Copyright 0 Munksgaard, 1996

AJRI 1996: 35:352-359 American Journal of Reproductive Immunology

Printed in the United States of America - all rights reserved. ISSN 8755-8920

Immunotherapy for Recurrent Pregnancy Loss:

Analysis of Results From Clinical Trials
CAROLYN
B. COULAM,
MARYSTEPHENSON,
J. JAROS
STERN,
AND DAVID
A. CLARK

Coulam CB, Stephenson M , Stern JJ, Clark DA. Immunotherapy for recurrent pregnancy Key words:
loss: Analysis of results from clinical trials. AJRI 1996; 35:352-359 0 Munksgaard, Recurrent spontaneous abortion,
Copenhagen immunotherapy, leukocyte
PROBLEM: Up to 80% of unexplained recurrent spontaneous abortions (RSA) are thought immunization, intravenous
to have an immunologic mechanism. Yet clinical trials using immunotherapy to treat women immunoglobulin, chromosomal
experiencing RSA have low treatment effects. The present study was undertaken to explain abnormalities
the low treatment effects.
METHODS: Results of clinical trials using allogeneic leukocyte immunization and intrave- CAROLYN B . COULAM
nous (IV) immunoglobulin (Ig) are compared. The mechanisms of pregnancy loss are re- Genetics and IVF Institute,
viewed in light of data on frequency of karyotype abnormalities in trophoblast of failing Fairfax, Virginia
pregnancies.
RESULTS: Results of two independent analyses using allogeneic leukocyte immunization MARY STEPHENSON
as immunotherapy for all women with RSA revealed live birth ratios of 1.16 (P = 0.03) and Recurrent Pregnancy Loss
1.21 ( P = 0.02). When the analysis was limited to primary aborters, the live birth ratio in- Program, Women’s Health Centre,
creased to 1.46 ( P = 0.006). Live birth ratio after immunotherapy for all RSA using IVIg University of British Columbia,
was 1.88 (P = 0.04). Because of low treatment effects, confounders to treatment success of Vancouver, B.C., Canada
maternal age and number of previous abortions were studied. Chromosomal abnormalities
have been identified in 55% of concepti from RSA. The frequency of chromosomal abnor- J. JAROS STERN
malities remained constant for up to six pregnancy losses. Women with a history of pri- Grupo De Reproduction y Genetica
mary compared to secondary RSA had a higher frequency of karyotypically abnormal AGN y Associados Hospital
concepti (x2
= 4.54, P c 0.05). Risk factors for RSA also include number of previous losses. Angeles Del Pedregal, Mexico,
CONCLUSION: Chromosomal abnormalities are a significant confounder when evaluating DF. Mexico
efficacy of immunotherapy for treatment of RSA. Some women with RSA have a high risk
of recurrent chromosomal problems. DAVID A. CLARK
Departments of Medicine,
Pathology, Obstetrics and
Gynecology, McMaster University,
Hamilton, Ontario, Canada

Presented at the Sixth International

Congress of Reproductive
INTRODUCTION Immunology, Washington, DC,
July 19-23, 1995.
The prenatal period holds by far the greatest risk to human life. At least 60% of
human embryos die before birth.’ The majority of deaths occur pre- or peri-implan- Address reprint requests to
tation’ and the remaining 15% occur po~timplantation~ after clinical recognition of Carolyn B. Coulam, M.D., Director
pregnancy. The losses result in substantial physical and emotional pain as well as of Reproductive Immunology,
medical expense. Particularly frustrating for patients and health care providers is Genetics and IVF Institute,
Fairfax, VA 2203 1.
the problem of recurrent pregnancy loss. As many as 5% of all couples conceiving
experience two consecutive spontaneous abortions and 2% have three or more
Submitted August 9, 1995;
10sses.~Effective treatment is needed for these individuals. Before effective treat- accepted August 25, 1995.
ment can be instituted, the cause of pregnancy loss must be determined. Little in-
formation on causes of death in utero is available. Chromosomal abnormalities have
Q MUNKSGAARD, COPENHAGEN
 IMMUNOTHERAPY FOR RECURRENT PREGNANCY LOSS /
been found in the majority of sporadic (non-recurrent)
abort use^,^ but literature is developing which suggests a
role of the immune system in the success of pregnancy.
In unsuccessful pregnancies manifest as recurrent sponta-
neous abortion, 50-80% of couples have been thought to
have evidence of an immunologic cause.*
Various forms of immunotherapy have been introduced
to treat couples suffering from recurrent unexplained abor-
tion~.'"~ However, the treatment effect of immunotherapy
in couples with unexplained recurrent spontaneous abor-
tion has been disappointingly low.'o The low treatment ef-
fect of immunotherapy could result from a treatment of low
efficacy for a widely prevalent problem in these individu-
als or from a highly effective treatment benefitting a small
subset of recurrent aborters. To determine which of these
possibilities is the most likely explanation and thus to pro-
pose ways in which treatment effects of immunotherapy
can be improved, the current study was undertaken. In this
study, the results of randomized clinical trials using im-
munization with allogeneic leukocytes and intravenous
(IV) immunoglobulin (Ig) are compared, and the mecha-
nisms of pregnancy loss are reviewed. Based upon under-
standing of mechanisms of pregnancy loss obtained from
these studies, methods to identify individuals most likely
to respond to immunotherapy, thus improving treatment
effects, is proposed.
RESULTS OF RANDOMIZED CLINICAL
TRIALS
Randomized clinical trials using immunotherapy in the
forms of immunization with allogeneic leukocytes'@14and
IVIg'53'6have been performed.
Allogeneic Leukocyte Immunotherapy for Recurrent
Spontaneous Abortion
Clinical trials evaluating allogeneic leukocyte immuniza-
tion have both and ~ h a l l e n g e d ' ~the
- ' ~ effi-
cacy method of immunotherapy for treatment of recurrent
TABLE I. Summary of Results of Clinical Trials Using lmmunotherapy for Treatment of Recurrent
Spontaneous Abortion
Treatment
Analysis (ref)
Patients (N)
Auto Ab in some patients
Antipaternal Ab in some patients
Prior LB in some patients
Relative treatment effect in all patients
Absolute increase in % LB in all patients
Number needed to treat for one additional LB
WBC = white blood count, IVlg = intravenous immunoglobulin, Ab = antibodies, LB = live birth.
353
spontaneous abortion. To address the uncertainties caused
by these conflicting results, a worldwide collaborative ob-
servational study and meta-analysis was performed." Fif-
teen collaborating centers participated in the study. Nine
randomized trials (seven double-blinded) were evaluated
independently by two data analysis teams to assure con-
clusions were robust. Although the independent analyses
used different definitions and statistical methods, the re-
sults were similar (Table I). The percent live birth ratios
(ratio of live births in treatment and control groups) with
95% confidence intervals were 1.16 (range 1.01-1.34; P
= 0.03) and 1.21 (range 1.04-1.37; P = 0.02). The abso-
lute differences in live birth rates between treatment and
control groups were 8% and 10%. These results were based
on a data set that included couples in whom the female
partner had autoimmune abnormalities (positive ANA,
positive anticardiolipin antibody) where treatment reduced
the probability of a live birth or pre-existing anti-paternal
leukocyte antibodies or one prior successful pregnancy
with their partner where the probability of success with-
out treatment was high." A logistic regression analysis in-
dicated negative prognostic variables of abnormal
autoimmunity and increasing numbers of losses with part-
ner and a positive prognostic variable of pretreatment pa-
ternal antibody. A supplementary analysis of primary
recurrent aborters (no prior live births), excluding those
with autoimmunity and pre-existing anti-paternal antibody
has been published subsequently." By selecting patients
most likely to benefit from immunotherapy, the outcome
may be improved by at least 50% (Table I, Analysis #3).
When the analysis was restricted to patients who became
pregnant after intervention, similar results were ~btained.'~
Intravenous Immunoglobulin Immunotherapy for
Recurrent Spontaneous Abortion
An alternative to leukocyte immunotherapy is intravenous
human immunoglobulin.15~'6Provided there is adequate
quality control, commercially available IVIg should be vi-
rus-free,18 decreasing the risk of viral transmission of dis-
WBC Immunization IVlg
#I (10) #2 (10) #3 (17) (16)
430 449 285 95
Yes Yes No No
Yes Yes No Yes
Yes Yes No Yes
1.16 1.21 1.46 1.82
8 10 16 28
13 10 6 4
AMERICAN JOURNAL OF REPRODUCTIVEIMMUNOLOGY VOL. 35,1996
354 / COULAM ET AL.
ease compared to leukocyte immunization.” Two random-
ized controlled trials of IVIg for treatment of recurrent
spontaneous abortion have been p ~ b l i s h e d . ” ~A’ ~Euro-
pean-based study showed a positive trend but did not
achieve statistical significance due to too few patients
for adequate statistical power given the magnitude of
the effect.’’ However, a second US-based trial did show
a significant benefit.I6 This prospective, randomized,
placebo-controlled trial showed the difference in live
birth rates between women receiving IVIg (62%) and pla-
cebo (34%) ( P = 0.04).16 As the US-based trial did not
have larger numbers than the European-based trial, the
positive result was due to an effect of greater magnitude.
Based upon the magnitude of effect in the US-based trial,
one needs to treat four women to achieve one additional
live birth, making IVIg therapy more effective than leu-
kocyte immunization in the treatment of recurrent sponta-
neous abortion (Table I). The greater magnitude of effect
in the US-based study compared to the European-based
trial could have arisen by chance or by use of a different
study design. One of the potentially significant differences
in study design between the two trials was timely initia-
tion of treatment. Patients began IVIg treatment prior to
conception in the US-based trial16 but after implantation
in the European-based trial.” Studies using other forms
of immunotherapy have shown a significant increase in
live birth rates when treatment was begun preconceptually
compared to postconceptually.” Another study using IVIg
to treat 11 women who had 4-8 unexplained pregnancy
losses reported a live birth rate of 85%.2’
MECHANISMS OF PREGNANCY LOSS
Early pregnancy loss can result from abnormalities within
the conceptus or the endometrium. It has been recognized
for some time that over 50% of early spontaneous abor-
tions are cytogenetically abnormal’; most of these data
TABLE II. Frequency of Abnormal Karyotypes Among Concepti From Women Experiencing Primary and Secondary
Recurrent Spontaneous Abortion Compared With Number of Prior Abortions
Number Prior Primary RSA
Abortions N (%) Abn
0-1 123 (61)
3 78 (67)
4 55 (53)
5 31 (29)
6 11 (55)
Total (3-6) 175
“Primary versus secondary RSA P < 0.05 (x’ = 4.54).
bSex ratio: xxlxy = 1.13.
CSuccessof karyotyping trophoblast 2 94%.
0 MUNKSGAARD, COPENHAGEN
have been obtained from women with I 2 abortions. Un-
til recently the extent to which chromosomal abnormali-
ties contributed to recurrent abortion was thought to be
relatively low.” Recently data have indicated that as many
as 60% of all abortuses from women experiencing recur-
rent spontaneous abortion may be chromosomally abnor-
mal.23 To further evaluate the frequency of abnormal
concepti among RSAs data were collected from three cen-
ters (Genetics and IVF Institute, USA; University of Brit-
ish Columbia, Vancouver, BC, Canada; Grupo De
Reproduction y Genetica AGN, Mexico City, Mexico) and
analyzed. The results are shown in Table 11. A total of 209
concepti from women with a history of RSA were in-
cluded: 175 from primary RSA and 34 from secondary
RSA. Fifty-five percent of women with primary RSA and
35% of women with secondary RSA have chromosomal-
ly abnormal embryonic trophoblasts. The difference in the
frequency of abnormal karyotypes from concepti of pri-
mary and secondary RSA is significant (x2 = 4.54, P <
0.05). That secondary recurrent aborters have a lower per-
centage of chromosomally abnormal concepti may account
for a higher success rate in women with one prior live birth
observed in the meta-analysis of immunotherapy using
husband leukocyte immunization for treatment of RSA.’’
Pregnancies with chromosomal abnormalities that abort
would not be expected to be rescued by immunotherapy.
The frequency of chromosomal abnormalities among re-
current spontaneous abortions could represent a significant
confounding variable in interpreting results from clinical
trials designed to test the efficacy of various forms of im-
munotherapy.
Data are accumulating to support an immunologic
mechanism contributing to the loss of chromosomally nor-
mal concepti that represent 45% of women with primary
recurrent miscarriage. The most extensive data has been
accumulated using genetically defined mice where several
models of partner-specific abortion were As
Abnormal Karyotypesb
Secondary RSA Combined
N (%) Abn N (%) Abn
9 (56) 132 (61)
7 (0) 85 (61)
8 (63) 63 (54)
14 (36) 45 (311
5 (40) 16 (50)
34 (35Ia 209 (52)
 IMMUNOTHERAPY FOR RECURRENT PREGNANCY LOSS /
in the human, it was confirmed that trophoblast cells are
resistant to transplantation immunity and deliberate immu-
nization against paternal antigens increased the probabil-
ity of success of pregnancy and not the probability of
fail~re.~’”~ The basis for pregnancy failure was found to
be the natural killer (NK) cell which recognized primitive
embryonic and tumor-like cells. NK cells cannot kill tro-
phoblast in vitro, but NK cells activated by cytokines such
as IL-2 may kill trophoblast as well as NK-resistant tu-
mor cells.26s28 Depletion of NK cells in vivo prevents abor-
tions in mice, whereas NK cell activators such as poly I
polyC- and y-interferon cause abortion^.^^-^^ Macrophages
have been proposed to play a role by interacting with NK
cells.28Cytokines facilitating this interaction, gamma in-
terferon and TNF-a, were also found to enhance the abor-
tion process, and antagonists of TNF-a prevented
abortions.28230 The fetus, removed from its trophoblas-
tic cocoon, acts as a typical allograft, but when within
is protected from classical transplant rejection type
i m m ~ n i t y ~ factors
’ . ~ ~ ; affecting trophoblast integrity play
a key role in determining the survival of the entire feto-
placental unit. Further studies have defined mechanisms
which may spontaneously activate the NK cell macroph-
age system, mechanisms involved in immunoprotection of
the implanted conceptus, and have confirmed homologies
between the findings in mice and in women.
In humans, there is evidence that CD8+ T cells may rec-
ognize trophoblast. These T cells carry the $-type recep-
tor rather than a0 T-cell receptor that requires HLA A, B,
C for efficient recognition and binding.33In normal suc-
cessful pregnancy or after T cell activation as occurs with
immunization, CD8+ cells are activated, express proges-
terone receptors, and in response to the hormone, secrete
a factor that inhibits activation of NK ~ e l l s . Other ~ ~ - ~ ~ ing a trisomic chromosomal abnormality is 67%.44Thus,
cytokines may also be produced by activated CD8+ T cells,
and it is hypothesized that some of these may enhance an-
tibody responses (IL-4), inhibit macrophages (IL- lo), and
facilitate activation of bone marrow-derived TGF-0-pro-
ducing suppressor cells (IL-3, GM-CSF) found in de-
~ i d u a . Women
~ ~ . ~ with~ recurrent miscarriage show a
deficient activation of their CD8+ cells in early preg-
nancy4’ and a relative loss of CD56+ and CD16- cells with
an excess of CD56+ CD16+ cells.41CD56+ CD16+ cells
represent conventional NK cells that are particularly ef-
fective in lysing trophoblastic target cells when activated
by IL-2.28An infiltrate of CD56+ CD16+ NK cells has
been noted in placental bed biopsies of incipiently abort-
ing patients with a history of recurrent spontaneous abor-
t i ~ n . Quantitation
~’ of CD56+ NK cells in peripheral blood
of patients with recurrent spontaneous abortion with a fail-
ing pregnancy has shown a significant elevation associated
with spontaneous abortion of a conceptus of normal karyo-
type, and a normal level associated with loss of embryos
that are karyotypically abn~rmal.~’ Further, high blood NK
levels in nonpregnant women have been found to predict
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 35,1996
355
abortive pregnan~ies.~~ Thus NK cells and failure to sup-
press NK cell activation plays an important role in immu-
nologically preventable spontaneous abortions. CD8+ cells
are required for immune protection against NK cell depen-
dent abortion in mice; these cells may carry UpT-cell re-
ceptor, however, rather than yGT-cell receptor (see Clark
et al., this issue).
IDENTIFICATION OF INDIVIDUALS LIKELY
TO RESPOND TO IMMUNOTHERAPY
Understanding the mechanisms involved in recurrent spon-
taneous abortion allows a more focused approach to treat-
ment. Since 55% of abortuses from women experiencing
previous recurrent spontaneous abortion are chromosomal-
ly abnormal (Table 11), differentiation of the 55% from the
remaining 45% is required. Table I11 outlines a model to
predict the risk of aborting a chromosomally abnormal
conceptus in subsequent pregnancies after three con-
secutive spontaneous abortions. Analysis of the model
suggests an increase in the percentage of chromosomal
abnormalities in abortuses as the number of pregnancies
increases. The majority of the abnormalities were tri-
somies (data not shown). Chromosomally abnormal
losses do not repeat 100% of the time, but rather the
overall risk of a repeated chromosomal abnormality is
35% after three losses and rises to 70% after six losses.
The high risk of recurrence of a chromosomal abnor-
mality as basis for nonimmunologically modifiable loss
suggested by this model is in agreement with data of
W a r b ~ r t o n If
.~~a woman aborts a karyotypically tri-
somic conceptus and subsequently conceives and aborts
a second pregnancy, the risk of the second abortus hav-
it appears that a subset of woman experiencing RSA are
at increased risk of recurrent chromosomal nondisjunc-
tion. Sensitive and specific markers for differentiating
these women from those likely to benefit from immu-
notherapy are needed.
Risk factors for chromosomal abnormalities in concepti
include maternal age43*4547 and history of previous tri-
The association of advancing maternal age
with increasing odds for embryonic trisomy has been well-
established for spontaneous abortions.46947 A number of
studies have reported advancing maternal age as a risk fac-
tor for RSA4547and several have found it not to be a risk
When multiple logistic analysis was used to
compare relative importance of maternal age of obstetric
history for prediction of embryonic karyotype, maternal
age was the only significant predictor of chromosomal ab-
n~rmality.~’ In this study, a history of previous live births
and number of previous miscarriages had no significant
relationship to the frequency of chromosomal errors in sub-
sequent miscarriages when maternal age was taken into
ac~ount.~’
356 I COULAM ET AL.

TABLE Ill. Model for Predicting Outcome of Pregnancies Among 1,000 Women With Primary RSA Who Have Three
Prior Unexplained Pregnancy Lossesa
Expected outcome Nature of failures Risk of a recurrent
Pregnancy Success Fail “Immunologically “C hromosomally chromosome
Number N N modifiable” (45%)b abnormal” (55%) abnormality
4 600 400 180 220 0.35
(43Y
5 172 228 103 125 0.4gd
(28)c
6 64 164 74 90 0.64
(16)c
7 36 128 58 70 0.70
“Theprobability of successful pregnancy is based upon previously published data.7
bBased on assumption that only chromosomally normal embryos can be “saved’ by immunotherapy, this is the maximum number of
modifables. As some represent karyotypically normal embryos from those who had prior loss of a chromosomally abnormal em-
bryo, some may succeed without any immunologic intervention.
‘Expected success rate without treatment based on meta-analysis data. The result is shown for the next pregnancy for those who
have failed the previous time.
dabnormal karyotypes - (10% genetic accidents) calculated from
chromosomal abnormalities
i.e. 125 - 18 - 107
220 - 220

The risk for spontaneous abortion in a subsequent preg-
nancy is increased when a normal embryonic karyotype is
found in abortus materia1.s’44’s2-54
When an embryonic chro-
mosomal abnormality was diagnosed, the risk of subse-
quent spontaneous abortion was 15-17%s,52compared with
23% when the embryonic karyotype was n ~ r m a l . ~Most .~‘
importantly, the subsequent losses after loss of karyotypi-
cally normal conceptus had a high probability of also be-
ing chromosomally n 0 r m a 1 . ~ Thus,
~ ~ ~ with
~ ~ ~a -history
~ ~ of
recurrent spontaneous abortion, demonstration or normal
embryonic karyotype may be the most important indica-
tion for diagnostic evaluation and possible treatment of
women experiencing recurrent spontaneous abortion. At
the present time, the only way to identify such women is
to have results of chromosome analysis of previous preg-
nancy losses available. However, recent data suggests it
may be possible to identify recurrent aborters at risk for
loosing a karyotypically normal conceptus without karyo-
typing products of conception. As previously discussed,
NK cells and failure to suppress NK cell activation plays
an important role in immunologically preventable sponta-
neous abortions.28An elevation in the percentage of cir-
culating CD56+ NK cells in women with reproductive
losses predicts the loss of a karyotypically normal concep-
tus while normal levels are associated with the loss of a
chromosomally abnormal c o n c e p t u ~A. ~more
~ striking as-
sociation has been reported when comparing losses with
abnormal and normal subsequent karyotypes by measur-
ing CD56+ NK cells, which also react with antibodies
raised against TJ6.” TJ6 is a 70-80 kDa protein found on
B lymphocytes during normal pregnancy.s6 During viable
pregnancies the percentage of circulating CD56+ cells that
express TJ6 was 28%.s7In nonviable pregnancies with nor-
mal karyotypes the percentage of circulating TJ6 CD56+
cells was significantly increased to 74% (P < 0.05) and
with abnormal karyotypes the percentage of circulating
TJ6+ CD56+ cells was significantly decreased to 10% ( P
< 0.05).57
Recent data suggest that IVIg therapy is useful in main-
taining pregnancies among women with a history of re-
current spontaneous abortion who loose karyotypically
normal embryos and who demonstrate elevated levels or
CD56+ NK cells greater than 12% of circulating white
blood cells in maternal blood.42A significantly higher pro-
portion of women with CD56+ cells > 12% who received
IVIg than those who did not receive IVIg had viable preg-
nancies ( P = 0.0002).42Interestingly, IVIg appears to de-
crease the percentage of circulating NK cells among
recurrent miscarriage patients.58 Thus, percentage of cir-
culating CD56+ NK cells is a marker of recurrent sponta-
neous abortions with an immunologic cause which is likely
to respond to immunotherapy with IVIg.
An immunologic cause of abortion may also mean an
immunologic abnormality (marker) is identified and is
thought to explain the spontaneous abortion. Some inves-
tigators have found increased frequencies of a variety of
autoantibodies in women with recurrent abortion.s9@These
women have been successfully treated using aspirin and
heparin:’ aspirin and prednisone:’ and IVIg” as immu-
notherapy. Other investigators have considered those
couples with three or more unexplained pregnancy losses,
not more than one live birth and no losses with another

0 MUNKSGAARD. COPENHAGEN
 IMMUNOTHERAPY FOR RECURRENT PREGNANCY LOSS /
partner as a category of immunologic pregnancy I O S S . ' @ ' ~
The rationale for including this latter category is historic
and was based upon theory subsequently proven not to be
completely correct. The theory stated that couples who
shared multiple HLA antigens abort due to failure of for-
eign paternal antigens to stimulate maternal immune re-
sponse necessary for maintenance of pregnancy.63The idea
of transfusing blood lymphocytes from the donor of for-
eign antigens on the "graft" (i.e., the father) to enhance
success, as in renal transplants, was tried.".633" The suc-
cess rate (live birth rate) in the pilot studies was 70% and
a randomized controlled trial was then performed, which
showed a 37% success rate in women injected with their
own blood lymphocytes and 77% in women injected with
their husband's cells. I ' Thus, a partner-specific immune
response defect hypothesis became an established expla-
nation for recurrent spontaneous abortions. Subsequent
data did not confirm generality of extensive HLA antigen
sharing and husband-specific hyporesponsiveness to pater-
nal MHC antigens in most couples with recurrent miscar-
~ - i a g eImmunotherapy
.~~ using allogeneic white blood cell
immunization was associated with a low (8-10%) treat-
ment effect when all women in this category were treated."
However, the treatment effect could be increased to 40-
50% by limiting the couples treated to those who had no
prior pregnancies beyond 20 wks gestation (primary abort-
ers) and whose female partner had absence of circulating
autoantibodies.'7.57This observation raises the question
whether secondary aborters (women experiencing recur-
rent spontaneous abortion after a previous livebirth or still-
birth) lose by a different mechanism than primary aborters.
Secondary aborters do not show a significant magnitude
of benefit following immunotherapy using husband's white
blood cell immunization." This is not due to an increased
frequency of chromosomal abnormalities among second-
ary aborters as shown in Table 11. Secondary aborters have
significantly fewer abnormal karyotypes than primary
aborters. However, their prognosis for a spontaneous suc-
cess is greater, perhaps because of the lower frequency of
chromosome abnormality or perhaps because they are more
likely to have had an immune response induced by expo-
sure for a sufficiently long time to conceptus antigens.67
The mechanism of loss among secondary aborters of
karyotypically normal embryos is not known. Preliminary
data suggest that whatever the mechanism, secondary
aborters respond as well as primary aborters to immuno-
therapy with IVIg (Coulam, unpublished data).
SUMMARY
Recurrent pregnancy loss is a health care concern. Safe and
effective treatments are necessary. Women experiencing
recurrent pregnancy loss are a heterogeneous population.
Chromosomal abnormalities are evident in 55% of primary
recurrent abortions. Women experiencing recurrent aneu-
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 35,1996
357
ploidy in their abortuses would not be expected to respond
to immunotherapy. To eliminate abnormal chromosomes
within concepti as a confounding variable for clinical tri-
als testing various forms of immunotherapy, specific mark-
ers are necessary to identify those who have abnormal
embryos. Currently available diagnostic markers include
karyotyping the trophoblast and percentage of circulating
CD56+ NK cells. Additional sensitive and specific tests
are needed. It may also be important to identify and ex-
clude from leukocyte immunotherapy patients with autoim-
mune markers. It is possible that IVIg may achieve better
results in treatment of RSA by virtue of an ability to ame-
liorate rather than to aggravate subclinical autoimmunity
that may be causing recurrent losses in some patients by
mechanisms yet to be defined.
REFERENCES
1. Edmonds DK, Lindsy KS, Miller JF, Williamson E, Wood
PJ. Early embryonic mortality in women. Fertil Steril 1982;
38:447-453.
2. Wilcox AJ, Weinberg CR, O'Connor JF, Bard DO,
Schlatterer JP, Canfield RE, et al. Incidence of early loss of
pregnancy. N Engl J Med 1988; 319:189-194.
3. Warburton D. Spontaneous abortion risks in man: Data from
reproductive histories collected in a medical genetics unit.
Am J Hum Gen 1964; 16:l-25.
4. Coulam CB. Epidemiology of recurrent spontaneous abor-
tion. Am J Reprod Immunol 1991; 26:23-27.
5. Boue J, Boue A, Lazar P. Retrospective and prospective epi-
demiological studies of 1500 karyotyped spontaneous hu-
man abortions. Teratology 1975; 12:11-1 6.
6. Wegmann TG, Lin H, Guilbert L, Mossman TR. Bio-
directional cytokine interactions in the maternal-fetal rela-
tionship: Is successful pregnancy a TH2 phenomenon?
Immunol Today 1993; 14:353-356.
7. Clark DA, Lea RG, Podor T, Daya S, Banwatt D, Harley
CD. Cytokines determining the success or failure of preg-
nancy. Ann NY Acad Sci 1991; 626526544.
8. McIntyre JA, Coulam CB, Faulk WP. Recurrent spontane-
ous abortion. Am J Reprod Immunol 1989; 21:lOO-104.
9. Coulam CB. Immunotherapy for recurrent spontaneous abor-
tion. Early Preg Biol Med 1995; 1:13-26.
10. Coulam CB, Clark DA, Collins JA, et al. (The Recurrent
Miscarriage Immunotherapy Trialist Group). Worldwide col-
laborative observational study and meta-analysis on alloge-
neic leukocyte immunotherapy for recurrent spontaneous
abortion. Am J Reprod Immunol 1994; 3255-72.
11. Mowbray JF, Liddel H, Underwood JL, Gibbins C, Reginald
PW, Beard RW. Controlled trial of treatment of recurrent
spontaneous abortion by immunization with paternal cells.
lancet 1985; 1:941-949.
12. Gatenby PA, Cameron K, Simes RJ, Adelstein S, Bennett
MJ, Jansen RPS, Shearman RP, Stewart GJ, Whittle M,
Doran TJ. Treatment of recurrent spontaneous abortion by
immunization with paternal lymphocytes: Results of a con-
trolled trial. Am J Reprod Immunol 1993; 29:88-94.
13. Ho HN, Gill TJ, Hsieh HJ, Jiang JJ, Lee TY, Hsieh CY. Im-
munotherapy for recurrent spontaneous abortion in a Chi-
nese population. Am J Reprod Immunol 1991; 25:lO-15.
14. Cauchi MN, Lim D, Young DE, Kloss M, Pepperell RJ.
Treatment of recurrent aborters by immunization with pa-
358 I COULAM ET A L .
temal cells-controlled trial. Am J Reprod Immunol 199 1 ;
25:16-17.
15. Mueller-Eckhardt G, Mohr-Pennert A, Heine 0, Neppert
J, Kunzel W, Mueller-Eckhardt C. Controlled trial on in-
travenous immunoglobulin treatment for prevention of re-
current spontaneous abortion. Br J Obstet Gynecol 1994;
10 1 :1072-1 077.
16. Coulam CB, Krysa L, Stem JJ, Bustillo M. Intravenous im-
munoglobulin for treatment of recurrent pregnancy loss. Am
J Reprod Immunol 1995; 34:333-337.
17. Daya S, Gunby J. The Recurrent Miscarriage Immuno-
therapy Trialist Group. The effectiveness of allogeneic
leukocyte immunization in unexplained primary recurrent
spontaneous abortion. Am J Reprod Immunol 1994;
3 2:294-3 02.
18. Kempf C, Jentsch P, Poirier B, et al. Virus inactivation dur-
ing production of intravenous immunoglobulin. Transfusion
1991; 31:423-427.
19. Soto B, Garcia-Bengoechea M, Riestra S, et al. Heterosexual
transmission of hepatitis C virus and the possible role of co-
existent human immunodeficiency virus infection in the in-
dex case. A multicentre study of 423 pairings. J Internal Med
1994; 236~515-519.
20. Kwak JYH, Gilman-Sachs A, Beaman KD, Beer AE. Re-
productive outcomes in women with recurrent spontaneous
abortions of alloimmune and autoimmune causes: Precon-
ception versus postconception treatment. Am J Obstet
Gynecol 1992; 166:1787-1795.
21. Christiansen OB, Mathiesen 0, Lauristen JG, Grunnet N.
Intravenous immunoglobulin treatment of women with mul-
tiple miscarriages. Human Reprod 1992; 7:7 18-722.
22. Coulam CB. Unexplained recurrent pregnancy loss; epi-
logue. Clin Obstet Gynecol 1986; 29:999-1004.
23. Stern JJ, Dorfmann A, Gutierrez-Najan AJ, Cerrillo M,
Coulam CB. Frequency of abnormal karyotypes among
abortuses from women with and without a history of recur-
rent spontaneous abortion. Fertil Steril 1996; in press.
24. Clark DA. Animal models of normal and aborting preg-
nancy, In Immunobiology of Normal and Diabetic Preg-
nancy (Chapter 2). di Mario U (ed). Chichester (UK): John
Wiley & Sons, 1990, pp. 23-37.
25. Chaouat G, Clark DA, Wegmann TG. Genetic aspects of
the CBA x DBA/2 and B10 x BIO.A models of murine
pregnancy failure and its prevention by lymphocyte
immunisation, I n Early Pregnancy Loss, Mechanisms and
Treatment. Beard RW, Sharp F (eds). Ashton-under-Lyne
(UK): Peacock Press Ltd., 1988, pp. 89-104.
26. Head JR. Can trophoblast be killed by cytotoxic cells? In
vitro evidence and in vivo possibilities. Am J Reprod
Immunol 1989; 2O:lOO-105.
27. Wegmann TG, Waters CA, Drell DW, et al. Pregnant mice
are not primed but can be primed to fetal alloantigens. Proc
Nat Acad Sci (USA) 1979; 76:2410-2414.
28. Clark DA. Controversies in reproductive immunology. Crit
Rev Immunol 1991; 11:214-247.
29. Menu E, Chaouat G, Kinsky R, et al. Alloimmunization
against a well-defined polymorphic major histocompatibil-
ity or Class I MHC-transfected L cell antigen can prevent
poly IC inducted fetal death in mice. Am J Reprod Immunol
1995; 33:200-211.
30. Chaouat G, Menu E, Clark DA, et al. Control of fetal sur-
vival in CBA x DBA/2 mice by lymphokine therapy. J
Reprod Fertil 1990; 89:447458.
31. Woodruff MFA. Transplantation immunity and the immu-
nological problem of pregnancy. Proc Royal SOCLond B
1958; 148168-75.
0 MUNKSGAARD. COPENHAGEN
32. Simmons RL, Russell PS. Immunologic interactions between
the mother and fetus. Adv Obstet Gynecol 1967; 1:38-43.
33. Heybome K, Fu Y-X, Nelson A, et al. Recognition of tro-
phoblasts by gamma-delta T cells. J Immunol 1994;
153:2918-2926.
34. Szekere-BarthoJ, Autran B, Debre P, et al. Immunoregulatory
effects of suppressor factor from healthy pregnant women’s
lymphocytes after progesterone induction. Cellular Immu-
nology 1989; 122:281-294.
35. Szekeres-Bartho J, Szekeres G, Debre P, et al. Reactivity of
lymphocytes to a progesterone receptor-specific monoclonal
antibody. Cellular Immunology 1990; 125:273-283.
36. Szekeres-Bartho J, Kinsky R, Chaouat G. The effect of a
progesterone-induced immunologic blocking factor on
NK-mediated resorption. Am J Reprod Immunol 1990;
24: 105-107.
37. Szekeres-Bartho J, Weill BJ, Mike G, et al. Progesterone re-
ceptors in lymphocytes of liver-transplanted and transfused
patients. Immunology Letters 1989; 22:259-261.
38. Erard F, Garcia-Sanz JA, Le Gros G. Switch of CD8 T cells
of noncytolytic CD8- CD4- cells that make TH2 cytokines
and help B cells. Science 1993; 260:1802-1805.
39. Moore SC, Soderberg LSF. Mouse bone marrow natural sup-
pressor cells: Induction and activity. FASEB J 1990; 4:435-
439.
40. Szekeres-Bartho J, Reznikoff-Etievant MF, Varga P, et al.
Lymphocytic progesterone receptors in normal and patho-
logical human pregnancy. J Reprod Immunol 1989;
16:239-247.
41. Michel M, Underwood J, Clark DA, Mowbray J, Beard RW.
Histologic and immunologic study of uterine biopsy tissue
of incipiently aborting women. Am J Obstet Gynecol 1989;
16 1 :409-414.
42. Coulam CB, Goodman C, Roussev RG, Thomason EJ,
Beaman KG. Systemic CD56+ cells can predict pregnancy
outcome. Am J Reprod Immunol 1995; 33:40-46.
43. Aoki K, Kajiura S, Matsumoto Y, Ogasawara M, Okada
S, Yagami Y, Gleicher N. Preconceptual natural killer cell
activity as a predictor of miscarriage. Lancet 1995;
345:1340-1342.
44. Warburton D, Kline J, Stein Z, Hutzler M, Chin A, Hassold
T. Does the karyotype of a spontaneous abortion predict the
karyotype of a subsequent abortion?-vidence from 273
women with karyotyped spontaneous abortions. Am J Hum
Genet 1987; 41:465468.
45. Cowchock FS, Gebas Z, Jackson LG. Chromosome errors
as a cause of spontaneous abortion: The relative importance
of maternal age and obstetric history. Fertil Steril 1993;
5911011-1014.
46. Warburton D. Chromosomal causes of fetal death. Clin
Obstet Gynecol 1987; 30:268-277.
47. Risch HA, Weiss NS, Clarke EA, Miller AB. Risk factors
for spontaneous abortion and its recurrence. Am J Epidemiol
1988; 12k420-424.
48. Lippman A, Ayme S. Fetal death rates in mothers of chil-
dren with trisomy 21 (Down syndrome). Ann Hum Genet
1984; 48:303-312.
49. Stene J, Stene E, Mikkelsen M. Risk for chromosome ab-
normality at amniocentesis following a child with a
noninherited chromosome aberration. Prenat Diagn 1984;
4:8 1-85.
50. Parazzini F, Acaia B, Ricciardeiello 0, Fedele L, Liata P,
Candiani GB. Short-term reproductive prognosis when no
cause can be found for recurrent miscarriage. Br J Obstet
Gynaecol 1988; 95:654-657.
51. Cowchock FS, Smith JB, David S, Scher J, Batzer F, Carson
 IMMUNOTHERAPY FOR RECURRENT PREGNANCY LOSS /
S. Paternal mononuclear cell immunization therapy for re-
peated miscarriage: Predictive variables for pregnancy suc-
cess. Am J Reprod Immunol 1990; 22:12-16.
52. Strobino B, Fox HE, Kline J, Stein Z, Susser M, Warburton
D. Characteristics of women with recurrent spontaneous
abortions and women with favorable reproductive histories.
Am J Public Health 1986; 76:986-994.
53. Morton NE,Chiu D, Holland C, Jacobs PA, Pettay D. Chro-
mosome anomalies as predictors of recurrence risk of spon-
taneous abortion. Am J Med Genet 1987; 28:353-360.
54. Hassold T. A cytogenetic study of repeated spontaneous
abortion. Am J Hum Genet 1980; 32:723-730.
55. Coulam CB, Beaman KD. Reciprocal alteration in circulat-
ing TJ6+ CD19+ and TJ6+ CD56+ leukocytes in early preg-
nancy predicts success or miscarriage. Am J Reprod
Immunol 1995; 34:2 19-224.
56. Nichols TC, Kang JA, Angkachatchai V, Beer AE, Beaman
KD. Expression of a membrane form of the pregnancy-as-
sociated protein TJ6 on lymphocytes. Cell Immunol 1994;
I55:2 19-229.
57. Clark DA, Coulam CB. Is there an immunological cause of
repeated pregnancy wastage? Adv Obstet Gynecol 1995; 3:in
press.
58. Kwak FMY, Kwak JYH, Beer AE. Peripheral blood natural
killer cells are effectively suppressed by immunoglobulin G
infusion in women with recurrent spontaneous abortions. Am
J Reprod Immunol 1995; 33:466.
59. Gleicher N, El-Roeity A, Confino E, et al. Reproductive
AMERICAN JOURNAL OF REPRODUCTIVEIMMUNOLOGY VOL. 35,1996
359
failure because of autoantibodies: Unexplained infertil-
ity and pregnancy wastage. Am J Reprod Gynecol 1989;
160: 1376-1 385.
60. Pratt D, Novonty M, Kaberlein G, Dudkiewicz A, Gleicher
N. Antithyroid antibodies and the association with nonorgan
specific antibodies in recurrent pregnancy loss. Am J Obstet
Gynecol1993; 168:837-841.
61. Lubbe WF, Liggins CG. Lupus anticoagulant and pregnancy.
Am J Obstet Gynecol 1985; 153:322-327.
62. Hasegawa I, Takakuwa K, Goto S, Yamada K, Sekizuka N,
Kanazawa K, Tanaka K. Effectiveness of prednison/aspirin
therapy for recument aborters with antiphospholipid anti-
body. Human Reprod 1992; 7:203-207.
63. Taylor C, Faulk WP. Prevention of recurrent abortion with
leukocyte transfusions. Lancet 1989; ii:68-69.
64. Beer AE, Quebbeman JF, Ayers JW, Haines RF. Major his-
tocompatibility antigens, maternal and paternal immune re-
sponses and chronic habitual abortions in humans. Am J
Obstet Gynecol 1981; 141:987-999.
65. Coulam CB. Immunologic tests in the evaluation of repro-
ductive disorders: A critical review. Am J Obstet Gynecol
1992; 167: 1844-1851.
66. McIntyre JA, Faulk WP, Nichols-Johnson VR, Taylor CG.
Immunological testing and immunotherapy in recurrent
spontaneous abortion. Obstet Gynecol 1986; 67: 169.
67. Clark DA, Coulam CB, Daya S, Gunby J. The modem pre-
ventive treatment of recurrent miscarriage. Brit J Obstet
Gynecol 1995; in press.