups, and the number of patients receiving donor leucocytes
was too small for an independent analysis with sufficient statistical power, the data from these groups
London
Taipei
Melbourne
Aalborg
Newtown
Paris
Edinburgh
Hamilton
Common odds ratio

0.01 0.1 1 10 100
Odds ratio for live birth
FIGURE 29.2 Odds ratio tree for immunotherapy trials in unexplained primary recurrent miscarriage. The odds ratio and
its 95% confidence interval are shown for each of the eight centers identified by their geographic location. The common
odds ratio and its 95% confidence interval is the overall estimate of the effect of treatment. (From Daya and Gunby [16]
with permission.)
were combined for the analysis. Similarly, in the control group, because no significant difference in live
birth rates was observed in patients receiving autologous leucocytes or saline, the data from these groups
were combined for the analysis. The odds ratio tree for the point estimates and their 95% confidence
intervals from each trial center that met the inclusion criteria are shown in Figure 29.2. No center’s
results were statistically significant and no significant heterogeneity in treatment effect was observed
among the centers (Breslow-Day statistic = 5.557, p = 0.592). The overall common odds ratio was
1.94 (95% CI 1.20−3.12; p = 0.007), indicating that immunotherapy significantly improved the live birth
rate compared to no treatment.
For the individual data meta-analysis, 285 subjects met the inclusion criteria and formed the basis for
the analysis. Immunotherapy was administered to 150 subjects resulting in live birth in 91 (60.7%). Out of
135 subjects in the control group, 60 achieved live birth (44.4%). The absolute treatment effect was 16.3%
(95% CI 4.8−27.8) and the number need to treat was calculated to be 6 (95% CI 4−21). Immunotherapy
significantly improved the probability of live birth (RR 1.46%; 95% CI 1.19−1.69). A significant negative
correlation was observed between the number of previous miscarriages and live birth rate (RR 0.77%;
95% CI 0.66–0.88). Thus, for each additional pregnancy loss beyond three, the likelihood of live birth
was reduced by 23%. Using logistic regression, the analysis was repeated to determine if there was an
interaction between immunotherapy and number of previous miscarriages. The interaction term was
statistically significant, indicating that as the number of previous miscarriages increased, the treatment
was observed to be more effective in improving the live birth rate. Immunotherapy and the number of
previous miscarriages were the only two variables to enter the final model that predicts the probability
of a successful outcome. Using this model, which included the interaction term, the treatment effect
can be seen to be greater with a higher number of previous miscarriages than with a lower number of
miscarriages (Figure 29.3). The absolute difference in live birth rates between treatment and control
groups was 16.3% [16], a figure which is much higher than the absolute treatment of effect of 8%–10%
observed in the original collaborative meta-analysis [13]. Thus, the outcome is improved by as much as
50% if patients selected for immunotherapy have unexplained primary recurrent miscarriage. Although
immunotherapy was shown to be effective in the original study, it appears that treatment efficacy is lower
in secondary aborters. Similarly, the presence of pretreatment antipaternal antibody appears to reduce
the effect of treatment.
The presence of autoimmunity appears to be a negative prognostic factor for immunotherapy. In
the original study, women with autoimmune abnormalities had a likelihood of live birth that was 62%
lower with immunotherapy compared to controls (RR 0.38%; 95% CI 0.16–0.77; p = 0.003) [13]. By
0.8
0.7
0.6
Probability of live birth

0.5
0.4
0.3
0.2
0.1
0
3 4 5 6 7 8 9
Previous abortions with present partner
FIGURE 29.3 Probability of live birth with and without immunotherapy in unexplained primary recurrent miscarriage.
The probabilities of live birth and their standard errors are shown using the final model that predicts a successful outcome
with immunotherapy (solid circles) and controls (open circles). (From Daya and Gunby [16] with permission.)
excluding these patients from the subgroup analysis, the treatment effect is enhanced. Collectively, these
observations indicate that the patient profile that has a high chance of success with immunotherapy is
one with unexplained primary recurrent miscarriages, no evidence of pretreatment antipaternal antibody,
and no autoimmune abnormalities. In such patients, the number needed to treat is six, indicating that for
every six patients treated compared to placebo, one additional live birth is obtained [16]. This magnitude
of treatment effect is impressive, and is persuasive that immunotherapy is very effective for this disorder.
Systematic Reviews of Immunotherapy

A Cochrane systematic review of published and unpublished trials was first performed in 2003, updated
in 2006, and updated yet again in 2014 [17]. The study subjects included women with three or more
miscarriages and no more than one prior live birth. In addition, their pretreatment evaluation required
normal karyotype in the couple, normal uterine anatomy, no evidence of antiphospholipid antibodies,
and no luteal phase deficiency or co-intervention. Twelve randomized trials (from 1985 to 2004) met
the inclusion criteria for this review. No new trials were identified for the most recent update. In 10
trials, immunotherapy was administered prior to pregnancy, in one trial treatment was administered
prior to pregnancy and again after conception, and in one trial treatment was administered only after
pregnancy was established. In total, the trials included 641 subjects: 316 immunized patients and 325
in the control group. The live birth rate in the experimental group was 64.9% (205 out of 316). The live
birth rate in the control group was 60% (195 out of 325). The results of the individual trials are shown in
the odds ratio tree (Figure 29.4). The overall common odds ratio estimate was 1.23 (95% CI 0.89−1.70;
p = 0.21), an effect that was not statistically significant. The display of the point estimates for each of
the trials demonstrated statistically significant heterogeneity in the effect of treatment across these trials
(heterogeneity Chi2 = 24.58; p = 0.01). Although the authors concluded that immunotherapy should
no longer be offered as treatment for unexplained recurrent miscarriage, the presence of heterogeneity
requires further discussion. A major source of heterogeneity was introduced by one trial in which women
who received immunotherapy had a higher rate of miscarriage than those in the control group [18]. The
observation from this trial led the Director of the Office of Therapeutics Research and Review, United
States Food and Drug Administration (FDA), to send a letter on January 30, 2002 to physicians believed
to be using leucocyte immunotherapy to prevent miscarriages, informing them that these products do not
have the required FDA approval and are considered investigational drugs that pose several safety concerns
Placebo Immunisation
Cauchi 1991
Christiansen 1994
Gatenby 1993
Ho 1991
Illeni 1994
Kilpatrick 1994
Mowbray 1985
Ober 1999
Panday 2004
Reznikoff 1994
Scott 1994
Stray-Pedersen 1994
Common OR
0.01 0.1 1 10 100
Odds ratio for live birth
FIGURE 29.4 Odds ratios for live birth in randomized trials comparing leucocyte immunization versus placebo in women
with recurrent miscarriage. (Data from Cochrane systematic review [17].)
[17]. Furthermore, administration of such cells or cellular products in humans was only to be performed
in the United States as part of clinical investigations, and then only if there is an Investigational New
Drug (IND) application in effect. All institutions, reproductive centers, and physicians were reminded
that they should not administer allogeneic cells or cellular products to miscarriage patients until an IND
has been submitted and reviewed by the Center for Biologics Evaluation and Research at the FDA. The
effect the conclusion of this trial had to clinical practice is significant and deserves further exploration.
The Recurrent Miscarriage Study

The Recurrent Miscarriage Study (REMIS) was a multicenter, randomized, double-blind trial to evaluate
the efficacy of paternal leucocyte immunization compared to saline as control intervention in women
with unexplained recurrent miscarriage [18]. The end point was a pregnancy that continued to at least 28
weeks of gestation. All results were reviewed by an independent data and safety monitoring committee,
with interim analyses planned after every 50 outcomes. Participants aged <41 years were included in one
of six centers if they had had three or more miscarriages, no more than one live birth with the current
partner, had no anti-HLA antibodies and no identifiable cause for their previous miscarriages. They were
randomly assigned at each center to either experimental or control groups. The randomization process
was stratified by center, with permuted blocks of size eight and ten. Subjects in the experimental group
received paternal leucocytes that had been separated by Ficoll gradient from one unit of blood the day
before immunization and had been stored overnight at 1°–6°C. To ensure participant blinding, the male
partners of subjects in the control group also provided a unit of blood which was discarded without
separation of the cells. Syringes and tubing used for immunization were prepared by blood bank personnel
and covered with opaque tape so that cloudy cell solutions could not be distinguished from clear saline
[18] in order to ensure double-blinding.
The day after the cell preparation, approximately 200 million leucocytes were suspended in 5 mL
normal saline and administered intravenously (3 mL), and 0.5 mL respectively in two subcutaneous and
two intradermal sites on the forearm. Subjects in the control group received identical volumes of saline
administered in the same fashion as in the experimental group. All subjects received their intervention
during the first 2 weeks of their menstrual cycle after a negative pregnancy test. After 6 months of
follow-up, subjects who were not pregnant received their respective interventions again.
The trial was terminated after the third interim analysis, well before the planned sample size had been
enrolled because the miscarriage rate in the treatment group was higher than that in the control group. At
the premature termination of the trial, in total 183 women had been randomly assigned to receive either
experimental (86 subjects) or control intervention (85 subjects). The groups were similar except that a
higher proportion of women in the experimental group had had a previous live birth (p = 0·054). In the
intention-to-treat analysis, the success rate was 36% (31 out of 86 treated with paternal leucocytes) in
the experimental group and 48% (41 out of 85 receiving saline) in the control group (OR 0.60; 95% CI
0.33−1.12; p = 0.108). When the analysis was adjusted for female age, number of previous miscarriages,
and previous live birth, the odds ratio was similar (OR 0.54; 95% CI 0.28−1.02; p = 0·056). Kaplan-
Meier-estimated pregnancy rates did not differ significantly between the groups: 78% in the treatment
group and 72% in the control group (log rank p = 0.232). When a subgroup analysis with adjustment for
the three important covariates was performed for subjects with primary recurrent miscarriage, the results
again were similar, with success rates in the experimental group of 18/59 (30%) compared to 32/70 (46%)
in the control group (OR 0.52; 95% CI 0.25−1.08; p = 0.082) [18].
Thus, in the REMIS trial, immunization with paternal mononuclear cells did not improve pregnancy
outcome in women with recurrent miscarriage. In contrast to most studies on this subject, the success
rate was higher in the control group than in subjects immunized with paternal leucocytes, even in
women with primary recurrent miscarriage (after excluding from the analysis those with a previous live
birth). The higher rate of pregnancy loss among subjects immunized with paternal cells suggested to
the investigators that immunotherapy may create more harm than benefit, leading them to recommend
against immunization for unexplained recurrent miscarriage [18]. An important point not appreciated
by the investigators in their attempt to reproduce the regimen of Mowbray et al. [5] was the manner in
which the prepared cells were handled. In the Mowbray trial, freshly separated leucocytes were used for
immunization, whereas in the REMIS trial, the leucocytes were stored overnight at 1°–6°C. The possible
adverse effect refrigeration may have on the immunogenicity of the cells is discussed in the next section.
Effect of Refrigeration on Immunogenic Potency of Leucocytes Used for Immunization

In the laboratory mouse model, mating of CBA/J female mice with allogeneic DBA/2 males generates
pregnancies susceptible to a high rate of spontaneous abortion (resorption) that is partner specific.
Changing either the female or male strain lowers the abortion rates to <10%. The high rate of
resorption in the CBA · DBA/2 model is correctable by immunization with cells bearing paternal major
histocompatibility (MHC) antigens [19]. The immunization strategy against abortion in the CBA · DBA/2
model has employed freshly isolated BALB/c cells (which have the same MHC as DBA/2). However,
when the allogeneic cells were stored overnight in tissue culture medium at 4°C, the beneficial effect was
abrogated, resulting in a resorption rate that was identical to that without immunization [20] (Figure 29.5).
The basis for this alteration was unclear. It was hypothesized that BALB/c lymphoid cells had to express
certain paternal alloantigens and a tolerance co-signaling molecule, CD200 (OX-2) to induce protection
[21]. Storage of BALB/c splenocytes causes loss of surface CD200 into the supernatant. Similarly, CD200
is lost if purified human blood mononuclear cells are stored. Thus, an intact cell membrane−bound
50
Implants/mouse
40 Abortion rate (%)
30
20
10 *
0
Control Fresh cells Stored cells
FIGURE 29.5 Effect of storing allogeneic leucocytes overnight on immunotherapy in the murine model. (Constructed
from data published in Clark et al. [20].)
TABLE 29.1
Comparison of Meta-Analyses with and without REMIS Trial Data
Cochrane Meta-Analysis Daya Updated Meta-Analysis
(Including REMIS [18]) (Excluding REMIS [18])
Number of subjects 641 510
Live birth rate
• Experimental group 64.9% 70.2%
• Control group 60% 58.8%
Absolute treatment effect 4.9% 11.4%
Odds ratio for live birth (95% confidence interval) 1.23 (0.89–1.70) 1.63 (1.13–2.36)
P value 0.21 0.009
Note: Cochrane meta-analysis was updated by the author of this chapter after excluding the data from the REMIS trial.
CD200 molecule is required for BALB/c splenocytes to immunize against abortions in the CBA · DBA/2
model. Fresh cells were required, and cells stored overnight, even at 4°C in serum-containing medium,
lost most of their activity.
The evidence presented from these murine studies provides a plausible explanation for the lack of
beneficial effect of paternal leucocyte immunization in the REMIS trial. A poll of investigators in
the worldwide collaborative study [13] indicated that only freshly isolated leucocytes were used for
immunization [20]. Thus, the method of preparing and storing the cell used in the REMIS trial nullified
the immunogenic activity of immunization. Hence, the trial could not adequately test the efficacy of
immunotherapy because the experimental intervention was akin to using a placebo. Consequently, the
inclusion of the REMIS trial in the Cochrane systematic review and meta-analysis [17] is invalid and
should be removed.
In the published Cochrane review, 12 trials were included comprising 641 subjects (316 in the
experimental group and 325 in the control group). The summary odds ratio of the effect of treatment was
1.23 (95% CI 0.89−1.70; p = 0.21). There was significant heterogeneity of the effect of treatment across
all trials. An updated (unpublished) meta-analysis was performed (by the author of this chapter) after
removing the REMIS trial, resulting in 510 subjects (248 in the experimental group and 262 in the control
group). The recalculated summary odds ratio is now 1.63 (95% CI 1.13−2.36; p = 0.009), indicating a
statistically significant effect in favor of immunotherapy. The test for heterogeneity of treatment effect
across all 11 trials is no longer statistically significant. Removing the REMIS trial from the meta-analysis
led to significant homogeneity of trials. The absolute treatment effect is 11.4% (experimental group
success rate = 70.2%; control success rate = 58.5%). The number needed to treat is nine. This estimate
is consistent with the two estimates in the worldwide collaborative study [13]. These comparisons in
outcomes between the published Cochrane meta-analysis and the current revised meta-analysis are
shown in Table 29.1. Clearly, the REMIS trial has negatively influenced the conclusions and opinions
regarding the value of leucocyte immunotherapy for recurrent miscarriage. It is now time to correct the
misperception that has prevailed for so many years so that women with unexplained recurrent miscarriage
can be offered immunotherapy that is efficacious.
Newer Systematic Reviews

More recently, two systematic reviews of immunotherapy for recurrent miscarriage have been published
[22,23]. The first report was limited to trials reporting random allocation to either leucocyte immunotherapy
or placebo in Chinese women with two or more miscarriages [22]. Trials in which patients receiving
lymphocyte immunotherapy before or after conception were included. After searching the literature, 14
published trials from 2006 to 2015 met the inclusion criteria and were selected. Methodological validity
was evaluated with the Jadad scale [24]. High-quality trials require a score from 4 to 7. Among the
trials selected, 13 out of the 14 achieved a Jadad score of 4, demonstrating high methodological quality.
No significant statistical heterogeneity was present demonstrating a consistency of treatment effect
across these trials. In total, there were 1271 subjects enrolled; 647 were randomized to receive leucocyte
immunization, and 624 were randomized to the control group. Successful pregnancies were observed
in 531 in experimental group (82.1%), and in 280 in the control group (44.9%) resulting in an absolute
treatment effect of 37.2%, which translates into a number needed to treat of approximately three. Thus,
in this systematic review, for every three women with recurrent miscarriage treated with immunization
with leucocytes, one additional successful pregnancy was achieved compared to placebo. The overall
odds ratio in favor of immunotherapy was 5.72 (95% CI 4.42−7.40; p < 0.00001). The magnitude of this
treatment effect is much larger than that seen in the previous systematic reviews and in the worldwide
collaborative study. It is not clear why such a difference in magnitude of treatment effect was observed,
and it may have to do with the fact that all trials were conducted on Chinese women, suggesting an
improved prognosis in this racial group.
A second systematic review was undertaken to assess whether the efficacy of leucocyte immunization
is influenced by whether it is performed before or after pregnancy has been established [23]. This study
is an update of a previous Cochrane review and used the same criteria to select trials for inclusion in the
analysis. The study included 13 trials from the English literature and 5 trials from the Chinese literature.
There were 1738 subjects enrolled; 739 were randomized to receive leucocyte immunization, and 999
were randomized to the control group. Successful pregnancies were observed in 575 in the experimental
group (77.8%), and in 461 in the control group (46.1%) resulting in an absolute treatment effect of 31.7%,
number needed to treat, approximately three. Thus, in this systematic review, for every three women with
recurrent miscarriage treated with immunization with leucocytes, one additional successful pregnancy
was achieved compared to placebo. The overall odds ratio in favor of immunotherapy was 3.74 (95%
CI 3.07−4.57; p < 0.00001). However, there was statistically significant heterogeneity in the effect of
treatment across the included trials. In addition, clinical variations in trial design included the dose
(number of leucocytes) used, route of immunization, and timing of immunization. Furthermore, there
was variability in the geographical location of the subjects included in the trials. Given such variation,
a further analysis was performed to examine the effect of subdividing trials into different groups based
on these variables. The results of the subgroup analyses are shown in Table 29.2. To study the effect
of timing of treatment on efficacy, the trials were divided into two groups: immunotherapy performed
before and during pregnancy, and immunotherapy performed only before pregnancy. The efficacy of
immunotherapy was significant in both subgroups, but the magnitude of the effect of treatment was larger
when immunotherapy was performed before and during pregnancy compared to only before pregnancy.
To evaluate the dose of immunotherapy, the trials were divided into two groups: low-dose (less than 100
million leucocytes or 100 mL of peripheral blood used to extract the leucocytes) and high-dose (more than
100 million leucocytes or more than 100 mL of peripheral blood used to extract the leucocytes). Efficacy
TABLE 29.2
Overall and Subgroup Analyses of Immunotherapy versus Control
Success Success
Rate in Rate in Absolute
Number of Number of OR for Live Treatment Control Treatment
Trials Included Subjects Birth (95% CI) Group (%) Group (%) Effect (%) P Value
Overall 18 1738 3.74 (3.07–4.57) 77.8 46.1 31.7 <0.0001
Treatment before 12 519 2.00 (1.39–2.88) 70.2 53.0 17.2 0.0002
pregnancy
Rx before and 8 747 4.67 (3.70–5.90) 81.8 44.5 37.3 <0.0001
during pregnancy
High dose 10 459 1.52 (1.04–2.22) 67.6 58.7 8.9 0.03
Low dose 8 1279 5.25 (4.16–6.64) 82.7 42.6 40.1 <0.0001
European and 10 410 1.45 (0.97–2.17) 64.3 53.5 10.8 0.07
American subjects
Asian subjects 8 1328 5.09 (4.05–6.40) 83.2 44.3 38.9 <0.0001
Note: The overall analysis and all subgroup analyses were performed for immunotherapy versus control intervention.
of immunotherapy was significant in both subgroups, but the magnitude of the effect of treatment was
larger when low-dose immunotherapy was used compared to high-dose immunotherapy. A third subgroup
analysis was performed to evaluate the effect of geographical location of the subjects included in the
trials. Although efficacy of immunotherapy was higher in both subgroups, the magnitude of the effect of
treatment was not statistically significant when trials included only European and American subjects. In
contrast, in trials that included Asian subjects, the magnitude of the effect of treatment was much larger
and was statistically significant.
Although performing such subgroup analyses provides an interesting hypothesis for further evaluation
of the covariates that may affect efficacy, they do not permit testing of treatment efficacy which can only
be performed within trials stratified for these subgroups rather than comparing their effect across trials.
Nevertheless, useful information has been provided to guide the design of future trials on optimizing the
efficacy of leucocyte immunotherapy in women with recurrent miscarriage.
Possible Adverse Effects from Immunotherapy

In the worldwide collaborative study, adverse effects observed or reported were collated, and represented
the first published data regarding the safety of immunotherapy with leucocytes [13]. In women who
received immunotherapy, maternal complications were observed in 2.1%, compared with 0.5% in the
control women. The maternal reports included viral infections (hepatitis and cytomegalovirus), flu-like
symptoms, and fever resulting from transfusion reaction. In a small number of subjects, antipaternal
erythrocyte antibodies or antipaternal platelet antibodies were observed. Adverse pregnancy outcomes
included preterm birth, intrauterine growth restriction, and fetal death. Adverse neonatal outcomes
included thrombocytopenia and congenital malformations. In total, the prevalence of adverse pregnancy
and neonatal outcomes were similar in the two groups (3% in the experimental group and 4% in the
control group) [13].
Although transfusions containing white blood cells carry a risk of graft-versus-host disease, there has
only been one case report of a cutaneous graft-versus-host–like reaction following immunization with
paternal leucocytes for recurrent miscarriage [25]. The patient developed two bullae at the injection site
that were successfully treated with steroids and analgesics.
In a comprehensive study [26] assessing the safety of intradermal immunotherapy with lymphocytes in
infertile women and those with recurrent miscarriage, local reactions were common. Almost all women
reported redness and itching at the injection site. Swelling was reported in 66% of the women and burning
sensation in 30% of the women. Blistering at the injection site was reported in 14% of the women, but it
was not clear whether these women misinterpreted swelling as blistering. Axillary lymphadenopathy was
observed in 8% of the women. Systemic complaints were less frequent and were reported in 8% of the
women. In most cases these complaints were nonspecific, such as fatigue, headache, dizziness, nausea,
vomiting, and diarrhea. Thus, adverse effects with immunotherapy occur with a low prevalence and with
limited severity and duration.
Conclusions
Although there are many proposed causes of recurrent miscarriage that can be treated, the management
of unexplained recurrent miscarriage has been challenging. The concept of allogeneic recognition failure
is well established and the approach of immunotherapy with paternal leucocytes was first introduced over
30 years ago. Unfortunately, this treatment strategy has witnessed controversies over the years stemming
from the manner in which treatment efficacy has been evaluated. To date, there is no randomized clinical
trial with sufficient power and stratification for important covariates, including numbers of previous
miscarriage, female age, and primary versus secondary recurrent miscarriage, to adequately determine
benefit. Also, the optimal number of leucocytes to be used, administration before or after pregnancy is
established, and the route of administration still require further research. Nevertheless, a critique of the
available information from randomized trials has demonstrated the treatment to be efficacious. This
conclusion was drawn in the 1990s after the worldwide collaborative study was performed. Unfortunately,
efficacy was questioned by the results of the REMIS trial, leading to the call for stopping immunotherapy
in the absence of methodologically valid trials. However, the REMIS trial, in using a strategy to store
overnight at low temperatures the prepared leucocytes, nullified the immunogenic activity of these cells.
The exclusion of the REMIS trial from all subsequent meta-analyses has consistently demonstrated
immunotherapy to be efficacious. More recent studies have shown the magnitude of the treatment effect to
be even higher and with more precision than that delivered by the original worldwide collaborative study.
Thus, even though paternal leucocyte immunization is a very effective method of managing unexplained
recurrent miscarriage, much remains to be done. Further research is required to determine the optimal
dose of treatment and the appropriate diagnostic tests that should be undertaken to identify those women
with recurrent miscarriage who will have the highest likelihood of success.
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30
IVIg Treatment for Recurrent Pregnancy Loss
Carolyn B. Coulam
Introduction
Does immunotherapy for treatment of reproductive failure enhance live births? The answer to this
question has been controversial. The reason for the controversy lies in the problem of patient selection
for a particular treatment. A treatment is more likely to work if it is given to those with a physiological
abnormality that the treatment can correct, and, if the treatment in fact corrects it [1]. Not all pregnancies
fail for the same reason. Causes for recurrent pregnancy loss have included chromosomal, anatomic,
hormonal, immunologic, and thrombophilic abnormalities [2]. Thus, one cannot use obstetrical history
alone to determine whether immunotherapy will be useful. Only patients experiencing reproductive
failure with an immunologic cause would be expected to respond to immunotherapy. The following
paragraphs will discuss how to identify those individuals most likely to respond to treatment with
intravenous immunoglobulin (IVIg), describe published success rates of IVIg therapy, and present
alternative treatments to IVIg.
How to Identify Those Individuals Most Likely to Respond to Treatment with IVIg
Of all of the causes of recurrent pregnancy loss, the ones that would be expected to respond to IVIg
treatment would be the etiologies that involve a mechanism that can be modulated by IVIg. The mechanisms
by which IVIg are believed to enhance live birth rates include [3]: decreased killing activity of NK cells,
decreased expression of proinflammatory T cell cytokines (Th-1), increased activity of regulatory T cells,
suppression of B cell production of autoantibody, IVIg contains antibodies to antibodies or anti-idiotypic
antibodies and IVIg acts on Fc receptors including binding of complement by the Fc component of IgG.
Based upon these mechanisms, IVIg would be expected to enhance live birth rates in individuals
who had elevated natural killer (NK) cells, activated T cell activity, excess of proinflammatory Th1-
type cytokines, diminished regulatory T cells, and elevated production of autoantibodies that can cause
endothelial damage and clotting and increase activation of complement. All of these findings have been
reported among women experiencing recurrent pregnancy loss [4–14]. Proinflammatory cytokines at the
maternal-fetal surface can cause clotting of the placental vessels and subsequent pregnancy loss. One
source of these cytokines is the NK cell. Biopsies of the lining of the uterus from women experiencing
recurrent pregnancy loss reveal an increase in NK cells [15]. Peripheral blood NK cells are also elevated
in women with recurrent pregnancy loss compared with women without a history of pregnancy loss in
some [7] but not all studies [17–19]. This discrepancy has three explanations: (i) Measurement of NK cells
in peripheral blood of women with a history of recurrent pregnancy loss has shown a significant elevation
associated with loss of a normal karyotypic pregnancy and a normal level associated with loss of embryos
that are karyotypically abnormal [20,21]. (ii) Focus on findings of peripheral NK cells versus uterine NK
cells predicting pregnancy outcome. Successful pregnancy begins at the uterine level. Peripheral NK cells
and uterine NK cells have completely different phenotypes and functions [22,23]. No correlation between
circulating NK cells and uterine NK cells has been found (Figure 30.1). (iii) Numbers of uUK cells versus
function of uNK cells. Unlike peripheral NK cells, uterine NK cells have little cytotoxic activity but are
a rich source of cytokines, particularly angiogenic ones, with possible roles in regulation of trophoblast
invasion and angiogenesis [24,25]. Uterine NK cell density has been reported to be both associated [26]
268
IVIg Treatment for Recurrent Pregnancy Loss 269
30
n = 100
r = 0.03; P = 0.752
25
Periph. blood: %CD56+ cells

20
15
10
0
0.1 1 10 100
Endometrium: CD56 mRNA relative expression
FIGURE 30.1 Correlation between peripheral blood NK (CD56+) cells and uterine NK (CD56+) cells.
and not associated [27] with infertility, suggesting that uterine NK function rather than number predicts
subsequent miscarriage in women with a history of recurrent miscarriage. Killer immunoglobulin-like
receptors (KIRs) determine NK function in the context of other receptor-ligand interactions [23,28].
Uterine NK cells express members of the killer immunoglobulin-like receptor (KIR) family that bind to
parental HLA-C molecules on the invading placental trophoblast cells. The maternal KIR genes and their
fetal ligands are highly variable, so different KIR/HLA-C genetic combinations occur in each pregnancy.
Some women only possess inhibitory KIR genes, whereas other women also express activating KIR genes.
The overall signal that NK cells receive from paternal HLA-C on trophoblast depends on the ratio of
activating and inhibitory KIR genes expressed by them. Therefore, NK cells provide a balance during
placentation to ensure maternal survival and an adequately nourished fetus.
Th1 cytokine expression has been shown to be increased in circulating T lymphocytes of women
experiencing recurrent pregnancy loss [7]. Regulatory T cells (Tregs) suppress immune responses of other
cells including T effector cells, thus helping to avoid unrestricted expansion of a T cell proinflammatory
response. IVIg has been shown to decrease Th1/Th2 cytokine ratios [8] and enhance T reg cells [29] as well
as to decrease NK cell killing activity [9–11]. All of these events are necessary for pregnancy to be successful.
IVIg would not be expected to be effective in enhancing live birth rates in women who had chromosomally
abnormal pregnancies or anatomic, hormonal, or thrombotic risk factors contributing to their losses.
Therefore, selection of the person most likely to respond to IVIg treatment would require documentation
of an immunologic risk factor and the absence of non-immunologic risk factors. Laboratory evaluations
to determine the presence of an immunologic risk factor could include the following:
• Blood drawn for antiphospholipid antibodies, antinuclear antibodies, antithyroid antibodies,

lupus-like anticoagulant, reproductive immunophenotype, NK activation assay, TH1/Th2 ratios
in peripheral lymphocytes, and Treg cells
• Endometrial biopsy for Endometrial Immune Profile, which is interpreted as hyperactive,
hypoactive, or normal [30]:
• Hyperactive
– Elevated IL-18/TWEAK ratios
– Elevated IL-15/Fn14 ratios
– Elevated CD56+
• Hypoactive
– Low IL-15/Fn14 ratio
– Low CD 56+
• Normal
– Normal IL-18/TWEAK ratios
– Normal IL-15/Fn14 ratios
– Normal CD56+
Examples of testing for risk factors not responsive to treatment with IVIg include:
• Chromosome analysis of previous pregnancy losses or possibly both partners

• Hysterosonogram, hysterosalpingogram, or hysteroscopy
• Thrombophilia panel
• Endometrial biopsy for herpes virus 6 (HHV-6) [31]
Success Rates of IVIg Therapy

Originally, IVIg therapy was used to treat women with post-implantation pregnancy losses who had not
been successful in pregnancies previously treated with aspirin and prednisone or heparin [32–36]. The
rationale for the use of IVIg in the original studies was the suppression of the lupus anticoagulant in a
woman being treated for severe thrombocytopenia. IVIg was often given with prednisone or heparin
plus aspirin. The estimated success rate of 71% for women at very high risk for failure with a history of
previous treatment failures suggested IVIg treatment was effective [32–36]. More recently, IVIg therapy
alone has been used to successfully treat women with antiphospholipid antibodies as well as women who
become refractory to conventional autoimmune treatment with heparin or prednisone and aspirin [37].
IVIg has been reported to successfully treat women with elevated circulating levels of NK cells, NK cell
killing activity with live birth rates between 70% and 80% [38].
IVIg has also been used to treat women with unexplained recurrent pregnancy loss. Ten controlled
trials of IVIg for treatment of recurrent pregnancy loss have been published [39–48]. Four of these report
significant enhancement in the live birth rate with IVIg treatment, and six were unable to show benefit
of treatment. The number of patients participating in each trial, the time of first IVIg administration
(pre- or post-conception), whether the patients were selected for treatment with IVIg based on obstetrical
history alone or obstetrical history and immunologic test results, and whether the trial showed benefit or
no benefit from treatment are summarized in Table 30.1 [39–48]. Five trials gave IVIg before conception
and four of the five showed significant benefit in enhancing live birth rates, whereas five trials delayed
treatment until pregnancy was established, and of these none demonstrated benefit of treatment (p = 0.04,
Fisher’s exact test). Among the trials showing benefit of treatment with IVIg, three out of four used
immune test results to select patients for IVIg treatment, and among trials showing no benefit from
TABLE 30.1
Classification of Outcome of Controlled Trials of IVIg in Recurrent Pregnancy Loss
Outcome Benefit
Trial N IVIg Started Selection (P < 0.05)
Moraru [38] 157 Pre-conception Immune testing Yes
Coulam [41] 95 Pre-conception Ob history Yes
Kiprov [43] 35 Pre-conception Immune testing Yes
Stricker [44] 47 Pre-conception Immune testing Yes
Stephenson [42] 39 Pre-conception Ob history No
Mueller-Eckhart [39] 64 Post-conception Ob history No
Christiansen [42] 34 Post-conception Ob history No
Christiansen [46] 58 Post-conception Ob history No
Perino [46] 46 Post-conception Ob history No
Jablonowska [47] 41 Post-conception Ob history No
TABLE 30.2
Summary of Published Meta-Analyses of Efficacy of IVIg for Treatment of Unexplained Recurrent
Reproductive Failure
OR (95% CI) OR (95% CI) OR (95% CI)
Study No. Trials No. Patients Overall Primary Ab Secondary Ab
Hutton 2007 [50] 8 442 1.28 (0.78–2.10) 0.66 (0.35–1.20) 2.71 (1.09–6.77)*
Daya 1999 [52] 6 240 1.08 (0.63–1.86) 1.04 (0.54–2.01) 1.18 (0.43–3.21)
Ata 2011 [52] 6 272 0.92 (0.55–1.54) 0.67 (0.32–1.39) 1.15 (0.47–2.84)
Clark 2011 [53] 5 210 2.10 (1.06–4.49)*
Li 2013 [54] 10 8207 1.62 (1.24–2.1)*
P < 0.05.
*
treatment, 0/6 selected patients for treatment using immune testing (p = 0.03). By waiting until 5–8 weeks
of pregnancy to begin treatment, women with pathology occurring earlier would have been excluded and
those pregnancies destined to succeed would be included, leading to selection bias. A negative correlation
with delay in treatment is significant. Only one study took into account the pregnancies lost as a result of
chromosomal abnormalities [49]. Approximately 70% of the pregnancies lost in the clinical trial would
be expected to have chromosomal abnormalities that would not be corrected by IVIg. It has also been
recently shown that some brands of IVIg can be as much as eight times for potent in suppressing NK cells
that were used in “negative” trials [50].
The aforementioned clinical trials have been included in four published meta-analyses summarized
in Table 30.2 [51–55]. None of the meta-analyses showed benefit of treatment with IVIg for primary
aborters. Two of the analyses demonstrated significant benefit only for secondary aborters (Table 30.2)
[51,54]. None of the studies included in the meta-analysis selected patients for inclusion based on
immunologic testing. All were included based on reproductive history alone. How can the effect of an
immunomodulatory treatment be evaluated if the subjects receiving the treatment were not determined
to have any detectable immune abnormalities that would merit their inclusion into the study? The sample
size required to show an effect would depend on the prevalence of immunologic problems among the
unselected patients. Indeed, IVIg was shown to increase the success rate in patients undergoing IVF
for treatment of unexplained infertility based on meta-analysis with a sample size of over 8000 patients
[54]. A number of clinical trials have demonstrated increased live birth rates after treatment with IVIg
when patients are selected based on immunologic testing provided treatment is given prior to conception
[3,11,38,48,49].
Alternative Treatment to IVIg in Patients with Elevated NK Cells

Since IVIg has been associated with significant cost and potential side effects, an alternative treatment
has been sought. Evidence from both animal [55] and human [48,56,57] studies suggest that intralipid
administered intravenously may enhance implantation and maintenance of pregnancy. Intralipid is a 20%
intravenous fat emulsion used routinely as a source of fat and calories for patients requiring parenteral
nutrition. It is composed of 10% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerine, and water.
Intralipid has been shown to decrease NK cytotoxicity both in vitro [56] and in vivo [57]. While the
mechanism by which intralipids suppress NK function is not known, effects of fatty acids have been
demonstrated to be mediated through receptors such as peroxisome proliferator-activated receptors
(PPARs) [58], G-protein-coupled receptors [59], and CD1 receptors [60]. Furthermore, intralipids have been
shown to stimulate the reticuloendothelial system and remove “danger signals” that can lead to pregnancy
loss [61]. Sedman et al. [62] have found a significant fall of NK activity and lymphokine-activated killer
activity after total parenteral nutrition regimens with long-chain triglycerides. Parenteral fat emulsions are
known to accumulate in macrophages and to impair various functions of macrophages and those of the
reticuloendothelial system. It was shown that the administration of fat emulsion, intralipid 20%, to recipient
mice can suppress NK cell activity probably through the impairment of macrophage function [63].
When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cells
treated with intralipid were compared with age- and indication-matched women treated with IVIg, no
significant differences were seen [48]. The overall live birth/ongoing pregnancy rate per cycle of treatment
was 61% for women treated with intralipid and 56% with IVIg [48]. Others have also reported intralipid
treatment increased live birth rate among women with recurrent reproductive failure and increased
CD56(+) cells [16]. The appeal of intralipid lies in the fact that it is relatively inexpensive and is not a
blood product.
Conclusions
Only patients experiencing reproductive failure with an immunologic cause would be expected to respond
to immunotherapy. Thus, IVIg is expected to enhance live births only in individuals who display elevated
uterine NK cells, activated uterine T cell activity, excess of uterine proinflammatory Th1-type cytokines,
diminished uterine regulatory T cells, and elevated antiphospholipid antibodies. Since intralipid is not
a blood product, has no reported side effects, and is inexpensive, it has been accepted as an alternative
treatment to IVIg among individuals exhibiting elevated decidual NK cells.
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31
The Role of Filgrastim
Fabio Scarpellini and Marco Sbracia
Introduction
Filgrastim, a recombinant human granulocyte colony-stimulating factor (G-CSF), has identical biological
activity with endogenous human G-CSF, but differs in containing an N-terminal methionine residue and is
not glycosylated [1]. G-CSF stimulates activation, proliferation, and differentiation of neutrophil progenitor
cells and it has been used in the treatment of patients with various neutropenic conditions [2–4]. G-CSF
mobilizes hematopoietic stem cells (HSCs) from bone marrow into the peripheral circulation [5] and
hence is used to increase the number of hematopoietic stem cells in the blood before collection for HSCs
transplantation [6]. G-CSF also exhibits significant neuroprotective effects in cerebral damage models [7].
G-CSF facilitates functional recovery in rats after stroke [8,9] and has an anti-apoptotic effect by activating
a variety of intracellular signaling pathways, including Janus protein tyrosine kinase/signal transducer
and activator of transcription (JAK/STAT) [8,10], extracellular-regulated kinase (ERK) [11,12], and
phosphatidylinositol 3-kinase/Akt (PI3 K/Akt) [13,14]. Available data indicate that filgrastim is generally
well tolerated; the side effects include fever, cough, chest pain, joint pain, vomiting, and hair loss. Rarer
and severe side effects are splenic rupture and allergic reactions [1,4]. The most frequent adverse reaction
is mild to moderate medullary bone pain, reported by approximately 20% of patients, although this can
generally be controlled using analgesics without the need to discontinue treatment [1,4].
G-CSF belongs to the group of colony stimulating factors (CSFs), macrophage colony-stimulating factor
(M-CSF or CSF1), granulocyte-macrophage stimulating factor (GM-CSF or CSF2), and granulocyte colony-
stimulating factor (G-CSF or CSF3). The CFSs are a group of glycoproteins that bind to specific receptors
on HSCs, promoting cell proliferation and differentiation into macrophages and granulocytes. They show
different structures, gene location, and different receptors. All CSFs are involved in the reproductive process
from ovulation to implantation and pregnancy [2]. G-CSF is a glycoprotein of 174–180 amino acids long
and with a molecular weight of 19,600 Dalton: its gene is located on the long arm of chromosome 17, in
region 17q11.2-q12.8 [15]. It binds to a specific receptor, the G-CSF R or CD114, encoded by a gene on the
short arm of chromosome 1 in the region 1p35–34.3. G-CSF is a protein 836 amino acids long and of 92,156
Daltons molecular weight [16]. The GCSF-R is associated with signal transduction through the JAK-STAT3
pathways. G-CSF and its receptor have been found on trophoblasts and in the decidua of several mammals,
including human placenta [17,18]. An anti-abortive role has been demonstrated for G-CSF in animal models,
and its depletion is indirectly involved in miscarriages [19,20]. It has also been shown that G-CSF has a
positive effect on trophoblast metabolism [21]. Furthermore, G-CSF is secreted in follicular fluid and its
levels correlated with oocyte competence and the implantation potential of corresponding embryos [22].
G-CSF and Recurrent Pregnancy Loss

More than 40% of recurrent pregnancy loss (RPL) cases remain unexplained [23]. Several causes have
been proposed to explain some of the cases of unexplained RPL, including an allo-immune response,
where RPL could be due to an imbalance in the Th1/Th2 cytokines, with a preponderance of Th1 cytokine
production instead of Th2 cytokine production (with an immunosuppressive role) [24]. Several treatment
modalities have been proposed to treat unexplained RPL, but the results are controversial [25].
275
Our team started using filgrastim in RPL in 1997, successfully treating a woman after five consecutive
miscarriages. We subsequently used G-CSF in several other women with encouraging results. The results
of a pilot study were first presented in 1998 at the annual American Society of Reproductive Medicine
(ASRM) meeting. A randomized controlled study was then performed, the results of which were published
in 2009 [26]. The inclusion criteria were: age <39 years, more than four previous miscarriages, failure of
previous therapy RPL, and negative results for all known causes of RPL, including normal karyotyping of
embryonic tissues in the previous miscarriage. Sixty-eight patients were included in the study: 35 women
underwent daily administration of recombinant filgrastim 1 µg (100,000 IU)/kg/day from the sixth day
after ovulation until menstruation or until 9 weeks of gestation. The control group consisted of 33 subjects
who were treated with saline solution. The live births in women treated with filgrastim were 82.8%,
whereas in the controls they were 48.5% (p = 0.0061). The number of patients needed to treat for one
additional live birth was 2.9. No infant showed any major or minor abnormalities. This study showed that
filgrastim may be a promising tool for the treatment of selected patients with unexplained RPL.
Subsequently data reported the use of filgrastim in women with recurrent implant failure (RIF),
showing good results in an uncontrolled study [27]. G-CSF seems to increase the chance of pregnancy in
patients with RIF. Therefore, our team started a controlled trial on RIF patients that is due to terminate
in 2019. Inclusion criteria included pregestational testing for aneuploidy (PGT-A). The preliminary data
seem to be encouraging (presented at the ASRM annual meeting in 2018).
Recently several authors have published reports about treatment with filgrastim in patients with
unexplained RPL [28] and RIF [29–31] showing the usefulness of this treatment in improving the outcome
of these reproductive disorders. Furthermore, several reviews and meta-analysis have been published in
the past 2−3 years showing the beneficial effects of filgrastim treatment in unexplained RPL and RIF after
IVF [32–35]. However, some of these papers reported data from patients with RPL or RIF who were not
well selected and may suffer from bias. Since the use of array-comparative genomic hybridization (CGH)
for genetic assessment of the abortus, the results are more accurate, less likely to suffer from culture
failure, and cost less than previous karyotyping. Consequently, we consider a euploid result in the previous
pregnancy should be mandatory before using filgrastim in unexplained RPL. In our clinical practice, we
only use filgrastim in women who have negative results for known causes of RPL and whose embryos
are euploid in the previous miscarriage. These strict criteria probably explain our results on more than
500 women treated in 20 years. Also, in RIF, we generally only treat with filgrastim when transferring a
single euploid blastocyst after PGT-A.
To the best of our knowledge, there are several centers of reproductive medicine using filgrastim for
these reproductive disorders with a beneficial effect, and other investigators are assessing filgrastim in
RPL and RIF. However, a multicenter controlled trial is warranted in order to establish the therapeutic
potential of filgrastim, and in which patients it may be beneficial.
Possible Mechanism of Action for G-CSF Treatment

in Recurrent Pregnancy Loss
The mechanism of action of filgrastim in RPL treatment is unclear, as there is no direct evidence of
the effect of filgrastim in pregnancy. However, there is indirect evidence regarding the interaction of
filgrastim with the trophoblast and immune system. The effects of G-CSF on trophoblast growth and
invasiveness have been reported by several studies. G-CSF and its receptor are expressed in trophoblast
cells throughout the pregnancy [17–21]. The G-CSF/G-CSFR axis was described in the placenta and
decidua in 1989 by Uzumaki et al. [17]. Several other authors have also shown its pivotal role in the
regulation of trophoblast invasiveness and development [18–21]. Several authors have reported that
granulite colony-stimulating factor receptor (G-CSFR) expressed in trophoblast cell lines activate
different signal transduction pathways, such as JAK/STAT, PI3 K, and MAPKs, which in turn increase
matrix metalloproteinase-2 and vascular endothelial growth factor secretion [36]. Furthermore, G-CSF
upregulates β1 integrin and increases the migration of human trophoblast cell line Swan 71 [37]. G-CSF
also increases the expression of mRNAs for several genes involved in the implantation processes in an in
vitro model taken from endometrial biopsies [38].
The Role of Filgrastim 277
Filgrastim is often used clinically to increase the number of stem cells after organ transplant or to activate
the reconstruction of the vascular bed after heart ischemia, and in neurology to treat patients with severe
degenerative diseases [1–14]. In our study, a significant increase of β-hCG levels was observed in ongoing
pregnancies from the fifth through the ninth gestational week in filgrastim-treated pregnancies when
compared to control pregnancies [26]. These data showed a direct effect of filgrastim on the trophoblast,
with the mobilization and activation of placental stem cells. Another mechanism of action may be the
effect of G-CSF on lymphocytes; several studies have shown that G-CSF promotes the mobilization and
proliferation of several lymphocyte and dendritic cells, in particular Treg and DC2 cells [39,40]. Our
unpublished data show that women with RPL treated with filgrastim had a significant increase in the
number of peripheral blood Treg cells when compared to normal pregnancy. Furthermore, in women with
RPL treated with filgrastim who subsequently miscarried again due to embryonic aneuploidy, there was
still an increase of Treg cells in the decidua compared to the controls. These data suggest that G-CSF may
mobilize and differentiate stem cells and immune cells enhancing trophoblast function.
It is well documented that G-CSF mobilizes mesenchymal stem cells from bone marrow into the
circulation; hence G-CSF is used to increase the stem cell concentration in the blood of donors when
stem cell transplantation is performed. Both stem cell and Treg cell mobilization seems to be due to
the regulation of chemokine CXCL12 and its receptor CXCR4. Several authors have described that
the inhibition of the CXCL12/CXCR4 axis is the key in G-CSF-mediated bone marrow stem cell
mobilization [41,42]. The CXCL12/CXCR4 axis is also involved in Treg mobilization, since G-CSF
decreases the expression of CXCL12 in these cells as well as the expression of the putative receptor
expression, CXCR4 [43].
All the above data suggest that G-CSF may promote the activation of two different mechanisms.
One is immunological, with the mobilization and activation of Treg cells with immunosuppressive
functions associated with the immune acceptance of pregnancy. The other mechanism is metabolic, with
the activation of trophoblast tissue and placental stem cells enhancing the invasiveness and growth of
trophoblastic tissue.
Safety
The safety of drugs in pregnancy is always of major concern. In our experience on more than 500
patients treated with G-CSF in the implantation period and during early pregnancy we have not observed
any major adverse effect in the mothers, fetuses, or infants. In our hands, this treatment is safe. Only
minor side effects have been observed such as local skin rash, which cleared in a few days, in 3.6% of
patients treated, fever in 2.6% of cases, and leukocytosis (above 25,000/mL) in 4.2% of patients. The
leukocytosis was lowered by suspending treatment for 2−3 days. However, it is important to remember
there are few data on possible filgrastim toxicity in pregnancy. Experimental data on animal models has
shown placental embolism only in rabbits [44], with a dosage 1000 times higher than we use in humans.
In rats, mice, and monkeys, no adverse effects have been observed [19,45]. In an early review by Dale
et al. in 2003 [46], involving patients who were under long-term treatment with filgrastim for chronic
neutropenia, no adverse effects on pregnancy or the fetus were reported in a series of 125 women. A
2013 review of data of patients treated with filgrastim by Pessach et al. [47] in hematopoietic stem cell
donation from healthy women donors during pregnancy and lactation concluded that filgrastim was safe.
Pessach et al. [47] observed that G-CSF crosses the placenta and stimulates fetal granulopoiesis, improves
neonatal survival in very immature infants, promotes trophoblast growth and placental metabolism, and
has an anti-abortive role. The information available indicates that administration of filgrastim is safe in
pregnancy. A recent paper by Boxer et al. in 2015 [48] reported no differences in pregnancy and neonatal
complications in women treated with filgrastim for chronic neutropenia during pregnancy compared to
controls, even when used in the first trimester.
Most data on filgrastim and pregnancy outcomes were obtained from patients or healthy donors
receiving dosages of filgrastim at least five times greater than used in our patients. Consequently, if
filgrastim is safe in pregnant women treated for chronic neutropenia, it should also be safe in women with
RPL, receiving only one-fifth of the dose.
Conclusions
The recombinant G-CSF, filgrastim, should be considered a safe and effective treatment for unexplained
RPL, in which the patient loses euploid embryos. Since embryonic aneuploidy is a major cause of pregnancy
loss and its frequency increases with maternal age, the genetic analysis of pregnancy tissue (preferably
with array-CGH) may help determine whether further investigations or treatments are required. Embryonic
aneuploidy testing should be mandatory before advising filgrastim treatment. The presence of a euploid
embryo in the previous miscarriage should also be considered mandatory before filgrastim treatment, as
filgrastim is expensive. Similarly, in RIF, PGT-A should be considered mandatory before treating with
filgrastim, and filgrastim should only be used when a healthy euploid blastocyst is transferred.
There are difficulties in the evaluation of the effectiveness of any treatment in RPL: confounding factors
such as maternal age, number of previous miscarriages, and embryonic aneuploidy, which increases with
maternal age, as described elsewhere in this book. In addition, there is a subsequent live birth rate of
40%–60% without treatment. A randomized controlled trial should be performed taking these covariates
into account. Such a study would need to recruit a large number of patients, and consequently would need
to be multicenter.
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toxicity via the ERK pathway. Neurobiol Aging. 2007;28(8):1258–69.
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role of a tyrosine kinase activity distinct from the Janus kinases. Blood. 2000;95:1656–62.
14. Komine-Kobayashi M, Zhang N, Liu M et al. Neuroprotective effect of recombinant human granulocyte colony-
stimulating factor in transient focal ischemia of mice. J Cereb Blood Flow Metab. 2006;26:402–13.
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stimulating factor. Nature. 1986;319:415–8.
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1992;79:1148–54.
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18. McCracken SA, Grant KE, MacKenzie IZ et al. Gestational regulation of granulocyte-colony stimulating factor
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improves neonatal rat survival after a lethal group B streptococcal infection. Blood. 1993;81:923–7.
20. Sugita K, Hayakawa S, Karasaki-Suzuki M et al. Granulocyte colony stimulation factor (G-CSF) suppresses
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The Role of Filgrastim 279
22. Lédée N, Lombroso R, Lombardelli L et al. Cytokines and chemokines in follicular fluids and potential of the
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32
Opinion: Immunotherapy Has No Place in the
Treatment of Recurrent Pregnancy Loss*
Micha Baum
In the second edition of this book, the case was made quite forcefully against the use of immunomodulation
in all its forms for women with recurrent miscarriage (RM). Unfortunately, in the last 6 years not much
has changed. In the current climate patients demand and expect a “treatment/cure” of their reproductive
failure. Hence, it is incumbent upon clinicians to show evidence that various regimens of treatment have
an effect and are free of side effects, rather than rely on anecdotal evidence, personal bias, and the results
of small, uncontrolled studies.
The concept of immune dysfunction as a basis for miscarriage is attractive. However, while pregnancy
has traditionally been viewed as a battle between the semi-allogenic fetus and the mother, in which the
fetus and surrounding trophoblast have to evade an immune response if that response is not suppressed.
However, an immune attack on the pregnancy has never been demonstrated. From an evolutionary
viewpoint it seems that the maternal immune cells and trophoblast cooperate rather than compete [1,2].
Indeed, there is no evidence of a classic graft-versus-host response in pregnancy. Pregnancy itself is
not an immune-suppressed state but one in which the maternal immune system is modulated without
suppression.
Much of the data pertaining to immune responses to the trophoblast have been obtained from murine
models, and the same mechanisms have been assumed to be relevant in humans. However, although the
modulation of the immune system into a cooperative response probably developed once in the evolution
of mammalian reproduction, there may be wide differences in the subsequent development of immune
modulation in different orders of mammals. Therefore, caution has to be applied to the extrapolation of
data from murine pregnancies to the human. In addition, the observed immune aberrations in pregnancy
failure may be a consequence rather than the cause of pregnancy loss.
Regardless, immunotherapy has been introduced into clinical practice as a treatment for RM based on
the hypotheses that either alloimmunity or autoimmunity is responsible for pregnancy failure. In order
to critically evaluate the use of paternal or third-party white cell immunization (active immunization),
intravenous immunoglobulin (passive immunization), or cytokine modulation as treatment for RM it is
necessary to examine the rationale for their use, and the results that are currently available.
Rationale (or Not) for Immunotherapy

Paternal White Cell Immunization
There have been a number of concepts suggested to explain the mechanism of action of active immunization.
None has stood the test of thorough investigation. The first concept of an alloimmune basis for RM was
based on an increased sharing of human leukocyte antigens (HLA) between both partners that prevents the
maternal production of a “blocking” antibody which protects the fetus against immunological attack [3].
Women with successful pregnancies were thought to produce this “blocking” antibody and those whose
* This chapter has been updated by Micha Baum from the original in the second edition by Raj Rai.
280
Opinion: Immunotherapy Has No Place in the Treatment of Recurrent Pregnancy Loss 281
pregnancy ends in miscarriage do not. White cell immunization has been reported to induce production
of the “blocking” antibody [4]. However, the “blocking antibody” hypothesis has never been validated,
and an increased sharing of HLA Class I alleles between partners has been refuted in a number of articles
and in Beydoun et al.’s [5] meta-analysis. Further, (a) production of “blocking” antibody is usually not
evident until after 28 weeks’ gestation and may disappear between pregnancies [6]; (b) miscarriage occurs
despite the presence of “blocking” antibody [7,8], and (c) women who exhibit no production of “blocking”
antibodies do experience successful pregnancies. Consequently, the clinical impact of such antibodies is
unclear [9]. Leucocyte immunization has also been reported to reduce natural killer cell numbers [10] and
modulate cytokine levels in favor of a Th-2 response. These mechanisms have also not been confirmed
in large studies, and have not been shown to be relevant to human pregnancies.
Intravenous Immunoglobulin/Intralipid
Current concepts on the etiology of RM focus on autoimmune-mediated pregnancy loss (such as
antiphospholipid syndrome), natural killer (NK) cells, a disordered cytokine balance at the feto-
maternal interface, Th-17 cells, and the role of T regulatory cells. Intravenous immunoglobulin (IVIg)
has a number of immunomodulatory effects on cytokine production, antigen neutralization, Fc receptor
blockade, alteration in the distribution and function of T cell subsets, antibodies, and autoantigens that
may potentially ameliorate a dysregulated immune response causal of pregnancy loss.
The relationship between peripheral blood NK (PBNK) cells and reproductive failure is one of the
most controversial fields in reproductive immunology. The levels and activation of NK cells is dependent
on other variables such as whether whole blood or fractionated mononuclear cells are used in the assay,
the time of day a sample is taken, whether any physical exercise has been performed, the parity of the
patient, and whether the samples have been previously frozen [11–15]. Different NK assays have also
been employed, and results may vary depending on whether the chromium-51 release cytotoxicity assay
or CD69 expression is assayed. Importantly, it is not known which in vitro assay most accurately reflects
in vivo function, and indeed what biological relevance such activity has. Furthermore, it is unclear what
an abnormal NK cell number is. While traditionally a peripheral NK cell level greater than 12% of all
lymphocytes has been regarded as the cutoff between a raised and a normal level [16], this figure is well
within the normal range (up to 29%) published by others [17]. Hence individuals with entirely normal
results are being labeled as having raised NK cell numbers. A fascinating study has cast further doubt on
the validity of PBNK cell testing in women with RM [18]. The authors reported that immediately after
insertion of an intravenous cannula for blood withdrawal, women with RM show an increased proportion
of NK cells within lymphocytes, elevated blood NK cell concentrations, and augmented NK activity
per milliliter of blood compared to a control group of women who have no known fertility problems.
However, these differences disappear after 20 minutes when blood is drawn again from the same cannula.
The authors concluded that the elevated NK indices previously observed in women with RM are due to a
transient increase in NK cell numbers, rather than a chronic state. Despite the above caveats and amidst
much publicity, PBNK cell testing is being promoted as a useful diagnostic test to guide the initiation of
a variety of immunosuppressive therapies among patients with either RM or infertility. Indeed, several
small observational studies reported enhanced PBNK cell activity with subsequent failure to conceive
or miscarry [16,19–24]. However, the largest single observational study of 552 women with a history of
between two and six miscarriages reported that PBNK cell cytotoxic activity was not correlated with
subsequent pregnancy outcome, and a meta-analysis of 22 studies reported no relationship between either
PBNK cell numbers or activity and pregnancy outcome [25].
Uterine NK (uNK) cells, which are phenotypically and functionally different from PBNK cells, and the
numbers of which are maximal during the window of implantation are perhaps of more interest. While
intra-cycle variation in uNK cell numbers has been documented [26], several studies have reported that
women with RM have a raised uNK cell level [27–29]. The largest reported prospective study reported
no correlation between uNK cell numbers and pregnancy outcome [27]. In addition, a prospective
randomized study designed to assess the efficacy of prednisolone suppression on “raised” uNK cell
numbers reported no significant difference in live birth rate between those treated with prednisolone
compared to those receiving placebo [29]. Is this surprising? Perhaps not. It is clear that interactions
between HLA-C and killer-immunoglobulin-like receptors (KIR) on decidual NK cells can influence the
success of early pregnancy events after implantation has occurred [30]. In addition, the name “natural
killer” cells is a misnomer for uNK cells, as these large, granular lymphocytes do not kill anything in vivo
[31]. Indeed, both genetic and functional studies support the view that activation of decidual NK cells by
MHC ligands on trophoblast has beneficial effects on pregnancy outcome [30].
As an alternative to IVIg, intralipid, which is a 20% intravenous fat emulsion that is usually used and
consists of soybean oil as well as egg yolk phospholipids, glycerine, and water, has been introduced into
the clinical arena. A single small non-randomized study, presented only in abstract form, reported a 50%
pregnancy rate and 46% clinical pregnancy rate among women with recurrent implantation failure who
had an elevated TH1 cytokine response. There are no published results in RM. The mechanism by which
intralipid modulates the immune system is still unclear. It has been proposed that fatty acids within the
emulsion serve as ligands to activate peroxisome proliferator-activated receptors expressed by the NK
cells. Activation of such nuclear receptors has been shown to decrease NK cytotoxic activity, enhancing
implantation [32]. Clearly, large randomized studies are needed [33].
Efficacy of Immunotherapy
The patient with RM is interested in the results regarding her subsequent pregnancy rather than the
theoretical basis. If the results of treatment show evidence of effect, the mechanism will eventually
be clarified. However, it is important that when evaluating the effect of any intervention proposed as a
treatment for RM to be cognizant of the fact that the two most important determinants of the outcome of a
particular pregnancy are the mother’s age and the number of miscarriages she has previously experienced.
The rate of sporadic fetal aneuploidy is in the region of 50% among women between 40 and 44 years of
age, rising to 75% among those older than 45 years. On the basis of a 15% clinical miscarriage rate, 35%
of women with three consecutive miscarriages will have done so purely by chance alone. Among such
women aged less than 39 years, a live birth rate of between 65%–70% with supportive care alone can be
expected [34]. However, 30%–35% of women with a recurring cause will miscarry again. It is against this
high spontaneous resolution rate that the efficacy of any putative treatment for RM has to be judged. It
has been claimed that immunotherapy may be effective in certain subgroups of women with RM, rather
than in all women with RM as a whole. However, these subgroups have not been well defined. The most
obvious subgroup is women losing genetically normal embryos. However, no studies have been performed
which are restricted to patients losing euploid embryos. If, however, randomization is properly performed,
no such restriction is necessary.
Paternal White Cell Immunization

A number of studies have examined the efficacy of paternal white cell immunization as a treatment for
RM. These studies, which have used differing methodologies, entry criteria, and analyses, have reported
conflicting results. The largest study (183 women), a double-blind, multicenter, randomized clinical trial,
reported that on an intention-to-treat basis, the success rate was 36% in the treatment group versus 48%
in the control group (odds ratio [OR] 0.60; 95% CI 0.33–1.12) [34]. If analysis was restricted to only those
who conceived, the corresponding success rates were 46% with immunization but 65% with placebo
saline injections (OR 0.45; 95% CI 0.22−0.91), suggesting that immunization may increase the rate of
clinically recognized pregnancy loss. Partly on the basis of this large study and the lack of scientific
validity underlying paternal white cell immunization, the FDA issued guidance in 2002 highlighting the
lack of efficacy of this treatment and reminding clinicians that it should only be offered in the context of
therapeutic studies and will require Investigational New Drug approval (http://www.fda.gov/CBER/ltr/
lit013002.htm) for use in the United States. In the 17 years since the FDA guidance, no U.S. center has
performed a subsequent study under the strict rules of the FDA.
The conclusions of several published meta-analyses have also been conflicting. A Cochrane review
published in 2014, based on 12 trials (641 women), reported an OR of 1.23 (95% CI 0.89–1.70) among
those administered paternal white cells compared with controls [36]. It has been suggested that leucocyte
immunization in the trial of Ober et al. [35] should be excluded from the meta-analysis, as Ober et al.
used refrigerated cells, whereas all other trials used fresh cells. The argument against using refrigerated
cells is based on work in laboratory mice (CBA/J female mice when mated with allogeneic DBA/2 males)
where there is a high incidence of embryo resorption. This resorption can be prevented by immunization
with paternal splenocytes. However, storage of the splenocytes causes loss of surface CD200 into the
supernatant [37], which abrogates the protective effect of immunization. However, the loss of CD200
may be relevant in muridae, but has never been investigated in humans. Therefore, in this author’s view,
there is no justification for removing Ober et al.’s [35] trial from any meta-analysis.
Before leucocyte immunization can be recommended, it is necessary to have a dose-finding study,
and then a properly randomized control study. In the meantime, it must be remembered that leucocyte
immunization has been used since 1985. In 25 years, there has been no conclusive evidence of effect.
Intravenous Immunoglobulin
Studies using IVIg have used different preparations, doses, starting times, frequency, and duration of
administration. In addition, differing entry criteria have been used. Some studies included those with
an autoimmune disturbance only, while others have included those with “unexplained” RM. Hence, at
present, the only reasonable basis for assessment of the efficacy of IVIg as a treatment for RM would be
to examine the results of meta-analyses. The Cochrane review [36] reports that irrespective of whether
analysis is performed on an intention-to-treat basis (OR 1.18; 95% CI 0.72–1.93) or not (0.98; 0.61–1.58),
IVIg does not improve pregnancy outcome among women with RM. The results of this analysis are
supported by two more recent publications which report that irrespective of the dose of IVIg, the time of
administration (pre-pregnancy, early pregnancy) or whether primary or secondary recurrent miscarriage
is examined, IVIg administration is not associated with an increase in the live birth rate [38,39].
Other Immunomodulators
Other agents have also been used to try to improve the live birth rate in RPL. Granulocyte colony-
stimulating factor (G-CSF) and anti-TNF-α agents are two examples. There are three trials of G-CSF,
and none on anti-TNF-α agents. The three trials on G-CSF have produced conflicting results. Scarpellini
and Sbracia [40] reported a statistically improved live birth rate after treatment (p = 0.0061), as did
Santjohanser et al. [41]. However, Zafardoust [42] was not able to demonstrate any benefit. Hence, further
trials have to be performed and evidence needs to accumulate before G-CSF or any other agent can be
recommended for routine use.
Conclusions
The lack of scientific rationale for immunotherapy has not stopped its introduction into clinical practice.
However, despite the limitations of meta-analyses, the use of either paternal white cell immunization or
IVIg as a treatment for RM has not been shown to be of benefit. The use of these immunomodulatory
agents should be resisted until adequately powered prospective randomized placebo-controlled studies in
defined populations of those with a specified immune disturbance have been conducted.
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Index
A hands and feet, 136, 137

heart four-chamber view, 136
Abdominal cerclage, 156 kidneys, 137
Abdominal metroplasty, 123 three-vessel view, 136, 137
Abnormal embryonic development, 49, 50, 54–55 Anti-β2-glycoprotein-I (aβ2-GP-I) antibodies, 215
Abnormal offspring, theoretical vs. empirical risks for, 38 Antibodies, 8
Abnormal placentation, 74–75 Anticardiolipin (aCL), 75, 215
aCGH, see Array comparative genome hybridization Anticoagulants, 223
aCL, see Anticardiolipin aspirin compared to, 227
ACOG, see American College of Obstetricians and biomarkers, 26–27
Gynecologists in patients with thrombophilia, 223–225
Activated partial thromboplastin time (aPTT), 71 treatment with, 226–227
Activated protein C (APC), 72 Antigen-presenting cells (APCs), 102, 106
Activated protein C resistance (APCR), 7, 81 Anti-HLA antibodies, 106–107
Active immunization, 280 Anti-Müllerian hormone (AMH), 64, 250–251
Activin A, 146 Antinuclear antibodies (ANAs), 107
AFC, see Antral follicle count Antiphospholipid antibodies (aPL), 70, 73, 75, 76, 79, 89,
Afibrinogenemia, 81 191, 215, 216
Aging gametes, 249 Antiphospholipid syndrome (APS), 26, 76, 102, 107,
AH, see Assisted hatching 185, 215
ALIFE study, 228 abnormal placentation, 74–75
Alloantibodies, 8 aPL positivity, 75
Allogeneic recognition failure, 266 arachidonic acid, 73
Alloimmune abortions, Th1-type immune response in, 91 defective endometrial angiogenesis, 74
Alloimmune pregnancy loss, 89 diagnosis of, 216
cytokine and hormonal network, 91 future directions, 219–220
regulatory T cells, 92–93 genetic predisposition, 71–72
Th1 abortogenic responses, 91–92 high risk, 219
Th17 inflammatory cells, 92–94 history of thrombosis, 217
Th1-type immune response, 91 inflammatory responses, 73–74
Th2-type immune response, 90 low-dose aspirin, 217, 218
α-fetoprotein (AFP), 165 low molecular weight heparin, 216, 218
Altruistic surrogacy, 253 management of, 216–218
American College of Chest Physicians, 227 molecular mimicry, 70–71
American College of Obstetricians and Gynecologists obstetric care in, 218–219
(ACOG), 30, 143, 167, 184, 224, 225, 227, 228 obstetric clinical criteria for, 215
American Society for Reproductive Medicine (ASRM), 1, optimal management of, 217
10, 57, 110, 126, 127, 128, 184, 186, 187, 188, patient risk stratification, 216–217
246 placental cells in, 75
AMH, see Anti-Müllerian hormone prostacycline, 73
Amniocentesis, 164, 167–168 and refractory cases, 219
ANAs, see Antinuclear antibodies related thrombosis, 215
Anembryonic pregnancies, 138 reproductive failure in, 71–75
Aneuploid embryos, 233, 234 study design limitations, 219–220
Aneuploidy thrombosis, 72–73
antenatal screening for, 164 treatment of, 217
incidence of, 239 Anti-PIF antibodies, 15
maternal serum markers of, 165 Antiplatelet agents, 223, 227–228
screening (11 to 14 weeks), 140–141 Antithrombotic agents, 223–228; see also
third party reproduction, 249, 250 Thrombophilias
Anomaly scan (10 to 14 weeks), 136 Antithyroid autoantibodies (ATA), 89
bladder, 137 Anti-TNF-α agents, 283
choroid plexus, 136
287
288 Index
Antral follicle count (AFC), 64 C

APCR, see Activated protein C resistance
APCs, see Antigen-presenting cells CA-125, 146
aPL, see Antiphospholipid antibodies C1D, 71–72
aPL syndrome, see Antiphospholipid syndrome CD44, 235
APS, see Antiphospholipid syndrome CD200, 283
aPT antibodies, 72 CD14+ cells, 19
aPTT, see Activated partial thromboplastin time CD69 expression, 17
Arabin pessary, 160, 161 CD1 receptors, 271
Arachidonic acid, 73 Cell free (cf)DNA screening, 164, 166
Arcuate uterus, 111, 119 contingent, 167
Array comparative genome hybridization (aCGH), 240 primary, 166–167
ART centers, 254 secondary, 167
ARTs, see Assisted reproductive technologies Center for Biologics Evaluation and Research, 262
ASIA, see Autoimmune syndrome induced by adjuvants Cervical cerclage, 121, 123, 153
Aspirin, 26–27, 223, 225 abdominal cerclage, 156
anticoagulants compared to, 227 comparison of treatment modalities, 162
in combination with anticoagulants, 225–226 history-indicated cerclage, 156–157
in recurrent pregnancy loss, 228 indicated cerclage, 159
ASRM, see American Society for Reproductive McDonald cerclage, 155
Medicine physical examination, 159
Assisted hatching (AH), 234–235 Shirodkar cerclage, 155–156
Assisted reproductive technologies (ARTs), 127, 209, 231, successful outcome after, 159
236–237, 243, 246, 249 ultrasound-indicated cerclage, 157–159
ATA, see Antithyroid autoantibodies Cervical incompetence, 121, 158
Autoantibodies, 8, 92 Cervical Incompetence Prevention Randomized Cerclage
Autoimmune diseases, 102 Trial (CIPRACT), 121
Autoimmune-mediated pregnancy loss, 281 Cervical insufficiency
Autoimmune-predisposing alleles, 104 congenital factors, 154
Autoimmune pregnancy loss, 89 defintion, 153
Autoimmune syndrome induced by adjuvants (ASIA), diagnosis of, 154–155
71, 76 etiology of, 154
Autoimmune thyroid disease, 63 mechanical dilation, 154
Autosomal monosomy, 34 obstetric trauma, 154
Autosomal trisomies, 33 pathophysiology, 153–154
Cervical intraepithelial neoplasia, 154
Cervical laceration, 154
B
Cervical mucus plug, 160, 161
Balanced translocation, 37–38 Cervical pessary, 160
BALB/c lymphoid cells, 263, 264 advantages of, 160–161
BCL2, 16 comparison of treatment modalities, 162
Bed rest, for threatened miscarriage, 148 correct placement, 161
β2GP1 molecule, 70, 72, 75 results, 161–162
βhCG, 134, 139 Cervix
Bicornuate uterus, 113, 114 functional defect in, 154
abdominal metroplasty for, 123 fundal pressure, 158
Strassman metroplasty, 121 length, 153–154, 157
Biochemical pregnancies, 4, 188 mechanical dilation of, 154
Biologic glue, 235 Cetuximab, 25
Biomarkers, 24–25 cfDNA, see Circulating free DNA
anticoagulants, 26–27 CGH, see Comparative genomic hybridization
defintion, 24 Chlamydia trachomatis, 91
pregestational testing for aneuploidy, 26 Chorionic villus sampling (CVS), 32, 164, 165, 167–168
in recurrent pregnancy loss, 25–28 Choroid plexus, 135, 136
uterine anomalies, 26 Chromosomal abnormalities, 128, 164–165, 169
Blastocyst morphology, 234 autosomal monosomy, 34
Blastocyst transfer, 233 autosomal trisomies, 33
Bleeding, 145 at different gestational ages, 32
Blighted ovum, 50 double trisomies, 33–34
Blood transfusion, 24 monosomy X, 34
British Medical Association, 254 in preimplantation embryos, 31–32
Index 289
recurrent aneuploidy, 35–36 Cumulative likelihood of pregnancy, 39

sex chromosomal polysomy, 35 CVS, see Chorionic villus sampling
spectrum of, 33–35 Cyclooxygenase-2, 209
tetraploidy, 34 Cytogenetic testing, 30–31
triploidy, 34 Cytokines, 85, 268
Chromosomal inversions, 39–40 Cytotoxic T lymphocyte-associated antigen 4
Chromosomal microarray (CMA), 30–31, 164, 168 (CTLA-4), 93
Chromosomal rearrangement, 38
Chromosome abnormalities, 8 D
Chromosome anomalies, 54
Chronic histiocytic intervillositis, 194 Dalteparin, 225
CIPRACT, see Cervical Incompetence Prevention DBA/2 model, 263
Randomized Cerclage Trial D&C, see Dilatation and curettage
Circulating free DNA (cfDNA), 31, 169, 170 DCs, see Dendritic cells
Clarification of Optimal Anticoagulation through Genetics DD, see DNA denaturation
(COAG) trial, 25 Decidual inflammation, 75
Class II HLA alleles, 8–9 Decidualization, 43–44, 74
Class II HYrHLA alleles, 105 Decidualized endometrium, 44–45
Cleft lip, 51–52 Decidual MΦ and DCs, 97
Cleft palate, 52 Decidual NK (dNK) cells, 94–95
Clinical associations, recurrent pregnancy loss, 6, 9 Decidual Tregs (dTregs), 93
CMA, see Chromosomal microarray Decidual vessels, thrombosis in, 82
CMV IgM, 70 Deep venous thrombosis (DVT), 215
CNVs, see Copy number variants Dendritic cells (DCs), 18
COAG trial, see Clarification of Optimal Anticoagulation DFI, see DNA fragmentation index
through Genetics trial Diabetes mellitus, pregnancy loss and, 64
Coagulation factors Didelphic uteri, 112
alterations in factor XIII, 80–81 “The Different Guidelines,” 187, 192
deficiencies of, 79 Differentiated decidual cells, 43
fibrinogen deficiency, 81 Dilatation and curettage (D&C), 174–175
hereditary factor FXIII deficiency, 79–80 Diminished ovarian reserve (DOR), 65
prothrombotic mechanisms, 85–86 Disorganized embryos, 49–51, 54
thrombophilias, 81–84 DNA damage, 129
Coagulation inhibitors, 79 DNA denaturation (DD), 127
Cochrane Register of Controlled Trials, 185 DNA fragmentation index (DFI), 250
Combined test, 165 Donut pessary, 160
Commercial surrogacy, 253 Doppler velocimetry, 75
Comparative genomic hybridization (CGH), 32, 128, DOR, see Diminished ovarian reserve
143, 192 Dose-dependent effect, 174
Conception, 13 Double trisomies, 33–34
ovulation, 235 Down syndrome (trisomy 21), 141, 164, 165, 167
timing of, 232, 235 Ductus venosus (DV), 165, 166
Conceptus, maternal immune response to, 107–108 DV, see Ductus venosus
Consecutive miscarriages, 2 DVT, see Deep venous thrombosis
Conservative myomectomy, 120 Dydrogesterone, 150, 198, 199, 204
Contingent cfDNA screening, 167 Dysfibrinogenemia, 81
Contingent test, 165 Dysmorphic uterus
Copy number variants (CNVs), 30, 55, 143, 168 hysteroscopic metroplasty in, 122
Corpus luteum, 16–17, 197 Müllerian tract defects, 113–114
deficiency, 202
hCG, 207, 209 E
Counselor, 56
in fetal structural malformations, 56–57 Early pregnancy factor (EPF), 14
information from more than one loss, 57 EBM, see Evidence-based medicine
information from one loss, 56–57 Ectopic pregnancy, 146
CRL, see Crown-rump length Edometrial stromal cells, 45
Crown-rump length (CRL), 134, 138, 139 Edwards syndrome, 165, 166
Cryoprecipitate, 81 EGFR, see Epidermal growth factor receptor
CTLA-4, see Cytotoxic T lymphocyte-associated Elevated FSH, and pregnancy loss, 64–65
antigen 4 EM, see Expectant management
C-type lectin-like receptors (CD94/NKG2A), 94 Embryo-derived signaling molecules, 14
290 Index
Embryo donation, 249 European Society for Gynaecological Endoscopy, 113

indications, 252 European Society of Human Reproduction and
issues with, 252 Embryology (ESHRE), 1, 10, 30, 37, 57,
Embryogenesis, paternal genome in, 126–127 184–185, 188, 189, 243, 246
Embryonic aneuploidy, 189 guideline, 186
Embryos, 13, 134–135, 145, 245 protocol, 187
aneuploid, 233, 234 Evidence-based medicine (EBM), 22
chromosomally abnormal, 249 drawbacks of, 23–24
disorganized, 49–51, 54 evidence-based approach, 22–23
embryoscopic lateral view of, 52 physician’s experience, 24
genetic assessment of, 189–190 randomized controlled trials, 22
implantation, 13, 19, 59 reducing selection bias, 23
live, 194 research question, 23
maternal communication, 14–15 trials, 24
morphology, 233–234 Expectant management (EM), 246
quality, 233–234 Extended cfDNA tests, 168–169
semi-allogeneic graft, 89 Extracellular-regulated kinase (ERK), 275
signaling, 13, 14 Extracellular vesicles, 96–97
Embryoscopy
as diagnostic modality, 48–49 F
technique, 49
Embryo selection hypothesis, window of implantation and, Factor H, 73
44–45 Factor V Leiden (FVL), 7, 82
Embryo-specific maternal communication Factor XIII (FXIII), 79–80, 80–81
preimplantation factor, 14–15 Familial aggregation, recurrent pregnancy loss, 5–6, 9
through immune system, 18–19 FDA, 282
Emergency cerclage, 159 Fertilization
Endocrinology, 59 delay between implantation and, 13–14
Endometrial angiogenesis, 74 paternal genome in, 126–127
Endometrial factor, 43–46 Fetal anomalies, recurrent pregnancy loss, 180
challenges and clinical implications, 46 Fetal death, 216
decidualization process, 43–44 Fetal heart rate (FHR), 140
embryo selection hypothesis, 44–45 Fetal structural malformations, 48
immunological factors, 45–46 diagnosed embryoscopically, 49–54
inadequate decidual responses, 44 disorganized embryos, 49–51
uterine natural killer, 45–46 early first trimester screening, 56
window of implantation, 44–45 embryoscopy, 48–49
Endometrial receptivity array (ERA) test, 236 genetic aberrations, 54–55
Endometriosis, 66, 102, 104 localized defects, 51–54
Endometrium, 16, 43, 231 nuchal translucency screening, 55
decidualized, 44–45 role of counselor, 56–57
selectivity of, 45 soft signs, 55–56
Endoscopic surgery, 118 testing, 57
Endothelial nitric oxide synthase (eNOS), 199 ultrasound, 55–56
eNOS, see Endothelial nitric oxide synthase Fetal thrombophilia, 86
Enoxaparin, 226, 227 Fetal tolerance, 95
EPCOT, see European Prospective Cohort on decidual MΦ and DCs, 97
Thrombophilias extracellular vesicles, 96–97
EPF, see Early pregnancy factor γ/δ Τ lymphocytes, 97
Epidermal growth factor receptor (EGFR), 22 hCG, 96
ERA test, see Endometrial receptivity array test matrix metalloproteinases, 97
ERK, see Extracellular-regulated kinase progesterone-induced blocking factor, 96
ESHRE, see European Society of Human Reproduction specific trophoblastic molecules, 96
and Embryology sperm, 96
Estrogen, 14, 43, 59 trophoblastic and maternal molecules, 96
Euploid embryos, 239 FHR, see Fetal heart rate
European Pharmacogenetics of Anti-Coagulant Therapy Fibrinogen
(EU-PACT) trial, 25 deficiency, 81
European Progestin Club, 200 hereditary abnormalities of, 81
European Prospective Cohort on Thrombophilias Filgrastim, 275–278; see also G-CSF
(EPCOT), 223–224 mechanism of action, 276
Index 291
recombinant, 276 Grade 3 (GD 3) embryo, 50

in recurrent pregnancy loss, 276, 277 Grade 4 (GD 4) embryo, 50
side effects, 275 Granulocyte colony-stimulating factor (G-CSF), 275, 283
toxicity in pregnancy, 277 mechanism of action, 276–277
First trimester scan, 134 recombinant, 278
4 to 5 weeks, 134–135 recurrent pregnancy loss, 275–276
structural abnormalities, 141–143 safety of drugs, 277
week 6, 135 Granulocyte macrophage colony-stimulating factor
week 8, 135 (GM-CSF), 90, 209
weeks 7 to 9, 135 Granulocyte-macrophage stimulating factor (GM-CSF or
First-trimester single-antigen bead (SAB), 223, 224 CSF2), 275
FISH, see Fluorescence in situ hybridization GS, see Gestational sac
FITC-PIF, 18 “Guidelines for Accreditation, Supervision and Regulation
Fluorescence in situ hybridization (FISH), 129, 240, 244 of ART Clinics in India,” 254
Folic acid supplementation, 226
Follicle-stimulating hormone (FSH), 64–65, 206 H
Free-β subunit of hCG (β-hCG), 165
FSH, see Follicle-stimulating hormone HA, see Hyaluronic acid
FVL, see Factor V Leiden HABENOX trial, 227, 228
FXIII-A, 79–80 Haplotypes, 104
FXIII-B, 80 Hashimoto thyroiditis, 62, 102, 107
hCG, see Human chorionic gonadotropin
G HCQ, see Hydroxychloroquine
Head defects, 51
γ/δ Τ lymphocytes, 97 Heartbeat, 135
Gastroschisis, 52–53, 141, 142 Heat shock proteins (HSPs), 15
G-banded karyotype, 30 HEEC, see Human endometrial endothelial cells
GCSF-R, 275 Hegar test, 155
Gefitinib, 22, 25 Hematoma, 139, 147–148
Genetic aberrations, abnormal embryonic development, and late obstetric complications, 147–148
54–55 subchorionic, 147–148
Genetic abortions, 187–188 Hematopoietic stem cells (HSCs), 275
Genetic anomalies, 55, 143 Heparin, 26, 216, 217, 218, 223, 224, 227
Genetic polymorphism, 9 Heparin-induced thrombocytopenia, 225
Genetic predispositions, 71–72, 104 Hereditary factor FXIII deficiency, 79–80
Genetics, 13, 143 Hereditary thrombophilias, 26, 79, 82, 192, 194, 223–226
Genetic screening, 164 anticoagulants, 223–225
amniocentesis, 167–169 antiplatelet agents, 225–226
cfDNA screening, 166–167 Heroic cerclage, 159
combined test, 165 hESC, see Human embryonic stem cell
contingent test, 165 History-indicated cerclage, 156–157
conventional screening modalities, 165–166 HLA, see Human leukocyte antigens
integrated test, 165–166 HLA-C, 269, 282
public health policies, 169 HLA-C allotypes, 104
quad test, 165 HLA class II homozygosity, 104
routine CVS, 167–169 HLA-C molecules, 95
routine screening, 164–165 HLA-DRB1 locus, 105
recurrent pregnancy loss couples, 169–170 HLA-G, 16–17, 105
Genetic testing, 57 Hodge pessary, 160
Gestation, 13 Hormones, 85–86
Gestational age, 49 HSCs, see Hematopoietic stem cells
Gestational carrier surrogacy, 253 HSPs, see Heat shock proteins
Gestational diabetes mellitus, 177, 179–180 Human chorionic gonadotropin (hCG), 14, 44, 85–86, 96,
Gestational sac (GS), 134, 138, 139, 140 165, 198, 206
GM-CSF, see Granulocyte macrophage colony-stimulating actions, 207
factor administration of, 206
GnRH, see Gonadotropin-releasing hormone assessment, 206
Gonadotropin-releasing hormone (GnRH), 60, 62 in assisted reproductive technologies, 209
G-protein-coupled receptors, 271 clinical studies on, 209–211
Grade 1 (GD 1) embryo, 50 Cochrane systematic review, 210
Grade 2 (GD 2) embryo, 50 corpus luteum actions, 207, 209
292 Index
Human chorionic gonadotropin (hCG) (Continued) determination of diagnosis, 107

in fetoplacental tissues, 207 immune tolerance, 102–103
future directions, 211 immunological rejection, 102–103
immune actions, 209 maternal immune response to conceptus, 107–108
intracellular signaling pathways, 207 reproductive failure, 103–105
isoforms, 207 reproductive immunology, 108
molecules, 206–207 selection of patients, 101–102
in pregnancy, 206–209 soluble factors, 106–107
pregnancy-promoting actions of, 208 Immunologic mechanisms in abortion, 92
recurrent pregnancy loss, 210 Immunomodulation, 280, 283
supplementation, 211 Immunophenotyping, 106
therapeutic use of, 211 Immunotherapy, 9, 250, 280; see also Leucocyte
in threatened miscarriages, 146, 148, 209–210 immunotherapy
uterine actions, 209 biomarkers, 27
Human embryonic stem cell (hESC), 16 efficacy of, 282–283
Human endometrial cycle, 43–44 intravenous immunoglobulin, 283
Human endometrial endothelial cells (HEEC), 74 intravenous immunoglobulin/intralipid, 281–282
Human leukocyte antigens (HLA), 107, 280, 281 other immunomodulators, 283
Human umbilical vein endothelial cells (HUVEC), 72 paternal white cell immunization, 280–281, 282–283
Humoral factors, 97 rationale (or not) for, 280–282
HUVEC, see Human umbilical vein endothelial cells of reproductive failure, 268
Hyaluronic acid (HA), 233, 235 systematic reviews of, 261–262
HY antigen, 105 “Immunotrophic” theory, 90
Hydropic fetus, 142, 143 Implantation
Hydrosalpinx, 122 fertilization delay between and, 13–14
Hydroxychloroquine (HCQ), 219 insufficient, 16
Hyperhomocysteinemia, 79 IMSI, see Intracytoplasmic morphologically selected
Hyperprolactinemia, 59, 60–62, 62 sperm injection
Hyperthyroidism, 62 IND, see Investigational New Drug
Hypocomplementemia, 73 Indoleamine 2,3-dioxygenase (IDO), 93
Hypofibrinogenemia, 81 Inherited thrombophilia, 225, 226
Hypothyroidism, 62–63 Inhibin A, 146, 165
Hysterosalpingography, uterine anomalies, 116, 117 Inhibins, 65–66, 207
Hysteroscopic metroplasty, 120, 122 Inhibitory checkpoint molecules, 97
Hysteroscopic myomectomy, 120 Inner cell mass (ICM), 32, 245
Hysteroscopic polypectomy, 118 INR, see International normalized ratio
Hysteroscopy, 49, 115, 116, 117, 192 Insulin-like growth factor binding protein-1 (IGFBP-1), 44
Insulin resistance, 64
I Integrated test, 165–166
Integrins, 16
ICSI, see Intracytoplasmic sperm injection Interleukin (IL)-3, 209
IGFBP-1, see Insulin-like growth factor binding protein-1 International Federation of Gynecology and Obstetrics
IgG anti-DmI antibodies, 75 (FIGO), 184, 185
IL-3, 74 International normalized ratio (INR), 25
IL-8, 223 Intracytoplasmic morphologically selected sperm injection
IL-10, 93 (IMSI), 233
IL17F, 16 Intracytoplasmic sperm injection (ICSI), 127, 129, 233
IL12RB2, 16 Intralipid, 271, 272, 282
Immune dysfunction, 280 Intramural myomas, 120
Immune-mediated pregnancy loss, 89 Intrauterine adhesions, 114–115
Immune responses, 280 Intrauterine growth restriction (IUGR), 6, 14, 16, 82, 177,
Immune system, embryo-specific maternal 178
communication, 18–19 Intrauterine hematomas, 139, 147
Immunobiology, 89 Intrauterine insemination (IUI), 232
alloimmune pregnancy loss, 89–94 Intrauterine polyps, 118
autoimmune pregnancy loss, 89 Intrauterine pregnancy, 146
fetal tolerance, 95–97 Intravenous immunoglobulin/intralipid, 281–282
NK cells, 94–95 Intravenous immunoglobulin (IVIg) threapy, 9, 192, 219,
Immunological “add-ons,” 235 268, 281, 282, 283
Immunological testing, 101 clinical trials, 271
cellular analysis, 105–106 controlled trials of, 270
Index 293
efficacy of, 271 L

with elevated NK cells, 271–272
identifying treatment with, 268–270 LA, see Lupus anticoagulant
immunologic risk factor, 269–270 Laceration, 154
live birth rates, 269 LAD testing, 103
recurrent pregnancy loss, 270 LAM, see Laparoscopic-assisted myomectomy
success rates of, 270–271 Laparoscopic-assisted myomectomy (LAM), 121
testing for risk factors, 270 Laparoscopy, uterine anomalies, 116–117
Investigational New Drug (IND), 262 Last menstrual period (LMP), 134, 138
Investigation protocol, 184 Lateral and median cleft lip, 51
ASRM guideline, 186, 187 LEEP, see Loop electrosurgical excision procedures
biochemical pregnancies, 188 Leucocyte immunization, 281
ESHRE guideline, 186, 187 Leucocyte immunotherapy, 257
factors affecting subsequent prognosis, 190–192 autoimmune abnormalities, 260–261
genetic assessment of embryo, 189–190 control vs., 265
good prognosis patients, 191 donor leucocyte groups, 259
guidelines, 184–187 effect of refrigeration on immunogenic potency,
medium prognosis patients, 191–192 263–264
poor prognosis patients, 192 efficacy of, 265, 266
pregnancy loss, 193–194 heterogeneity, 259
RCOG guideline, 185–186, 187 intradermal immunotherapy, 266
resistant patient, 192–193 live birth rate, 260, 261
two or three losses, 187–188 Mowbray trial, 257–258
upper limit of pregnancy loss, 188–189 newer systematic reviews, 264–266
In vitro fertilization (IVF), 13, 17–18, 60, 120, 129, 231, odds ratio tree, 260
236, 250 possible adverse effects from, 266
assisted hatching, 234–235 for recurrent miscarriage, 258–261
assisted reproductive technique, 231, 236–237 Recurrent Miscarriage Study, 262–264, 267
biologic glue, 235 subgroup analysis, 259–261
embryo quality, 233–234 systematic reviews of, 261–262
immunological “add-ons,” 235 worldwide collaborative prospective study, 258–259
improving implantation, 234–235 Leukemia inhibitory factor (LIF), 67
improving synchronization, 235–236 Leukocyte Ig-like receptor (LILRB1), 94
sperm selection techniques, 233 LH, see Luteinizing hormone
subsequent live birth rate, 231–232 LIF, see Leukemia inhibitory factor
time to conceive, 232 Lifestyle factors, recurrent pregnancy loss, 6, 9–10
IRAKBP1, 16 LILRB1, see Leukocyte Ig-like receptor
IUGR, see Intrauterine growth restriction Limb defects, 53
IUI, see Intrauterine insemination Lipopolysaccharide (LPS), 15
IVF, see In vitro fertilization LIT, see Lymphocyte immunotherapy
IVF and embryo transfer (IVF-ET), 250 Live birth, 187–188, 227, 231, 239
IVF-ET, see IVF and embryo transfer odds ratios, 262
IVF/intracytoplasmic sperm injection (ICSI), 60 ratios, 259
IVF outcomes, 244 Live embryos, losses of, 194
IVF-PGD/preimplantation genetic screening (PGS), 255 LIVE-ENOX trial, 225
IVF surrogate gestational program, 254 LMP, see Last menstrual period
IVIg therapy, see Intravenous immunoglobulin (IVIg) LMWH, see Low molecular weight heparin (LMWH)
therapy Localized defects, 51–54
head, 51
lateral and median cleft lip, 51–52
J limb, 53
JAK/STAT, see Janus protein tyrosine kinase/signal neural tube, 51
transducer and activator of transcription trunk, 52–53
Janus protein tyrosine kinase/signal transducer and umbilical cord, 54
activator of transcription (JAK/STAT), 275 Loop electrosurgical excision procedures (LEEP), 154
Lotus 1 trial, 199
Low-dose aspirin (LDA), 217, 218, 219
K
Low molecular weight heparin (LMWH), 74, 216, 217,
Killer-immunoglobulin-like receptors (KIR), 94, 104, 218, 219, 223, 224, 225, 226, 227
269, 282 LPD, see Luteal phase defect
KRAS gene, 25 LPS, see Lipopolysaccharide; Luteal phase support
294 Index
Luminex single-antigen bead (SAB) assay, 107 single-gene causes of, 40

Lupus anticoagulant (LAC), 72, 215, 216, 218, 219 threatened, 139
Luteal phase defect (LPD), 60, 236 MMP, see Matrix metalloproteinases
Luteal phase deficiency, 59–60 Molar pregnancy, 146
Luteal phase support (LPS), 60 Molecular mimicry, 70–71
Luteal support, 199 Monosomy X, 34
Luteectomy-induced progesterone, 197 Mosaicism, 34
Luteinizing hormone (LH), 206, 207 Motile sperm organelle morphology examination
Lymphocyte immunotherapy (LIT), 103 (MSOME), 233
Lymphocyte lineage profiling, 105 Mowbray trial, 257–258
MRC RCOG, see Medical Research Council/Royal
M College of Obstetricians and Gynaecologists
MSOME, see Motile sperm organelle morphology
Macrophage colony-stimulating factor (M-CSF or examination
CSF1), 275 MTHFR, see Methylenetetrahydrofolates reductase gene
Magnetic resonance imaging (MRI), uterine anomalies, Müllerian tract defects, 110–115
116, 117 arcuate uterus, 111
Major histocompatibility (MHC) antigens, 263 bicornuate uterus, 113
Male factor, 126 classification of, 111
chromosomal abnormalities, 128 dysmorphic uterus, 113–114
funding support, 131 intrauterine adhesions, 114–115
laboratory evaluation, 127–130 myomas, 114
paternal genome, 126–127 polyps, 114
semen parameters, 127 subseptate uterus, 111
sperm aneuploidy, 129 T-shaped uterus, 113–114
sperm DNA quality, 129–130 unicornuate uterus, 111–112
Mammals, zygote, 14 uterus didelphis, 112–113
Maternal age Murine gestation, 14
autosomal trisomies, 33 Murine pregnancies, 280
recurrent pregnancy loss, 4, 7 Myomas, 114, 120–121
of trisomy, 21, 36
Maternal cell contamination (MCC), 143 N
Maternal factor abortions, 187–188
Maternal immune response to conceptus, 107–108 Nasal bone (NB), 165, 166
Maternal serum cytokines, 106 Nasal bone length (NBL), 166
Maternal spiral arteries, 82 National Health Service (NHS), 25, 184
Matrix metalloproteinases (MMP), 74, 97, 209 National Institute of Child Health and Human
MCC, see Maternal cell contamination Development (NICHD), 30
McDonald cerclage, 155, 156 Natural cytotoxicity receptors (NCR), 94
Medical Research Council/Royal College of Obstetricians Natural killer (NK) cells, 8, 17, 27, 94, 105, 190,
and Gynaecologists (MRC RCOG), 156 268–269, 281
Mendelian genes, 30 allorecognition system, 94
Menstrual cycle, 44 decidual NK (dNK) cells, 94–95
Metalloproteinases, 16 in miscarriage, 95
Metformin, 64 peripheral blood, 268
Methylenetetrahydrofolates reductase (MTHFR) gene, 79 preimplantation factor and, 17–18
Metroplasty, 111 uterine, 268–269
MHC antigens, see Major histocompatibility antigens NB, see Nasal bone
MHC genes, 104 NBL, see Nasal bone length
Mickey Mouse sign, 141, 142 NCR, see Natural cytotoxicity receptors
Microparticles, 85 Neural tube defects, 51
Microscissor, 51 Next-generation sequencing (NGS), 32, 143, 192, 234, 243
Miscarriage (or abortion), 138–139, 185, 190, 192–193, 198 NF, see Nuchal skinfold
definition, 1 NGS, see Next-generation sequencing
diagnosis of, 138–139 NHS, see National Health Service
incidence of, 147 NICHD, see National Institute of Child Health and Human
management of, 139 Development
maternal age, 23 NICHD Stillbirth Collaborative Research Network, 30
NK cells in, 95 NIPS, see Noninvasive prenatal screening
previous, 2–4, 7 NIPT, see Noninvasive prenatal test
rates, 111 NKa, 106
Index 295
NOH-APS observational study, 219 Pericentric inversions, 39

Non-apoptotic sperm, 233 Perinatal mortality, 181
Noninvasive prenatal screening (NIPS), 31, 140–141, 142 Peripheral blood mononuclear cell (PBMC), 17
Noninvasive prenatal test (NIPT), 239 Peripheral blood NK (PBNK) cells, 281
Nonviable pregnancy, 138–139 Peroxisome proliferator-activated receptors (PPARs), 271
Normal pregnancy Personalized medicine, 24
cytokine network, 91 biomarkers, 24–25
immunologic mechanisms in, 90 drawbacks of, 25
T cells (Treg), 92–94 PGD-A, see Preimplantation diagnosis of aneuploidy
Th2-type immune response in, 90 PGDIS, see Preimplantation Genetic Diagnosis
Norwegian Mother and Child Cohort Study, 175 International Society
NT, see Nuchal translucency PGS, see Preimplantation genetic screening
Nuchal skinfold (NF), 166 PGT, see Pregestational testing
Nuchal translucency (NT), 55, 141, 165 PGT-A, see Pregestational testing for aneuploidy
PGT for monogenic/single gene defects (PGT-M), 243
O PGT-M, see PGT for monogenic/single gene defects
Phosphatidylinositol 3-kinase/Akt (PI3 K/Akt), 275
OAT, see Oligoasthenoteratozoospermia Phosphatidylserine, 233
Obesity, recurrent pregnancy loss and, 175, 177 Phospholipids (PL), 70
Obstetric care, in antiphospholipid syndrome, 218–219 Physiological intracytoplasmic sperm injection
Obstetric complications (PICSI), 233
hematoma and, 147–148 Physiological midgut, 52
thrombophilias in, 83 PIBF, see Pregnancy-induced blocking factor;
Obstetric outcomes, 172 Progesterone-induced blocking factor
fetal anomalies, 180 PICSI, see Physiological intracytoplasmic sperm injection
gestational diabetes mellitus, 177, 179–180 (PICSI)
intrauterine growth restriction, 177, 178 PIF, see Preimplantation factor
perinatal mortality, 181 PL, see Phospholipids
placental abruption, 180–181 Placental abruption, 180–181
preeclampsia, 175–177 Placental apoptosis, 85
pregnancy-induced hypertension, 175–177 Placental infarction, 75
small for gestational age, 177, 178 Plasmacytoid dendritic cells (pDC), 18
spontaneous preterm labor, 172–175 Plasminogen activator inhibitor-1 (PAI-1), 64
Oligoasthenoteratozoospermia (OAT), 250 Plasminogen activator inhibitor (PAI), 79
Oligozoospermia, 251 Platelet-activating factor (PAF), 14
Oocyte, 126 POC, see Products of conception
Oocyte donation, 249, 255 Polycystic ovary syndrome (PCOS), 10, 59, 64, 102, 104
indication, 251 Polydactyly, 53
issues with, 252 Polymorphisms, 130
for recurrent pregnancy loss, 252 Polypectomy, 118
Polyps, 114, 117
P Postpartum hemorrhage (PPH), 79
PPARs, see Peroxisome proliferator-activated receptors
PAF, see Platelet-activating factor PPH, see Postpartum hemorrhage
PAF-R, 14 Practice Committee of the American Society for
PAI, see Plasminogen activator inhibitor Reproductive Medicine, 235
Paracentric inversions, 39, 40 Precision medicine, 24
Partner specificity, recurrent pregnancy loss, 6 Preeclampsia, 147, 175–177, 216, 219
Passive immunization, 280 Pregestational testing (PGT), 57
Patau (trisomy 13) syndrome, 164, 166 Pregestational testing for aneuploidy (PGT-A), 35, 191,
Paternal genome 192, 193, 231, 232, 234, 236, 249, 250, 252
in embryogenesis, 126–127 biomarkers, 26
in fertilization, 126–127 Pregnancies of unknown location (PULs), 1
Paternal leucocyte immunization, 257–258 Pregnancy-associated plasma protein (PAPP)-A, 165
Paternal meiotic errors, 33, 34 Pregnancy failure, risk of early, 139
Paternal seminal fluid cytokines, 106 crown-rump length, 139
Paternal white cell immunization, 280–281, 282–283 fetal heart activity, 140
PBMC, see Peripheral blood mononuclear cell gestational sac, 139, 140
PCOS, see Polycystic ovary syndrome prediction models, 140
pDC, see Plasmacytoid dendritic cells sonographic markers, 140
PDI-T, see Protein disulfide isomerase/thioredoxin yolk sac, 139–140
296 Index
Pregnancy-induced blocking factor (PIBF), 27–28 Progesterone (progestogens), 14, 43, 59, 85, 96, 158, 252
Pregnancy-induced hypertension, 147, 175–177 biomarkers, 27–28
Pregnancy loss, 1, 2, 7, 30, 59, 239 clinical data, 203–204
bleeding diatheses leading to, 79–81 deficiency, 197
diabetes mellitus and, 64 dydrogesterone, 198, 199
elevated FSH and, 64–65 evidence of effect, 198–200
endometriosis, 66–67 first trimester of pregnancy, 200
hyperprolactinemia and, 60–62 immune response, 202
inhibins and, 65–66 immunomodulatory effect of, 197–198
insulin resistance, 64 luteal support, 199
luteal phase deficiency and, 59–60 micronized, 198, 199, 203
mixed pattern of, 194 midluteal serum, 202
negative prognostic effect of, 3 other, 204
polycystic ovary syndrome and, 64 for pregnancy development, 16
progesterone resistance, 66–67 preimplantation factor and, 16–17
prothrombotic mechanisms of, 85–86 PRISM trial, 204
specific forms of, 193–194 production, 197
thrombophilias in, 82–83 PROMISE trial, 198–199, 200, 203–204
thyroid abnormalities and, 62–63 and recurrent miscarriage, 200
upper limit of, 188–189 in recurrent pregnancy loss, 197
Pregnancy outcome resistance, 66–67
hydrosalpinx affect, 122 secretory effects, on endometrium, 199
uterine septum, 119–120 serum, 198
PREGNANTS study, 219 in threatened miscarriages, 61, 146, 149
Preimplantation diagnosis of aneuploidy (PGD-A), 243 Progesterone-induced blocking factor (PIBF), 59, 96, 97,
Preimplantation embryos, chromosomal abnormalities in, 147, 197–198, 202
31–32 Prognosis patients
Preimplantation factor (PIF), 13, 14–15 good, 191
autocrine effect, 15 medium, 191–192
effect of, 16 poor, 192
embryo-specific maternal communication, 14–15 PROK1, 44
as monotherapy, 19 Prolactin (PRL), 44, 60
natural killer cells, 17–18 PROMISE trial, 60, 198–199, 200, 203–204
and progesterone, 16–17 commencement of therapy, 204
receptors to, 15 subgroup analysis, 203
safety, 19 PROMISSE study, 219
trophoblast invasion, 16 Prophylactic cerclage, 121
Preimplantation Genetic Diagnosis International Society Prophylactic cervical cerclage, 111–112
(PGDIS), 245 Prostacycline, 73
Preimplantation genetic screening (PGS), 26, 231, 243 Prostaglandin E2, 207, 209
accuracy and precision of, 244–246 Protamines, 127
history of, 243–244 Protein disulfide isomerase/thioredoxin (PDI-T), 15
hypothesis of, 243 Prothrombin (PT), 72
second-generation, 244 Prothrombotic mechanisms
Preimplantation genetic testing for aneuploidy (PGT-A), 243 cytokines, 85
general considerations, 239–240 fetal thrombophilia, 86
history of, 243–244 hormones and thrombosis, 85–86
for recurrent pregnancy loss, 240, 246 microparticles, 85
Preimplantation genetic testing, for monogenic disorders SNPs, 86
(PGT-M), 32 Psoriasis, 102
Preimplantation genetic testing for structural Psychological support, threatened miscarriage, 150
rearrangement (PGT-SR), 39 PT, see Prothrombin
Prenasal thickness (PT), 166 PTB, see Preterm birth
Prenatal chromosomal microarray, 168 Public health policies, 169
Preterm birth (PTB), 154, 158, 159, 160, 174 PULs, see Pregnancies of unknown location
Preterm deliveries, 174
Preterm premature rupture of membranes (PPROM), 172 Q
Primary APS (PAPS), 70
Primary cfDNA screening, 166–167 qPCR, see Quantitative polymerase chain reaction
PRISM trial, 149, 204 Quad test, 165
Products of conception (POC), 102, 239 Quantitative polymerase chain reaction (qPCR), 240
Index 297
R risk factors, 8
secondary, 4–5
RA, see Rheumatoid arthritis serum, 15
Randomized controlled trials (RCTs), 22, 23, 60, 235 spontaneous preterm labor and, 172–175
RCOG, see Royal College of Obstetricians and subgroups of, 4–5, 7–8
Gynaecologists superfertility in, 45
Receptive uterine environment, 15–16 surrogacy for, 254–255
Recurrent aneuploidy tertiary, 5
biological basis of, 35 Recurrent second trimester fetal deaths, 193–194
clinical management of, 36 Recurrent spontaneous miscarriage, 257
with higher-order losses, 35–36 Red deer (Cervus elaphus), 14
Recurrent miscarriage (RM), 35–36, 189, 202 Relaxin, 207–208
defintion, 1 REMIS trial, see Recurrent Miscarriage Study trial
preeclampsia and, 175 Replacement therapy, 81
prevalence of, 2 Reproduction, receptive uterine environment for, 15–16
progestogens and, 198, 200 Reproductive failure, 101
theoretical vs. empirical risks for, 38 cellular analysis, 105–106
Recurrent Miscarriage Immunotherapy Trialists Group determination of diagnosis, 107
(RMITG) trial, 48, 180, 191 immunological, 102
Recurrent Miscarriage Study (REMIS) trial, 262–263, maternal and paternal genetic testing, 104–105
264, 267 personal and family history, 104
Recurrent pregnancy loss (RPL), 1, 22, 169 soluble factors, 106–107
antibodies, 8 workup of, 103
in aspirin, 228 Reproductive immunology, 108
cause of, 197 Rescue cerclage, 159, 160
class II HLA alleles, 8–9 Resistant patient, 192–193
clinical associations, 6, 9 Retroplacental hematoma, 139, 147, 148
couples, choices for individual, 169–170 Rheumatoid arthritis (RA), 103
endocrinology of, 59–67 Ribosomal DNA, 37
endometrial factor in, 43–46 Ring pessary, 160
epidemiologic parameters, 2–6 RM, see Recurrent miscarriage
familial aggregation, 5–6, 9 RMITG trial, see Recurrent Miscarriage Immunotherapy
fetal structural malformations, 48–57 Trialists Group (RMITG) trial
heparin in, 227 Roseburia intestinalis, 71
as homogeneous condition, 184 Royal College of Obstetricians and Gynaecologists
human chorionic gonadotropin, 206–211, 210 (RCOG), 30, 37, 57, 138, 184, 227
inadequate decidual responses, 44 guideline, 185–186
incidence of, 2, 10 protocol, 187
intravenous immunoglobulin (IVIg) therapy, RPL, see Recurrent pregnancy loss
268–272
investigation protocol, 184–194
S
lifestyle factors, 6, 9–10
maternal age, 4, 7 SAB testing, 108
maternal causes of, 250 SAPS, see Secondary antiphospholipid syndrome
miscarriage risk in, 10 SART, see Society for Assisted Reproductive Technology
NK cell, 8 SCCA, see Sperm chromatin condensation assay
obesity and, 175, 177 SCD, see Sperm chromatin dispersion assay
obsteric outcomes after, see Obsteric outcomes Screening, 164
occurrence, 2, 7 amniocentesis, 167–169
partner specificity, 6, 9 aneuploidy, 169
perinatal mortality, 181 antenatal, 164
placental abruption, 180–181 cfDNA, 166–167
preeclampsia and, 175–177 for chromosomal abnormalities, 164, 169–170
pregnancy complications, 181 modalities, 165–166
pregnancy-induced hypertension, 175–177 public health policies, 169
pregnancy losses in, 1 routine, 164–165
preimplantation genetic testing for aneuploidies Secondary antiphospholipid syndrome (SAPS), 70, 73
for, 240 Secondary cfDNA screening, 167
prevalence of, 2 Secondary yolk sac (SYS), 139–140
previous miscarriages, 2–4, 7 Second-generation PGS, 244
primary, 4–5 Segmented filamentous bacteria (SFB), 71
298 Index
Semen parameters, 127 Structural abnormalities, in first trimester scan, 141–143

Semipermeable zona pellucida, 13 gastroschisis, 141, 142
Septate uterus, 119, 120 hydropic fetus, 142, 143
Septum, 120, 191 malformations, 141
Serum- and glucocorticoid-inducible kinase 1 (SGK1), 44 Mickey Mouse sign, 141, 142
Serum markers, threatened miscarriages, 146 Subchorionic hematoma, 139, 147–148
Serum progesterone levels, 60 late obstetric complications, 147–148
17β -estradiol, 209 progestogens in, 149
Sex chromosomal polysomy, 35 Submucous myomas, 114, 117
SFB, see Segmented filamentous bacteria Subseptate uterus, 111
SGA, see Small for gestational age Subsequent live birth rate, 231–232
SGK1, see Serum- and glucocorticoid-inducible kinase 1 Substitution therapy, 81
Shirodkar cerclage, 155–156 Superfertility, in recurrent pregnancy loss, 45
Single-nucleotide polymorphism (SNP), 86, 143 Superficial venous thrombosis, 215
SLE, see Systemic lupus erythematosus Surrogacy, 249, 253
Small amniotic sac syndrome, 249–250 altruistic, 253
Small for gestational age (SGA), 84, 177 classification of, 253
SMFM, see Society for Maternal Fetal Medicine gestational carrier, 253
SNP, see Single-nucleotide polymorphism guidelines for, 254
Society for Assisted Reproductive Technology (SART), 246 indications, 253
Society for Maternal Fetal Medicine (SMFM), 30, 143 issues with, 253–254
Soft markers, 166 for recurrent pregnancy loss, 254–255
Sonohysterography, uterine anomalies, 116, 117 traditional, 253
SORBS1 expression, 16 types of, 253
SORBS2 expression, 16 Sydney criteria, 185
Specific trophoblastic molecules, 96 Syncytial knots, 75
Sperm, 96 Syncytiotrophoblasts, 207
aneuploidy, 129 Syndactyly, 53
chromatin, 127 SYS, see Secondary yolk sac
quality, 233 Systemic lupus erythematosus (SLE), 73, 102, 103, 107
selection techniques, 233
Spermatogenesis, 251 T
Spermatozoa, 235
Sperm chromatin condensation assay (SCCA), 130 TAFI, see Thrombin-activatable fibrinolysis inhibitor
Sperm chromatin dispersion assay (SCD), 130 TAI, see Thyroid autoantibodies
Sperm DNA fragmentation, 130 TE mosaicism, 244, 245
Sperm DNA quality, 129–130 Tetraploidy, 34
Sperm donation, 249 TFPI, see Tissue factor pathway inhibitor
indication, 251 Th1 abortogenic responses, 91–92
role of, 251 autoantibodies, 92
Sperm FISH assays, 129 infections, 91–92
Spina bifida, 52 maternal genes, 92
Spontaneous abortions/miscarriage, 226, 239, 249, 250 stress, 91
cell-free DNA analysis, 31 Th1 cytokines, 18, 59, 90, 202, 209, 268, 269
chromosomal abnormalities, 31–36 Th2 cytokines, 18, 27, 59, 90, 202, 209
chromosomal inversions, 39–40 Th17 cytokines, 94
cytogenomic testing for, 30–31 Th17 inflammatory cells, 93–94
genetic factors, 30 Third-party leucocytes, 257
laboratory methods in evaluating, 30–31 Third party reproduction (TPR), 249
recurrent aneuploidy, 35–36 classification, 249
single-gene causes of, 40 embryo donation, 249, 252
structural chromosomal rearrangements, 37–40 embryonic causes, 249–250
translocation heterozygotes, 37–39 oocyte donation, 249, 251–252
Spontaneous preterm birth (sPTB), 154, 172–175 parental causes, 250–251
Sporadic miscarriages, 7 sperm donation, 249, 251
sPTB, see Spontaneous preterm birth surrogacy, 249, 253–255
Small for gestational age (SGA), 177, 178 Threatened miscarriage, 139
Stillbirth Collaborative Research Network’s definition, 145
multicenter, 216 human chorionic gonadotropin, 146, 209–210
Strassman metroplasty, 121 late obstetric complications, 147
Stroke, 215 natural history, 145
Index 299
progesterone-induced blocking factor, 147 Transvaginal cerclage, 155

prognostic factors, 146–147 Transvaginal sonography (TVS), 115, 134, 138
psychological support, 150 Transvaginal sonohysterography (SHG), 116
safety and side effects, 149–150 Transvaginal ultrasonography, 153, 159
serum markers, 146 Treg cells, 92–93, 107, 108, 269
subchorionic hematoma, 147–148 decrease of, 94
treatment, 148–150 Th17 imbalance, 94
ultrasound, 146 in women, 93–94
3D ultrasound, 115 Treg-Th17 balance, 93
Thrombin, 81 TRH, see Thyroid-releasing hormone
Thrombin-activatable fibrinolysis inhibitor (TAFI), 79 Tricuspid regurgitation (TR), 165, 166
Thrombogenesis, 72 Triploidy, 34
Thrombophilias, 81 Trisomies, cell-free DNA analysis to detect, 31
anticoagulants in, 223–225 TRL5, 16
antiplatelet agents, 227–228 Trophectoderm biopsy (TEB), 244–246
cohort studies, 83–84 Trophoblast
hereditary, 81, 223–226 debris, 74
inherited, 225, 226 immune responses to, 280
in late obstetric complications, 83 invasion, 16, 74
patients without, 226–228 Trunk defects, 52
in pregnancy loss, 82–83 TSH, 63, 64, 107, 206
thrombosis, 82 T-shaped uterus, 113–114
treatment options, 84 Tumor necrosis factor (TNF) α production, 223
Thromboprophylaxis, 225 TUNEL assay, 130
Thrombosis Turner syndrome, 166, 167, 251, 252
in decidual vessels, 82 TVS, see Transvaginal sonography
hormones and, 85–86 22q11.2 deletion syndrome, 168
Thyroid abnormalities Twin pregnancy
hyperthyroidism, 62 cerclage in, 159–160
hypothyroidism, 62–63 pessary in, 161
thyroid autoimmunity, 63 2D sonography, 117
Thyroid autoantibodies (TAI), 63, 107
Thyroid peroxidase antibodies (TPOAb), 62, 63 U
Thyroid-releasing hormone (TRH), 62
Thyroxine, 63 UK’s National Institute for Health and Clinical
TIA, see Transient ischemic attack Excellence, 25
Time-lapse system embryoscopy, 234 Ultrasonography, 117
Time-lapse systems (TLS), 234 Ultrasound, 192
TIMP, see Tissue inhibitor of metalloproteinase to detect anomalies, 48
Tissue factor pathway inhibitor (TFPI) antibodies, 72, 86 as diagnostic modality, 55
Tissue inhibitor of metalloproteinase (TIMP), 16 early first trimester screening, 56
Tissue plasminogen activator (tPA), 79 nuchal translucency screening, 55
TLRVYK sequence, 71 soft signs, 55–56
TLS, see Time-lapse systems threatened miscarriage, 146
T lymphocytes, 27, 46 uterine anomalies, 115
TNF-related apoptosis-inducing ligand (TRAIL), 96 Ultrasound follow-up, 134
Toll-like receptor 4 (TLR4), 74 aneuploidy screening (11 to 14 weeks), 140–141
TORCH IgM, 70 early anomaly scan (10 to 14 weeks), 136–138
tPA, see Tissue plasminogen activator in first trimester, 134–138
TPOAb, see Thyroid peroxidase antibodies 4 to 5 weeks, 134–135
TPR, see Third party reproduction genetics, 143
TR, see Tricuspid regurgitation nonviable pregnancy, 138–139
Traditional surrogacy, 253 risk of early pregnancy failure, 139–141
TRAIL, see TNF-related apoptosis-inducing ligand structural abnormalities, 141–143
Transducers, 134 subchorionic hematoma, 139
Transient ischemic attack (TIA), 215 threatened miscarriage, 139
Translocation heterozygotes, 37 week 6, 135
abnormal offspring, 38 week 8, 135
balanced translocation, 37–38 weeks 7 to 9, 135
frequency management of, 37–39 Umbilical cord defects, 54
live births in, 39 Unconjugated estriol (uE3), 165
300 Index
Unexplained recurrent spontaneous abortions (URSA), 89 Uterine morphology, 117

Unicornuate uterus, 110, 111–112 Uterine natural killer (uNK) cells, 45–46, 103, 281, 282
United States Collaborative Perinatal Project, 174 Uterine septum, pregnancy outcome, 119–120
United States Food and Drug Administration (FDA), 22, Uteroplacental arteries, 82
261–262 Uterus didelphis, 112–113
URSA, see Unexplained recurrent spontaneous abortions
Uterine actions, human chorionic gonadotropin, 209 V
Uterine agenesis, 110
Uterine anomalies, 110 Vaginal bleeding, 139, 145
arcuate uterus, 111 Vaginal micronized progesterone, 158, 159, 162
bicornuate uterus, 113 Vaginal progesterone, 149
biomarkers, 26 Vasculosyncytial membranes, 75
cervical cerclage, 121 VD deficiency (VDD), 250
dysmorphic uterus, 113–114 VEGF, 74
hysterosalpingography, 116 Venous thromboembolism (VTE), 223, 225, 226
hysteroscopy, 116 Vertical fusion, 110
imaging modalities, 117 Vitamin D, 107, 250
imaging uterine morphology, 117 VTE, see Venous thromboembolism
intrauterine adhesions, 114–115
intrauterine polyps, 118 W
laparoscopy, 116–117
magnetic resonance imaging, 116 WES, see Whole exome sequencing
malformations, 122 WHO, see World Health Organization
Müllerian tract defects, 110–115 Whole exome sequencing (WES), 55, 57
myomas, 114 Window of implantation (WOI), 44–45, 235–236
polyps, 114 WOI, see Window of implantation
sonohysterography, 116 Wolffian ducts, 110
Strassman metroplasty, 121 World Health Organization (WHO), 1, 24
subseptate uterus, 111
surgical intervention, 118 Y
treatment, 118–122
T-shaped uterus, 113–114 Y chromosome, 128
ultrasound, 115 Yolk sac, 134, 139–140
unicornuate uterus, 111–112
uterus didelphis, 112–113 Z
Uterine artery embolization, 121
Uterine fibroid embolization, 121 Zona pellucida, 15
Uterine malformations, 192 Zygote, 14, 15, 19, 126

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Part I Basic Principles

3. Recurrent Pregnancy Loss­from Evidence-Based to Personalized Medicine........................... 22

Part II Etiology

5. The Endometrial Factor in Recurrent Pregnancy Loss.............................................................. 43

6. Fetal Structural Malformations and Recurrent Pregnancy Loss.............................................. 48

7. The Endocrinology of Recurrent Pregnancy Loss....................................................................... 59

8. The Etiology of the Antiphospholipid Syndrome......................................................................... 70

9. Defects in Coagulation Factors Leading to Recurrent Pregnancy Loss................................... 79

10. The Immunobiology of Recurrent Miscarriage........................................................................... 89

11. Immune Testing in Recurrent Pregnancy Loss..........................................................................101

12. Uterine Anomalies and Recurrent Pregnancy Loss...................................................................110

13. The Male Factor in Recurrent Pregnancy Loss......................................................................... 126

Part III The Developing Pregnancy

15. Threatened Miscarriage and Recurrent Pregnancy Loss.........................................................145

16. The Role of Cerclage and Pessaries..............................................................................................153

17. What Genetic Screening Is Appropriate in Recurrent Pregnancy Loss?............................... 164

18. Obstetric Outcomes after Recurrent Pregnancy Loss...............................................................172

Part IV Management

22. Human Chorionic Gonadotropin Supplementation in Recurrent Pregnancy Loss............... 206

23. Antiphospholipid Syndrome: Management of the Obstetric Patient........................................215

24. Can Recurrent Pregnancy Loss Be Prevented by Antithrombotic Agents?........................... 223

28. Third Party Reproduction in Recurrent Pregnancy Loss........................................................ 249

30. IVIg Treatment for Recurrent Pregnancy Loss........................................................................ 268

31. The Role of Filgrastim.................................................................................................................. 275

32. Opinion: Immunotherapy Has No Place in the Treatment of Recurrent

Howard J.A. Carp, MB BS, FRCOG

Nasser Al-Asmar D. Ware Branch

Gautam Nand Allahbadia Jeffrey Braverman

Salim Daya Akanksha Allahbadia Gupta

Ashok Kumar Rubina Merchant

Dolores J. Lamb Pere Mir

C.V. Rao Carlos Simon

Lorena Rodrigo Joe Leigh Simpson

Yehuda Shoenfeld Akhila Vasudeva

Epidemiologic Parameters Relevant for RPL

Number of Previous Miscarriages

3 misc. 4 misc. 5+ misc.

Not pregnant Miscarriages Births

Integration of Epidemiologic Knowledge in Research and Management of RPL

Class II HLA Alleles

Why Is There a Delay between Fertilization and Implantation?

Embryo-Specific Maternal Communication—PIF

PIF Autocrine, Autotrophic, and Protective Actions

Receptive Uterine Environment Is Critical for Reproduction

Implantation Is Insufficient: Effective Trophoblast Invasion Is Also Required

PIF Interaction and Synergy with Progesterone and HLA-G

PIF Interaction and Effect on NK and Dendritic Cells in RPL

Embryo-Maternal Communication through the Immune System

Howard J.A. Carp

Reducing Selection Bias

Articulating the Research Question

Drawbacks of Personalized Medicine

Biomarkers in Recurrent Pregnancy Loss

Pregestational Testing for Aneuploidy (PGT-A)

Aspirin Control Weight OR

3. Recurrent Pregnancy Lossfrom Evidence-Based to Personalized Medicine........................... 22