Journal of Reproductive Immunology 123 (2017) 78–87

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Journal of Reproductive Immunology

journal homepage: www.elsevier.com/locate/jri

Review article

Antiphospholipid syndrome and recurrent miscarriage: A systematic review MARK

and meta-analysis
Thaís da Silva Santosa, Andressa Lorena Iequeb, Hayalla Corrêa de Carvalhoa, Ana Maria Sellf,
Maria Valdrinez Campana Lonardonid, Izabel Galhardo Demarchid, Quirino Alves de Lima Netoe,
⁎
Jorge Juarez Vieira Teixeirac,
a
Postgraduate Program in Biosciences and Physiopathology, State University Maringa, 5790 Colombo Avenue, 87020-900, Maringa, Brazil
b
Postgraduate Program in Health Sciences, State University Maringa, 5790 Colombo Avenue, 87020-900 Maringa, Brazil
c
Postgraduate Program in Biosciences and Physiopathology, Department of Clinical Analyses and Biomedicine, State University Maringa, 5790 Colombo Avenue, 87020-
900 Maringa, Brazil
d
Department of Clinical Analyses and Biomedicine, State University Maringa, 5790 Colombo Avenue, 87020-900 Maringa, Brazil
e
Department of Basic Health Sciences, State University Maringa,5790 Colombo Avenue, 87020-900 Maringa, Brazil
f
Department of Cell Biology and Genetics, Center of Biological Sciences, State University Maringa,5790 Colombo Avenue, 87020-900 Maringa, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Antiphospholipid syndrome (APS) is an autoimmune condition that is associated with thrombosis and morbidity
Antiphospholipid antibody in pregnancy. The exact mechanisms by which these associations occur appear to be heterogeneous and are not
Antiphospholipid syndrome yet well understood. The aim of this study was to identify and analyze publications in recent years to better
Habitual abortion understand the diagnosis and its contribution to monitoring APS among women with recurrent miscarriage
Pregnancy complications
(RM). This systematic review and meta-analysis was conducted using the PubMed and Web of Knowledge da-
tabases, with articles published between 2010 and 2014, according to the PRISMA statement. Of the 85 iden-
tified studies, nine were selected. Most of the studies reported an association between recurrent miscarriage and
specific antiphospholipid antibodies, as anticardiolipin antibodies (aCL), lupus anticoagulant (LA), anti-β2-
glycoprotein I antibodies (aβ2GPI) and antiphosphatidylserine (aPS), which showed a relationship with RM. The
main result of the meta-analysis revealed association between antiphospholipid antibodies (aPLs) and/or APS
compared to the patients with RM (OR: 0.279; 95% CI: 0.212-0.366) and APS cases compared to the patients
with RM (OR: 0.083; 95% CI: 0.036-0.189). High heterogeneity among these studies (I2 = 100.0%, p < 0.001)
was observed. In addition, there was no significant publication bias across studies according to Begg's test
(p = 0.230), although Egger's test (p = 0.037) suggests significant publication bias. The funnel plot was slightly
asymmetrical. Systematic review and meta-analysis demonstrated a positive association between antipho-
spholipid antibodies and/or antiphospholipid syndrome in patients with recurrent miscarriage.

1. Introduction thrombosis or morbidity in pregnancy. Morbidity in pregnancy includes

Antiphospholipid syndrome (APS) is an autoimmune condition that
is characterized by the production of antiphospholipid antibodies
(aPLs) and associated with thrombosis and morbidity in pregnancy
(Kutteh and Hinote, 2014). The major aPLs that is found in APS are
anticardiolipin antibodies (aCLs), lupus anticoagulant (LA) and anti-β2-
glycoprotein I antibodies (aβ2GPI) (Kutteh and Hinote, 2014; Miyakis
et al., 2006). The international Sapporo criteria (1999), revised in 2006,
emphasizes the diagnosis of APS when at least one of the major aPLs is
detected on two or more occasions in a 12-week interval between
measurements and associated with a clinical condition, such as
unexplained stillbirths at ≥10 weeks of gestation, preterm delivery due
to eclampsia, preeclampsia, or placental insufficiency, and three or
more consecutive miscarriages (Miyakis et al., 2006).
The APS is associated with recurrent miscarriage (RM) (Kutteh and
Hinote, 2014), defined by the American Society for Reproductive
Medicine as two or more spontaneous pregnancy losses (Practice
Committee of the American Society for Reproductive Medicine, 2013).
The incidence of aPLs in RM patients is between 15% and 20% (Kutteh
and Hinote, 2014). Given the complexity and heterogeneity of RM, the
etiology remains unknown in about 50% of couples (American Society
for Reproductive Medicine, 2016). Research on APS has decreased in

⁎
Corresponding author at: Department of Clinical Analyses and Biomedicine, State University Maringa, 5790 Colombo Avenue, 87020-900, Maringa, Brazil.
E-mail address: jjvteixeira@gmail.com (J.J.V. Teixeira).

http://dx.doi.org/10.1016/j.jri.2017.09.007
Received 18 January 2017; Received in revised form 15 September 2017; Accepted 18 September 2017
0165-0378/ © 2017 Elsevier B.V. All rights reserved.
T.d.S. Santos et al.
such indeterminate cases (Cecatti et al., 2000). There is a discussion in
the literature about the definition of RM. The commonly accepted and
widely adopted definition of recurrent miscarriage is three or more
consecutive miscarriages (Diejomaoh, 2015; Stirrat, 1990). However, in
practice, many researchers and clinicians have now revised the defi-
nition to two or more consecutive miscarriages, a less rigorous defini-
tion (American Society for Reproductive Medicine, 2016; Diejomaoh,
2015; Stirrat, 1990; Sugiura-Ogasawara et al., 2014). Without treat-
ment, APS can contribute to 90% of recurrent miscarriage (Rai et al.,
1995). The clinical treatment of choice among patients with APS is the
use of heparin and low-dose aspirin (Kwak-Kim et al., 2013). However,
therapy should be tailored to the needs of the patient. The identification
of additional risk factors should be considered, as well as the dis-
semination of information to patients about morbidity during and after
pregnancy and the positive outcome after appropriate treatment (Ruiz-
Irastorza and Khamashta, 2007).
Despite the complexity of APS and its being responsible for a high
rate of obstetric complications some advances have been made in our
understanding of the pathogenesis of the disease. However, many gaps
in our knowledge remain, especially regarding diagnosis and treatment
(Branch et al., 2010). Given the large production of research that has
related aPLs to RM, we performed a systematic review and meta-ana-
lysis to identify and analyze recent publications on the syndrome to
better understand the diagnosis and its contribution to monitoring APS
among women with history of obstetric complications.
2. Materials and methods
2.1. Search strategy
The research was conducted in accordance with the PRISMA state-
ment (Shamseer et al., 2015) to search for articles that were published
from January 2010 to December 2014. The first stage of the research
included a definition of search descriptors by three researchers (ALI,
HCC, TSS) and an expert (JJVT). The next step was a summary of re-
search in the PubMed and Web of Knowledge databases. We used
PubMed MeSH terms and Web of Knowledge topics (TS). The de-
scriptors remained the same for both databases and were divided into
two blocks: Block 1: “Antiphospholipid Syndrome” OR “Antibodies,
Antiphospholipid”; Block 2: “Abortion, Habitual”- major term (to en-
sure greater search accuracy). Afterward, both blocks were combined to
retrieve the greatest number of abstracts (Supplementary file 1).
2.2. Study selection
According to previously defined strategies, the research was per-
formed using our own filters of the databases: summary available,
human, English, and publication period (January 1, 2010, to December
31, 2014). All of the abstracts that approached the topic of interest were
selected by three researchers (ALI, HCC, TSS) by consensus. For the
selection of articles, we included studies that used different definitions
for RM (2 or more abortions; 3 or more abortions) (American Society
for Reproductive Medicine, 2016; Stirrat, 1990). Due to literature de-
scription that other aPLs and clinical manifestations are strongly asso-
ciated with APS, and the international consensus recognizes these as-
sociations (Miyakis et al., 2006), we excluded other probable causes for
abortion, such as cytogenetic, anatomical, hormonal, and metabolic
abnormalities and infections. The articles were retrieved in full-text
PDF format and reanalyzed by the same three researchers to ensure
higher accuracy of selection. The articles were organized and structured
into three blocks of items and randomly assigned to the other judges
(AMS, IGD, MVCL and QALN) for independent review. After reading
and analyzing the articles in the block, each judge exchanged his block
with another judge for validation. Doubts encountered in the sample
selection were resolved by consensus among the judges. The exclusion
criteria were reviews, letters, editorials, news, commentaries,
79
Journal of Reproductive Immunology 123 (2017) 78–87
guidelines, and duplicate publications. Studies that included popula-
tions with other diseases related to RM or that did not study the re-
lationship between RM and aPLs were also excluded (Supplementary
file 1).
2.3. Quality assessment
Steps which involved selection of items and researchers were per-
formed randomly. To improve and increase the sensitivity of the sys-
tematic review, the researchers also conducted additional searches of
the original articles’ reference lists. The goal was to identify additional
references that were not found in the initial stages of the search.
2.4. Data extraction
The data was organized in tables containing the most important
variables, including the prevalence of aPLs in patients with RM, the
type and number of patients with aPLs for each study, and the positive
cutoff values. These features were analyzed according to the following
topics: type of study, study period, population, age (mean ± standard
deviation), inclusion and exclusion criteria, author biases, study lim-
itations, and outcomes. Three researchers (ALI, HCC, TSS) extracted the
data, and then four reviewers (AMS, IGD, MVCL and QALN) in-
dependently validated the data that was extracted. Various consensus
meetings were convened to agree with the final structure of the tables
and define the most relevant results of the search.
2.5. Statistical analysis
The forest plan was structured with odds ratio (OR) values with 95%
confidence interval between grouped variables (Table 1). Various fre-
quency measurements were analyzed, such as aPLs and/or APS and RM
cases, aPLs and/or APS cases about the definition used for RM, and APS
among RM cases. The evaluation of the heterogeneity among studies
was done by using χ2 test, according to the Higgins and Thompson I2
statistic classification: low (25%), moderate (50%), and high (75%)
(Higgins et al., 2003), the estimate was analyzed in random-effects. For
potential publication bias, Begg's (Begg and Mazumdar, 1994) and
Egger's tests (Egger et al., 1997) with significance p < 0.05 were used,
as well as the funnel plots for symmetry analysis. All statistical analyzes
for meta-analysis were carried out using the STATA 9.0® software
package (Stata Corporation, College Station, TX, USA) with significance
for p < 0.05
3. Results
3.1. Selection and characteristics of the studies
The search strategy yielded a total of 85 articles identified with the
use of filters. Of these, 76 were excluded because they did not meet the
objectives of the study, so the final sample contained nine articles
(Fig. 1). The main features of the included studies in the review show
that the majority of studies on APS are developed in Europe and are
case-control designs (Table 1).
The age ranged from 20 to 45 years, the most prevalent age range,
corresponding to the fertile age of the women included in studies. All of
the authors used inclusion criteria to select their samples, with the
common intention to include only patients without a definite cause of
RM. For sample selection, three authors used 3 or more consecutive
miscarriages as definition for RM (Chen et al., 2012; Motak-pochrzęst
and Malinowski, 2013; Sater et al., 2012) and six articles used 2 or
more consecutive miscarriages (Cohn et al., 2010; Lončar, 2010;
Obayashi et al., 2010; Roye-Green et al., 2011; Subrt et al., 2013; Van
Den Boogaard et al., 2013) (Table 1). Seven studies of nine included in
the meta-analysis showed an association between RM and aPLs (Chen
et al., 2012; Cohn et al., 2010; Lončar, 2010; Motak-pochrzęst and
 T.d.S. Santos et al.

Table 1
Characteristics of included studies in systematic review.

Search Design Period Patient/ RM cases(%)/ aPLs or APS APS Cases MD; SD (range) Definition RM IC EC AB AAB Outcome (RM)
Population Population cases (%)/ for sample
Population selection

Motak-pochrzęst and CC 2009–2012 155 RM (46 155 (76%)/ 46 (22%)/205 25 Pa: 31 (22 − 45) C: 29 3 or more Y Y N N RM patients compared with controls was
Malinowski aPLs); 50C 205 significant for aCL, LA and others
(2013) POL alloimmune factors. The heterogeneity of
immunological risk factors shown in this
study indicates the usefulness of detecting
alloimmune factors and autoantibodies in
patients with RM of unknown etiology.
Subrt et al. (2013) CC 2004–2006 157 RM (84 157 (68%)/ 84 (36%)/231 NR Pa: 32 ± 4.3 (23- 44) C: 33 (27–42) 2 or more Y Y N Y aPLs positivity is truly independent risk
CZE aPLs); 74C 231 factor in multifactorial model of RM.
Van Den Boogaard CS-R 1988–2006 312 RM 1719 (100%)/ 312 (18%)/ 312 Pa: 32.6 ± 5.8 2 or more Y Y Y N Study showed that patients with RM and
et al. (2013) UK + APS; 1407 1719 1719 aPLs did not differ for clinical obstetric
UNRM parameters compared with patients with
UNRM.
Chen et al. (2012) CCeOP 2007–2010 109 aPLs 109 (69%)/ 109 (69%)/158 NR Pa: 29.4 (20–38) C: 29.1 (22–35) 3 or more Y Y Y Y aPLs-RM patients demonstrated
CHN + RM; 49C 158 significantly decreased endometrial and
subendometrial vascularity as assessed by

80
3D-PDA during natural midluteal phase.
Sater et al. (2012) CC-R 02/ 277 RM; 288C 277 (49%)/ NR NR Pa: 31.6 ± 5.4C: 31.7 ± 4.5 3 or more Y Y Y Y Elevated aCL IgM and IgG, and aPS IgG
BHR 2010−10/ 565 antibodies are positively associated with
2010 RM.
Roye-Green et al. CC NR 50 RM (23 50 (27%)/185 23 (12%)/185 NR Pa: 32 (21 − 40) C: 36 (20–42) 2 or more Y Y N Y aPLs are the only autoantibodies which
(2011) JAM aPLs); 135C showed association with RM in Jamaica.
Cohn et al. (2010) CS 1987–2006 693 RM (176 693 (100%)/ 176 (25%)/693 42 Pa: 32 ± 5.6 2 or more Y Y Y Y The overall prognosis of a successful
UK aPLs; 517 693 pregnancy in women with RM and aPLs
UNRM) treated with heparin and aspirin is good.
Obayashi et al. CS-P 1999–2007 367 RM (149 367 (100%)/ 149 (40%)/367 13 Pa: 31.9 ± 4.3 2 or more Y Y Y Y It was not obtained any evidence that aPE
(2010) JPN aPLs) 367 predicts further miscarriage in patients with
RM.
Lončar (2010) SRB CC 2004–2008 60 RM; 60C 60 (50%)/120 NR NR Pa: 29.09 ± 3.2C: 27.1 ± 2.95 2 or more Y Y Y N In PW with RM compared to healthy PW a
slightly positive correlation of LA with aCL
of both IgM and IgG antibodies, as well as a
positive correlation of aCL with aβ2GPI IgG
exist.

3D-PDA: three-dimensional ultrasonography and power Doppler angiography; AB: Author’s bias- Citation in article about bias of the author himself; AAB: Another author’ bias- Citation in article about bias of other authors; aβ2GPI: β2-glycoprotein
I antibodies; aCL: anticardiolipin antibodies; aPLs: antiphospholipid antibodies; aPE: antiphosphatidylethanolamine antibodies; APS: antiphospholipid syndrome; aPS: phosphatidylserine antibodies; BHR: Bahrain; C: control; CC: case-control study;
CS: cohort study; CHN: People's Republic of China; CZE: Czech Republic; EC: Exclusion criteria; IC: Inclusion criteria; JAM: Jamaica; JPN: Japan; LA: lupus anticoagulant; MD: Median age in years; N: no; NR: not reported; O: observational; P:
prospective; Pa: patients; PAI-1: Plasminogen Activator Inhibitor-1; POL: Poland; PW: pregnant women; R: retrospective; RM: recurrent miscarriage; SD: standard deviation; SRB: Serbia; UNRM: unexplained recurrent miscarriage; UK: United
Kingdom, Y: yes.
Journal of Reproductive Immunology 123 (2017) 78–87
T.d.S. Santos et al.
Malinowski, 2013; Roye-Green et al., 2011; Sater et al., 2012; Subrt
et al., 2013) (Supplementary file 2-Table 6). As expected, the age var-
iation among the patients corresponded to the fertile age of women. All
authors used inclusion criteria and statistical analysis. Most authors
used the definition 2 or more consecutive miscarriages for RM.
It is recommended to calculate the cutoff value for enzyme-linked
immunosorbent assays (ELISA) tests and this calculation was performed
in some of the studies that were included in this review (Cohn et al.,
2010; Obayashi et al., 2010; Roye-Green et al., 2011; Sater et al., 2012;
Van Den Boogaard et al., 2013). The positivity cutoff value for aCL IgM
ranged from 6 to 40 MPL-U/ml and for aCL IgG ranged from 9.29 to 40
GPL-U/ml. Van Den Boogaard et al. (2013) and Cohn et al. (2010)
stated that lower cutoffs were used than those that are recommended
for the diagnosis of APS (Miyakis et al., 2006; Wilson et al., 1999).
Other authors also used low cutoff values (Lončar, 2010; Motak-
pochrzęst and Malinowski, 2013; Roye-Green et al., 2011; Sater et al.,
2012). Regarding the LA ratio, a variation of ≥1.10 to ≥1.30 is ob-
served (Table 2). Therefore, five of the nine included articles in review
performed the calculation of the cutoff value and six authors used low
cutoff values.
Only one of the studies used quantitative sandwich enzyme im-
munoassay and others did not report the type of ELISA (Chen et al.,
81
Journal of Reproductive Immunology 123 (2017) 78–87
Fig. 1. Flow diagram of articles included to sys-
tematic review − PRISMA statement (Shamseer
et al., 2015).
2012; Cohn et al., 2010; Lončar, 2010; Motak-pochrzęst and
Malinowski, 2013; Roye-Green et al., 2011; Subrt et al., 2013). Related
to LA research some of the studies used simplified dilute Russell’s viper
venom time (Chen et al., 2012; Cohn et al., 2010; Obayashi et al., 2010;
Van Den Boogaard et al., 2013) with a neutralization process using
frozen and thawed platelets (Cohn et al., 2010; Van Den Boogaard et al.,
2013), while others used Kaolin cephalin clotting time (Cohn et al.,
2010; Lončar, 2010) and the time of activated partial thromboplastin
time (Lončar, 2010; Obayashi et al., 2010; Roye-Green et al., 2011). It is
recommended to use more than one method to detect LA, which has
been done by some studies (Cohn et al., 2010; Lončar, 2010; Obayashi
et al., 2010). Motak-pochrzęst and Malinowski (2013) reported the use
of confirmatory tests for LA, but the tests were not specified. The la-
boratory criteria of APS establish the repetition of laboratory tests for
confirmation of the diagnosis. Four studies stated repetition (Chen
et al., 2012; Cohn et al., 2010; Motak-pochrzęst and Malinowski, 2013;
Van Den Boogaard et al., 2013), other did not report (Lončar, 2010;
Roye-Green et al., 2011; Sater et al., 2012; Subrt et al., 2013) and only
one stated not performing repetition (Obayashi et al., 2010). Three
articles performed the repetition of the laboratory tests in 6 weeks
(Chen et al., 2012; Cohn et al., 2010; Van Den Boogaard et al., 2013)
and only one author repeated it in the interval of 12 weeks (Motak-
T.d.S. Santos et al. Journal of Reproductive Immunology 123 (2017) 78–87

*dilute Russell’s viper venom time; aβ2GPI: anti-β2-glycoprotein I antibodies; aCL: anticardiolipin antibodies; aPE: antiphosphatidylethanolamine antibodies; aPS: antiphosphatidylserine antibodies; aPT: antiprothrombin antibodies; AU: unit
arbitrary; CPV: Cutoff positive value; D: dependent; GPL: IgG phospholipid units; GPU: IgG phospholipid units; I: independente; IgM: immunoglobulin M; IgG: immunoglobulin G; LA: Lupus anticoagulant; MPL: IgM phospholipid units; MPU: IgM
pochrzęst and Malinowski, 2013) (Table 3). Four of nine studies used

aPS/aPT/aPE
simplified dilute Russell’s viper venom time (Chen et al., 2012; Cohn
et al., 2010; Obayashi et al., 2010; Van Den Boogaard et al., 2013),

NA

NA
NA

NA
NA

NA
AU
NR

NR
three used the time of activated partial thromboplastin time (Lončar,
2010; Obayashi et al., 2010; Roye-Green et al., 2011) and two authors
used Kaolin cephalin clotting time (Cohn et al., 2010; Lončar, 2010).

SGU/SMU
Only three authors used more than one method to detect LA (Cohn
aβGPI

U/mL
NA

NA
NA

NA
AU
NR

NR
et al., 2010; Lončar, 2010; Obayashi et al., 2010).

GPU/MPU 3.2. The relationship between antiphospholipid antibodies and recurrent

GPL/MPL

GPL/MPL
GPL/MPL
GPL/MPL

miscarriage
U/mL

U/mL
Unit

aCL

NA
NR

The main aPLs included were aCLs, aβ2GPI, and LA. The positivity
of aPLs among the included studies ranged from 18% to 54.6%
P+ > 0.44; P- > 1.0

phospholipid units; NR: Not reported; NA: Not applicable; P+: Plasma protein-dependent; P-: Plasma protein-independent; SGU: IgG phospholipid units; SMU: IgM phospholipid units; U: unit. (Table 4). The studies that detected aCLs and LA found these antibodies
in the analyzed samples, ranging from 1.8% to 36.5% and 2% to 88.1%,
respectively (Table 4). The studies that detected IgG and IgM antibodies
aPE IgM

to aCL found these antibodies in the analyzed samples (Chen et al.,

2012; Roye-Green et al., 2011; Sater et al., 2012), ranging from 4% to
NA

NA
NA
NA
NA
NA

NA
NR

36.5% and 1.8% to 26%, respectively. Only one article did not attempt
to identify aCLs (Obayashi et al., 2010), and two studies did not attempt
P+ > 0.32; P- > 0.45

to identify LA (Sater et al., 2012; Subrt et al., 2013) (Table 4). Four of
nine studies that analyzed the association between aCLs and RM found
significant associations in their statistical analysis (Lončar, 2010;
Motak-pochrzęst and Malinowski, 2013; Roye-Green et al., 2011; Sater
aPE IgG

et al., 2012). Only one of three studies in their statistical analysis found
association between aβ2GPI and RM (Lončar, 2010). Two of three ar-
NA

NA
NA
NA
NA
NA

NA
NR

ticles that evaluated LA and RM found in their statistical analysis a

significant association between them (Lončar, 2010; Motak-pochrzęst
aPT IgM

> 13.7

and Malinowski, 2013) (Table 1).

NA
NA
NA
NA

NA
NA
NA
NA

Some of the articles investigated other aPLs besides aCLs, LA and

aβ2GPI (Obayashi et al., 2010; Sater et al., 2012; Subrt et al., 2013).
≥10.87
aPS IgG

Two studies investigated antiphosphatidylethanolamine (aPE)

(Obayashi et al., 2010; Subrt et al., 2013) and antiphosphatidylserine
NA

NA
NA

NA
NA
NA
NA
NR

(Sater et al., 2012; Subrt et al., 2013). One study investigated anti-
phosphatidylinositol (aPI), antiphosphatidylglycerol (aPG) and phos-
≥ 16.86 IgM; ≥ 7.60 IgG

phatidic acid, but the statistical data related to these antibodies were
not reported (Table 5) (Subrt et al., 2013). In their statistical analysis
D ≥ 1.9; I ≥ 6.3
Cutoff positive value, ratio positive value LA and unit used in tests of aCL, aβGPI, aPE, aPS and aPT.

only one significant association was found between antipho-

sphatidylserine IgG and RM (Sater et al., 2012) (Table 1).
Three studies investigated association of number of miscarriage
D > 20
aβGPI

among patients with aPLs (Motak-pochrzęst and Malinowski, 2013;

NA

NA
NA

NA
NR

NR

Subrt et al., 2013; Van Den Boogaard et al., 2013), but did not find
differences in frequency of aPLs (Motak-pochrzęst and Malinowski,
aCL IgM

≥ 9.94

2013), genetic polymorphism of plasminogen activator inhibitor-1

> 40

> 10

> 13
>6

>6
≥6

(PAI-I) (Subrt et al., 2013) and patients with APS in relation the con-
NA
NR

trols (Van Den Boogaard et al., 2013).

aCL IgG

≥ 9.29
> 12

> 40

> 10
> 11

> 20
≥ 11

3.3. Meta-analyze: subgroup analysis and publications bias

CPV

NA
NR

The meta-analysis demonstrated a significant association between

> 1.3*
≥1.20

≥1.10
≥1.20

≥1.10

aPLs and/or APS compared to the patients with RM, with an overall
≥1.3
Ratio

combined OR of 0.279 (95% CI: 0.212-0.366) (Supplementary file 2-

NA

NA
NR
LA

Fig. 2). The seven studies included in the meta-analysis evidenced high
Motak-pochrzęst and Malinowski (2013)

heterogeneity, suggesting a random effect model. In addition, there was

no significant publication bias across studies according to Begg's test
(p = 0.230), although the funnel plot was slightly asymmetrical
Van Den Boogaard et al. (2013)

(Supplementary file 2- Fig. 6). However, The Egger's test (p = 0.037)

suggests significant publication bias.
Roye-Green et al. (2011)

Obayashi et al. (2010)

In the subgroup analysis of the frequency of only APS cases in re-

Subrt et al. (2013)

Cohn et al. (2010)

lation to RM, there was high heterogeneity (I2 = 100.0%, p < 0.001)
Chen et al. (2012)
Sater et al. (2012)

among the four studies included (Cohn et al., 2010; Motak-pochrzęst

Lončar (2010)

and Malinowski,2013; Obayashi et al., 2010; Van Den Boogaard et al.,

Reference

2013) and it was demonstrated a significant association between APS

Table 2

cases compared to the patients with RM, with an overall combined OR

of 0.083 (95% CI: 0.036-0.189) (Supplementary file 2- Fig. 5).

82
T.d.S. Santos et al. Journal of Reproductive Immunology 123 (2017) 78–87

Table 3
Comparison between tests described for aCL, aβ2GPI and LA.

Reference Test used for aCL and aβ2GPI (aPLs) Kit-commercial used (companies) Repeat in 6 weeks or 12
LA assay type weeks the tests

Motak-pochrzęst and NR ELISA LA: Instrumentation Laboratory; aPL: Pharmacia Deutschland GmbH 12 weeks
Malinowski (2013)
Subrt et al. (2013) NA ELISA NR NR
Van Den Boogaard et al. (2013) dRVVT NR NR 6 weeks
Chen et al. (2012) dRVVT ELISA aPLs: Aesku Diagnostics; dRVVT: Dade Behring 6 weeks
Sater et al. (2012) NA Quantitative sandwich aPLs: REAADS kits-Corgenix NR
ELISA
Roye-Green et al. (2011) aPTT ELISA aCL- APL Diagnostics Inc.; aβ2GPI- Inova Diagnostics Inc., aPTT: NR
Sigma Diagnostics
Cohn et al. (2010) dRVVT and ELISA NR 6 weeks
KCCT
Obayashi et al. (2010) aPTT and NR aPTT: Automated aPTT, Organon Teknica; dRVVT: Gradipore Ltd. NA
dRVVT
Lončar (2010) KCCT and ELISA aβ2GPI- Aniara Corporation; aCL- Institute for the Application of NR
aPTT Nuclear Energy − INEP; aPTT: Instrumentation Laboratory

aβ2GPI: anti-β2-glycoprotein I antibodies; aCL: anticardiolipin antibodies; aPLs: antiphospholipid antibodies; aPTT: activated partial thromboplastin time; dRVVT: dilute Russell's viper
venom time; ELISA: Enzyme-linked immunosorbent assay; KCCT: Kaolin Cephalin Clotting Time; LA: Lupus anticoagulant; NR: Not reported; NA: Not applicable.

Table 4
Percentage of patients with aPLs total, aCL, aβGPI, LA.

Reference aPLs total aCL IgG aCL IgM aCL IgG/IgM aβGPI LA

Motak-pochrzęst and Malinowski (2013) NR NR NR 16.1% NA 13.5%

Subrt et al. (2013) 52.2% (3–8)ӿ 54.6% (2) NR NR NR NR NA
Van Den Boogaard et al. (2013) 18.0% NR NR NR NA NR
Chen et al. (2012) NR 11.0% 1.8% 1.8% NA 88.1%
Sater et al. (2012) NR 36.5% 10.1% NR NR NA
Roye-Green et al. (2011) NR 4.0%* 26.0%* 0% 14.0% ¥** 2.0%
Cohn et al. (2010) 25.0% NR NR NR NA NR
Obayashi et al. (2010) NR NA NA NA 2.5% ** 2.7% + 13.6%***
Lončar (2010) NR NR NR NR NR NR

ӿ amount of recurrent miscarriages;¥ Sum IgG (n:6%), IgM (n:2%), IgG and IgM (n:6%); *Sum médium/high positive IgG (2%) IgM (16%) and low positive IgG (2%) IgM (10%); **Anti-
β2GP1- aCL dependent antibodies; *** Sum of confirmatory methods for Lupus anticoagulant: dilute Russell's viper venom time (2.7%) and dilute activated partial thromboplastin time
(13.6%); aβ2GPI: anti-β2-glycoprotein I antibodies; aCL: anticardiolipin antibodies; IgG: immunoglobulin G; IgM: immunoglobulin M; LA: Lupus anticoagulant; NR: Not reported; NA: Not
applicable.

Table 5 heterogeneity among these studies (I2 = 100.0%, p < 0.001).

Distribution of patients with aPE, aPS and aPT. However, for the group with definition of 3 or more recurrent mis-
carriages, composed of the inclusion of two articles in the meta-analysis
Reference aPE IgM aPE IgG aPS IgM aPS IgG aPT IgM
(Chen et al., 2012; Motak-pochrzęst and Malinowski, 2013), there was
Motak-pochrzęst and NA NA NA NA NA no significant association with aPLs and/or APS, with an overall com-
Malinowski bined OR of 0.393 (95% CI: 0.131-1.183) (Supplementary file 2- Fig. 4).
(2013) The group with the definition of 2 or more recurrent miscarriages, with
Subrt et al. (2013) NR NR NR NR NA
Van Den Boogaard NA NA NA NA NA
five articles included in the meta-analysis (Cohn et al., 2010; Obayashi
et al. (2013) et al., 2010; Roye-Green et al., 2011; Subrt et al., 2013; Van Den
Chen et al. (2012) NA NA NA NA NA Boogaard et al., 2013), found significant association, with an overall
Sater et al. (2012) NA NA NR 30% 12% combined OR of 0.243 (95% CI: 0.179-0.330) (Supplementary file 2-
Roye-Green et al. NA NA NA NA NA
Fig. 3).
(2011)
Cohn et al. (2010) NA NA NA NA NA
Obayashi et al. (2010) P+: P+: 10.1%; NA NA NA 4. Discussion
6.3%; P-: P-: 3.8%
1.6%
Lončar (2010) NA NA NA NA NA Seven of nine studies with analysis statistically found a relationship
between aPLs and RM (Chen et al., 2012; Cohn et al., 2010; Lončar,
aPE: antiphosphatidylethanolamine antibodies; aPS: antiphosphatidylserine antibodies; 2010; Motak-pochrzęst and Malinowski, 2013; Roye-Green et al., 2011;
aPT: antiprothrombin antibodies; IgG: immunoglobulin G; IgM: immunoglobulin M; LA: Sater et al., 2012; Subrt et al., 2013). Other two studies did not find
Lupus anticoagulant; NR: Not reported; NA: Not applicable; P+: Plasma protein-depen-
significance in their analysis, but one of the authors did not investigate
dent; P-: Plasma protein-independent.
specifically the relationship between aPLs and RM (Van Den Boogaard
et al., 2013) and the other author mainly investigated the relationship
According to Begg's test (p = 0.734) and Egger's test (p = 0.223), there
between antiphosphatidylethanolamine and RM in aPLs which are not
was no significant publication bias, although the funnel plot was
conventional (Obayashi et al., 2010). These findings are consistent with
asymmetrical (Supplementary file 2- Fig. 7).
other studies and further strengthen the involvement of antibodies in
In the subgroup analysis of the frequency of aPLs and/or APS cases
RM (Galarza-Maldonado et al., 2012; Kutteh and Hinote, 2014; Porter
in relation to the definition used for RM, the two groups showed high
and Scott, 2005). However, one study of Israeli women did not find an

83
T.d.S. Santos et al.
association between aPLs and RM (Marai et al., 2004). The discrepancy
may be explained by differences between studies with regard to eth-
nicity, sample size and selection criteria (Sater et al., 2012). Among the
studies included in our meta-analysis, we confirmed an association
between aPLs and/or APS and RM or only APS cases and RM, even
though the frequencies of aPLs and/or APS or only APS cases are low
among RM patients (28% and 8%, respectively). However, this is close
to the frequency that other studies have found when researching the
most common causes of RM, which ranged from 3% to 33%
(Diejomaoh, 2015; Sugiura-Ogasawara et al., 2014).
Some authors sought to relate the number of RM with the presence
of aPLs and/or APS (Motak-pochrzęst and Malinowski, 2013; Subrt
et al., 2013; Van Den Boogaard et al., 2013), but no differences were
found between groups of patients with APS compared to genetic poly-
morphism or unexplained RM, which is consistent with Jaslow et al.
(2010). Based on these data, Van Den Boogaard et al. (2013) suggested
updating the classification criteria previously used and that APS testing
should be considered when there are two or more miscarriages for all
women (American Society for Reproductive Medicine, 2012; Miyakis
et al., 2006; The American College of Obstetricians and Gynecologists,
2012). Six of the nine included articles defined RM as 2 or more con-
secutive miscarriages for sample selection, only one had no significant
association in their analyzes (Obayashi et al., 2010). Three studies
which used the definition to 3 or more for consecutive miscarriages had
a significant association in their analyzes with aPLs and RM (Chen
et al., 2012; Motak-pochrzęst and Malinowski, 2013; Sater et al., 2012).
In our meta-analysis, high heterogeneity was noted in both definitions
of RM. However, for a more rigorous definition (3 or more), there was
no significant association of the definition of RM with aPLs and/or APS,
but it should be noted that only two articles were meta-analyzed for this
definition. Despite this finding, it is not possible to infer that the choice
of the definition to RM influences the significance of the results in re-
lation to aPLs and RM. The RM defined as 3 or more consecutive mis-
carriages had been widely adopted, but many authors have justified
using the definition to 2 or more consecutive miscarriages because of
the decline in fertility with the recent increase in the prevalence of
childless couples and increasing maternal age (Diejomaoh, 2015;
Sugiura-Ogasawara et al., 2014). More studies need to be evaluated so
that a more appropriate definition is achieved, but, in order to avoid
discrepancies and lack of uniformity in the definition of RM, which
create difficulties for researchers in the comparison of results and
clinicians in patient care (Diejomaoh, 2015). It seems appropriate to
use two or more miscarriages as definition for RM, such as suggested by
Van Den Boogaard et al. (2013), the American Society for Reproductive
Medicine (2016) and as used by most articles selected in this systematic
review.
Subrt et al. (2013) compared the frequency of PAI-I with aPLs in
patients with RM and found that the homozygote genotype of PAI-I
(-675) 4G/4G correlated with an increased risk of RM, independent of
the frequency of aPLs. Studies that were published after those that were
included in our study found a correlation between PAI-I and APS
(Aisina et al., 2014; Singh et al., 2013). In addition, there is evidence of
familial genetic predisposition to APS in population studies. The
Human Leukocyte Antigen (DRw53, DR7 and DR4) (Sebastiani et al.,
1996), polymorphism of β2-glycoprotein I antigens, polymorphism of
genes encoding platelet glycoproteins, mediators and genetic defects of
the immune system have also been described in patients with APS
(Meroni, 2008). Studies related to genetic polymorphism can favor a
better understanding of pathophysiology of RM or APS, demonstrating
factors of susceptibility and/or protection (Nowak et al., 2016;
Sebastiani et al., 1996). Thus, it would be possible to better manage
pregnancies in patients with RM or autoimmune diseases, promoting
early interventions and specific treatment, contributing to reduce ma-
ternal-fetal morbidity and mortality in these patients (Alves et al.,
2007).
We observed a large variation in the results of the laboratory tests
84
Journal of Reproductive Immunology 123 (2017) 78–87
(Table 4). A possible explanation may be the high variety of commercial
kits that are available, which may lead to a lack of interlaboratory re-
producibility and influence the reported prevalence. To avoid large
interlaboratory variation, it is recommended the calculation of cutoff
point from population controls of studies (Miyakis et al., 2006; Tincani
et al., 2004), which likely influenced the reported frequency of aCLs
and other studied antibodies.
The correlation of LA and aCL antibodies in the same patient range
50% to 75% (Nahass, 1997). Because of this variation, the patient is
positive for aPLs and negative for another antibody, and the laboratory
diagnosis of APS should consider using a large variety of tests (Levine
et al., 2002). The sensitivity and specificity of the tests were also dif-
ferent. Lupus anticoagulant and aβ2GPI are more specific to APS,
whereas aCL is more sensitive (Levine et al., 2002; Miyakis et al., 2006;
Perches et al., 2009; Reber et al., 2004). The specificity of aCL can
change, depending on the Ig class, and is highest in titers of IgG (Levine
et al., 2002). Regarding the diagnosis of APS, one study demonstrated
the sensitivity of 18.7% and specificity of 92.8% in LA tests. In aCL
assays, IgM reported a sensitivity of 92% and specificity of 1.2% and the
IgG showed the sensitivity of 40% and specificity of 82.5% (Perches
et al., 2009). Possibly, the differences between the classes of im-
munoglobulins are due to the fact that IgM is related to infections or
diseases in the acute phase while the IgG to chronic diseases.
4.1. Association between anticardiolipin antibodies and recurrent
miscarriage
Four studies of nine that analyzed the association between aCLs and
RM found significant associations in their statistical analysis (Lončar,
2010; Motak-pochrzęst and Malinowski, 2013; Roye-Green et al., 2011;
Sater et al., 2012), which is consistent with the literature (Opatrny
et al., 2006). Some studies found a high interlaboratory variation for
this antibody (Favaloro and Silvestrini, 2002; Tincani et al., 2001).
Ulcova-Gallova et al. (2005) argued that the high proportion of aCL
positivity may be related to cross-reactions because of the nature of
their epitopes. Correlations were found between the IgG and IgM
classes of aCLs and RM, but Roye-Green et al. (2011) did not find a
significant association for the IgM class, similar to another study
(Alijotas-Reig et al., 2010). Anticardiolipin antibody IgM can occur in
other diseases and result in false positives in the presence of rheumatoid
factors or cryoglobulins (Miyakis et al., 2006; Opatrny et al., 2006).
Despite all the interference, IgM research is still recommended (The
American College of Obstetricians and Gynecologists, 2012).
4.2. Association between aβ2GPI and recurrent miscarriage
Of three studies that analyzed the association between aβ2GPI and
RM only one article found a significant association on their analysis
(Lončar, 2010). The other included in the present study did not observe
this association (Roye-Green et al., 2011; Sater et al., 2012). The lit-
erature is controversial regarding the relationship between this anti-
body and RM. Some studies indicated a relationship between aβ2GPI
and morbidity in pregnancy (Alijotas-Reig et al., 2010; Faden et al.,
1997; Forastiero et al., 1997; Lee et al., 2001), but other studies did not
find such (Lee et al., 1999; Opatrny et al., 2006) or even refuted it
because of the lack of standardization in the tests and the different
methodologies (Galli et al., 2003a). Some studies cited specific re-
lationships between aβ2GPI IgM (Forastiero et al., 1997), IgG (Alijotas-
Reig et al., 2010), IgA (Lee et al., 2001) and RM. These controversies in
the literature can be explained by interlaboratory variability that still
exists in the study of aβ2GPI. However, its interlaboratory reproduci-
bility is considered good and better than the reproducibility of aCL
assays (Reber et al., 2002).
Similar to aCLs, aβ2GPI research also has methodological limita-
tions and issues with standardization (Reber et al., 2002). It also suffers
from possible interference from cryoglobulins and rheumatoid factors
T.d.S. Santos et al.
in the interpretation of aβ2GPI IgM. Since 2006, aβ2GPI has been in-
cluded in the laboratory classification criteria for APS (Miyakis et al.,
2006). Roye-Green et al. (2011) suggested aCL screening in habitual
abortion and aβ2GPI antibodies tests to identify patients with higher
risk. However, the standardization of aβ2GPI tests needs to be im-
proved to allow comparisons between studies to RM
4.3. Association between lupus anticoagulant and recurrent miscarriage
Three articles that evaluated LA and RM and two articles found a
direct relationship between LA and RM on their statistical analysis
(Lončar, 2010; Motak-pochrzęst and Malinowski, 2013). Despite the
low number of articles that compared this antibody with RM, LA ap-
pears to be correlated with morbidity in pregnancy and thrombosis
(Galli et al., 2003b; Opatrny et al., 2006). However, the interlaboratory
reproducibility of LA tests is also poor when considering the number of
assays that are available commercially (Jennings et al., 2002). The
sensitivity and specificity of LA tests are principally affected because of
the choice of reactants and variation between laboratories (The
American College of Obstetricians and Gynecologists, 2012). As de-
scribed by Jennings et al. (2002), laboratories often use various com-
binations of reagents and methods for detecting LA. This was not dif-
ferent in the articles included in this review. There are no accepted
recommendations concerning the choice of LA tests. Because there is no
fully sensitive test, the international consensus of 2006 recommended
the use of two or more tests before considering the absence of LA
(Miyakis et al., 2006). Three articles in the present systematic review
adhered to this recommendation (Cohn et al., 2010; Lončar, 2010;
Obayashi et al., 2010).
4.4. Other antiphospholipid antibodies not included in classification criteria
and recurrent miscarriage
In addition to classic studies with well-defined antibodies, other
authors investigated different aPLs in an attempt to diagnose APS. A
significant association was found between the IgG class of antipho-
sphatidylserine and RM (Sater et al., 2012). Subrt et al. (2013) found a
high prevalence of non-common antibodies (antiphosphatidylinositol
and antiphosphatidylserine), but without statistical results. Antipho-
sphatidylethanolamine was investigated by Obayashi et al. (2010) and
Subrt et al. (2013), but no correlation was found.
Sater et al. (2012) found an independent association between an-
tiphosphatidylserine IgG and RM, which was confirmed by other stu-
dies (Branch et al., 2001; Ulcova-Gallova et al., 2005; Yamada et al.,
2003). Studies in mice indicated that the presence of aPS causes ob-
stetric morbidities, because they act on the trophoblasts (Tong et al.,
2014), and one study with immunized mice with aPS IgG detected
clinical signs of APS, including an increased rate of fetal resorption
(Yodfat et al., 1996).
Some studies indicated that antiphosphatidylethanolamine is asso-
ciated with such APS events as pre-eclampsia, preterm delivery
(Yamada et al., 2009), RM (Gris et al., 2003; Sugi et al., 2004), in-
fertility (Sanmarco, 2009), thrombosis, neurological features, and li-
vedo reticularis (Karmochkine et al., 1993). This suggests that anti-
phosphatidylethanolamine can increase the detection of aPLs
(Sanmarco, 2009). Branch et al. (2001) detected antipho-
sphatidylserine, antiphosphatidylinositol, antiphosphatidylglycerol and
antiphosphatidylethanolamine during pregnancy and found a re-
lationship between these antibodies and preeclampsia and intrauterine
growth restriction. The modest positive predictive value should low
specificity and not have prognostic value in studying these aPLs in the
evaluation of these morbidities during pregnancy (Branch et al., 2001).
Despite one of the articles included in this review found an asso-
ciation between antiphosphatidylserine and RM, further studies are still
necessary for considering the addition of this other antiphospholipid in
laboratory’s routine as criteria of APS diagnosis. Although the
85
Journal of Reproductive Immunology 123 (2017) 78–87
recommendations of international consensus of 2006 consider these
aPLs (antiphosphatidylserine, antiphosphatidylinositol, antipho-
sphatidylglycerol and antiphosphatidylethanolamine) to present high
sensitivity, their specificity is low for the diagnosis, which justifies the
exclusion of these antibodies in the laboratory criteria of APS (Miyakis
et al., 2006).
4.5. Antiprothrombin and recurrent miscarriage
Sater et al. (2012) found higher titers of antiprothrombin (aPT) IgM
associated with RM, but without significant association between aPT
and RM. Bertolaccini et al. (2005) found that the prevalence of aPT was
higher among patients with morbidity during pregnancy compared
with pregnancy with no complications. Although the studies did not
observe association, other studies indicate an association between aPT
and others manifestations of APS (Hoxha et al., 2012; Sherer et al.,
2003).The laboratory diagnosis of aPT does not have good standardized
tests yet, using in-house ELISA, which may be a reason for the dis-
crepancies when comparing interlaboratory tests (Galli, 2000; Galli
et al., 2003a). Thus, the tests show low sensitivity mainly for IgM, but
the specificity features best results (Galli, 2000). There is still no data
that allows recommendations regarding the detection of aPT in patients
with APS (Galli, 2000; Hoxha et al., 2012), although such tests can be
beneficial when laboratory diagnostic tests for APS are negative
(Bertolaccini et al., 2005). Considering that the data of the articles in-
cluded in this review and the controversy still existing between the
relationships of aPT and APS, we consider the evaluation of the inter-
national consensus to be prudent, which considered that the inclusion
of aPT in the classification criteria for APS is premature (Miyakis et al.,
2006).
4.6. Strengths and limitations of the study
This meta-analysis synthesizes high robustness and considerable
precision in the search for articles that are related to miscarriage and
APS/antibodies. Numerous meetings between researchers and experts
took place on the best definition of MeSH terms and the selection of
publications by consensus and, whenever necessary, the randomized
form was chosen. All the research findings are represented by several
tables included in the manuscript and a supplementary file.
Some limitations, however, must be noted: the use of only two da-
tabases, notwithstanding the considerable number of articles that are
included in these; the high heterogeneity between studies and the small
number of papers included in the meta-analysis, although the random-
effects model was used in order to minimize the effects of hetero-
geneous variability; the inclusion of only English-language articles and
relatively short 5-year publication timeframe, despite our goal was to
find only recent publications. These factors may contribute to the ex-
istence of potential selection bias. Nonetheless, our review revealed
methodological problems and biases that occur in the research of this
issue. One methodological problem that was found in some of the ar-
ticles was that they did not report or did not apply the strategy of re-
peating the diagnostic tests. Four of the nine included studies repeated
laboratory tests at week’s intervals, three at 6-week intervals and only
one at 12-week intervals. Three authors who repeated laboratory tests
and analyzed the relationship between aPLs and RM showed on their
studies a statistically significant association (Chen et al., 2012; Cohn
et al., 2010; Motak-pochrzęst and Malinowski, 2013). We cannot infer
from these articles that the difference of week’s intervals or the lack of
repetition may interfere with the association of aPLs and RM, but more
studies are needed to clarify whether it interferes with this association
or not. The repeated examinations are based on expert opinions and
help to ensure that the antibodies detected are relevant to the diagnosis
of APS (Miyakis et al., 2006), since the transient presence of aCL, LA
and aβ2GPI is reported (Male et al., 2005). Another methodological
problem that we can highlight is the absence of reference to adhesion to
T.d.S. Santos et al.
the recommendation of the use of two or more tests before considering
the absence of LA by some studies. Three authors used more than one
method to detect LA, and, of these, two presented a significant asso-
ciation in their searches between aPLs and RM (Cohn et al., 2010;
Lončar, 2010).Due to the number of articles, we cannot infer that not
using or using more than one method to detect LA may interfere with
the association of aPLs and RM, more studies are needed for such. This
recommendation is made because there is no fully sensitive test for LA
(Miyakis et al., 2006).
5. Conclusion
Systematic review and meta-analysis demonstrated a significant
association between antiphospholipid antibodies and/or antipho-
spholipid syndrome or only antiphospholipid syndrome cases compared
to the patients with recurrent miscarriage. We found recurrent mis-
carriage is related to the specificity of antiphospholipid antibodies, with
relationships to anticardiolipin antibodies, lupus anticoagulant, anti-
β2-glycoprotein I antibodies and antiphosphatidylserine. More studies
are needed to increase the accuracy of findings and confirm which the
best antiphospholipid antibodies relationships with recurrent mis-
carriage. The number of recurrent miscarriage does not appear to be
influenced by the presence of antiphospholipid antibodies. The lack of
standardization between antiphospholipid antibodies tests, not ap-
plying the strategy of repeated test for diagnostic and not using two
tests for confirming the lupus anticoagulant are factors which may
hinder the diagnostic of antiphospholipid syndrome determination.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.jri.2017.09.007.
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