Abstract
Purpose: Increased levels of antiphosholipid antibodies (aPLs) are associated with the autoimmune disorder antiphospholipid syndrome (APS) and are known to play a role in infertility. We investigated the possible effect of prolonged use of hormonal contraception (HC) on autoimmunity after discontinuing HC in women with infertility problems. Material and Methods: We analyzed hormonal status including ovulation and the humoral autoimmune response to eight phospholipids detected by ELISA in 1190 infertile women aged 21–46 (mean age: 29.3) years. Results: Long-term HC did not affect ovulation, but in the absence of ovulation HC masked the hormonal disorders. The majority of patients taking HC for more than 10 years with fertility failure had significantly higher levels of aPLs of the IgG isotype against phosphatidylinositol and phosphatidylserine (p > 0.001) than against β2-glycoprotein I and annexin V. Discussion: Thus, high levels of aPLs are significant for the anticoagulant treatment before and during pregnancy to prevent pregnancy loss.
Key words: infertility, hormonal contraception, antiphospholipid antibodies, antiphospholipid syndrome
Abstract
Zusammenfassung
Zielsetzung: Das erhöhte Auftreten von Antiphospholipid-Antikörpern im Blut ist mit der Autoimmunerkrankung Antiphospholipid-Syndrom (APS) assoziiert und spielt bekanntlich eine Rolle bei Infertilität. In Frauen mit Fertilitätsproblemen wurde die mögliche Auswirkung der Langzeiteinnahme hormoneller Empfängnisverhütungsmittel auf die Autoimmunität untersucht, die auch nach Absetzen des Mittels fortbesteht. Methoden: In 1190 unfruchtbaren Frauen im Alter von 21–46 (Durchschnittsalter: 29,3) Jahren wurden Hormonstatus und Eisprung sowie die durch ELISA ermittelte humorale Immunreaktion auf 8 Phospholipide untersucht. Ergebnisse: Die Langzeiteinnahme hormoneller Empfängnisverhütungsmittel hatte keine Auswirkung auf den Eisprung, aber bei Ausbleiben des Eisprungs hat die hormonelle Empfängnisverhütung die Hormonstörung überdeckt. Die Mehrheit der Patientinnen mit Fertilitätsproblemen, die Hormonpräparate zur Empfängnisverhütung über einen Zeitraum von mehr als 10 Jahren einnahmen, wiesen signifikant höhere Antiphospholipid-Antikörper-Spiegel des IgG-Isotyps gegen Phosphatidylinositol und Phosphatidylserin (p > 0,001) auf als gegen β2-Glykoprotein I und Annexin V. Diskussion: Hohe Antiphospholipid-Antikörper-Spiegel erweisen sich also als signifikant für die antikoagulatorische Therapie vor und während der Schwangerschaft, um einem frühzeitigen Ende der Schwangerschaft vorzubeugen.
Schlüsselwörter: Infertilität, hormonelle Empfängnisverhütung, Antiphospholipid-Antikörper, Antiphospholipid-Syndrom
Introduction
Hormonal contraception (HC) is a birth control method that prevents ovulation by suppressing gonadotropins and changing the properties of cervical mucus. The most common combined contraceptives contain the both hormones, estrogen and progestin. HC has various well-known side effects such as bleeding, elevated liver tests, increased risk of cardiac disease, psychological and sexual changes, and deep vein thrombosis 1, 2. Although the history of HC commenced in 1921, with studies done in a rabbit model 3, the first combined HC was used in Great Britain in 1957. Since then, a large number of women of reproductive age have used HC with or without side effects 4. Heterogeneous autoantibodies specific for negatively charged membrane phospholipids were detected in 1906 by Wassermann 5 in patients with syphilis, and immunochemically isolated by Pangborn 6. Antiphospholipid antibodies (aPLs) are detectable in almost 80 % of patients with autoimmune disease. Furthermore, they are also present in patients with reproductive failure, with a wide range of potential complications before spontaneous and/or in vitro fertilization (IVF), during problems encountered in early or late pregnancy (including spontaneous miscarriage, missed abortion, fetal death, intrauterine growth retardation, prematurity, and preeclampsia), and especially in primary or secondary antiphospholipid syndrome (APS) 7, 8, 9, 10. Antiphospholipid antibodies can interfere with normal conception by binding to phospholipids in the ovary, thus preventing formation and release of a functional ovum, or in normal fertilization by binding to the surface of either the ova or the spermatozoa with cytoplasmic, mitochondrial, endothelial and trophoblast epithelial cell membranes and platelets 11, 12. aPLs can negatively influence the remarkable tolerance mediated by complex immunoregulatory mechanisms which exists between mother and fetal trophoblast 13, 14. Fertility problems caused by microthrombosis of the utero-placental vasculature are the result of endothelial cell activation, inhibition of protein complex formation and fibrinolysis, and annexin V displacement 15, 16, 17, 19, 20; other mechanisms for non-thrombotic fetal damage have been also considered 16, 17, 18, 21. During our routine practice in counseling infertile couples, we observed an increase in serum autoantibodies against phospholipids in oral contraceptive users who decided to discontinue HC and attempt to conceive. Problems with fertility were evident (no spontaneous pregnancy and unsuccessful IVF, often combined with spontaneous miscarriage or missed abortion). The aim of our present study was to establish a screening profile for all aPLs (aPLs of the IgG, IgM, IgA isotypes specific for cardiolipin, phosphatidyl (ph)-serine, ph-ethanolamine, ph-inositol, ph-glycerol, phosphatidic acid, annexin V and β2-GPI) in selected infertile women with reproductive failure and to evaluate the possible side effects of prolonged HC on their autoimmunity.
Material and Methods
Patients
The study group consisted of 1190 patients, aged 21–46 years (mean age: 29.3) (Table 1) who were referred for pregnancy planning to the Division for Infertility and Immunology of Reproduction at the Department of Obstetrics and Gynecology, Charles University and Faculty Hospital, Pilsen, Czech Republic between 2008 and 2013. All women of reproductive age were divided into three main groups according to the use and the length of HC intake (Table 1). The 1st group included 220 women who had never used HC and served as a control group. The 2nd group (221 women) were on HC for a maximum period of two years, and the 3rd group included 749 patients who had used HC for 3 to 20 years. We also studied a 4th subgroup, selected from the 3rd group of infertile women, who had taken HC for 10 and more years. The common goal of all patient groups was successful conception after repeated fertility failures. Their partners had normal spermiograms evaluated according to WHO criteria 22. Medical history indicated a good health status for both partners.
Table 1 Fertility data of 1 190 patients after HC use with analysis of ovulation at 6 months after termination of HC.
| Group 1 | Group 2 | Group 3 | Subgroup 4 from group 3 | |
|---|---|---|---|---|
| RPL – repeated pregnancy lossNP – never pregnant | ||||
| N | 220 | 221 | 749 | 213 |
| Age range (mean) | 20–39 (28.7) | 21–44 (27.8) | 22–46 (31.4) | 25–46 (35.6) |
| Complications | ||||
|
2 (0.9 %) | 45 (20.4 %) | 65 (8.7 %) | 4 (1.9 %) |
|
1 (0.4 %) | 4 (1.8 %) | 6 (0.8 %) | 0 |
|
64 (29.0 %) | 120 (54.3 %) | 540 (72.1 %) | 190 (89.2 %) |
|
153 (69.7 %) | 46 (20.8 %) | 134 (17.9 %) | 19 (8.9 %) |
|
0 | 6 (2.7 %) | 4 (0.5 %) | 0 |
| Physiological ovulation | 202 (92 %) | 102 (46 %) | 157 (21 %) | 23 (11 %) |
Blood samples were obtained from each woman between 7:00–10:00 a. m. All obtained serum samples were frozen and stored at − 18 °C in our serum bank. All sera were screened for the presence of eight aPLs. No female patient had a history of vascular thrombosis, thrombocytopenia, autoimmune or infectious disease.
The study was approved by the local research ethics committee of the Charles University Medical Faculty Hospital, Pilsen, Czech Republic. Written informed consent was obtained from all female participants.
Antiphospholipid antibodies (aPLs)
Enzyme-linked immunosorbent assay (ELISA) was used for the detection of aPLs of the IgG, IgA and IgM isotypes against ph-serine, ph-ethanolamine, ph-inositol, ph-glycerol, phosphatidic acid, annexin V, β2-GPI and cardiolipin levels as previously described 19. Briefly, serially diluted sera were incubated with plates coated with highly purified phospholipids. After washing the plates, anti-human immunoglobulins conjugated to horseradish peroxidase were incubated with the antigen-antibody complex. Unbound conjugate was then washed away. Addition of an enzyme substrate generated an enzymatic colorimetric reaction, which was stopped by diluted acid. The intensity of the color, which was directly proportional to the concentration of antibodies, was measured photometrically.
Statistical analysis
Statistical analysis of all laboratory results was done using Mann-Whitney U-Test for Independent Samples in Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA), with p-values ≤ 0.05 considered statistically significant.
Results
Table 1 shows the medical history of 1190 women before immunological examination. Long-term use of HC interferes with physiological ovulation but also depends on the age of patients. Normal ovulation was present in 92 % of women who never used HC. In the 2nd and 3th groups with long-term HC use, ovulation was restored in 46 % (2nd group), 21 % (3rd group), and 11 % in the 4th subgroup (Table 1).
Serum samples from 1190 infertile patients were evaluated for antibodies of the IgG, IgM and IgA isotypes specific for eight different phospholipids. We assessed individual aPLs with respect to their specificity, prevalence (given as a percentage) and significance. The results in Table 2 showed overall positivity for aPLs in 78 % of women who used HC for more than 2 years (p ≤ 0.001) and also demonstrated significantly higher levels of antibodies against ph-inositol and ph-serine (p ≤ 0.001). The antibodies predominantly present were of the IgG isotype. Results also demonstrated significant differences between patients who never used HC (group 1) and those who had used HC for more than 10 years (subgroup 4). Increased aPLs positivity was found in 89 % of patients, with higher levels of aPLs against ph-inositol (p ≤ 0.001), ph-serine (p ≤ 0.001), ph-ethanolamine (p ≤ 0.01), β2-GPI and annexin V (p ≤ 0.01). Our detailed analysis suggests that the prevalence of positive levels of aPLs against five different phospholipids was much higher in the group of HC users than in non-HC users, where we found only very low levels of such antibodies.
Table 2 Analysis of antiphospholipid antibodies in women without and with long-term HC use (2008–2013).
| Group 1 | Group 2 | Group 3 | Subgroup 4 from Group 3 | p-value | ||
|---|---|---|---|---|---|---|
| NS – not significantGroup 1 – not on HC; Subgroup 4 (HC use of between 10 years and 20 years) | ||||||
| N | 220 | 221 | 749 | 213 | ||
| Mean age (years) | 28.7 | 27.8 | 31.4 | 35.6 | NS | |
| Presence of antiphospholipid antibodies against: | 56 (25 %) | 95 (43 %) | 588 (78.5 %) | 189 (89 %) | 0.001 | |
|
IgG | 3 (1.4 %) | 4 (1.8 %) | 18 (2.4 %) | 5 (2.3 %) | NS |
| IgM | 2 (0.9 %) | 3 (1.3 %) | 0 (0 %) | 5 (2.3 %) | NS | |
|
IgG | 54 (24.5 %) | 58 (26.2 %) | 273 (36.4 %) | 212 (99.5 %) | 0.001 |
| IgM | 2 (0.9 %) | 1 (0.4 %) | 5 (0.6 %) | 5 (2.0 %) | NS | |
|
IgG | 19 (8.6 %) | 21 (9.5 %) | 321 (42.8 %) | 208 (98.0 %) | 0.001 |
| IgM | 0 (0 %) | 4 (1.8 %) | 8 (1.0 %) | 6 (2.8 %) | NS | |
|
IgG | 11 (5.0 %) | 8 (3.6 %) | 57 (7.60 %) | 41 (19.0 %) | NS |
| IgM | 0 (0 %) | 4 (1.8 %) | 12 (1.6 %) | 8 (4.0 %) | NS | |
|
IgG | 12 (5.4 %) | 8 (3.6 %) | 61 (8.1 %) | 51 (24.0 %) | 0.01 |
| IgM | 1 (0.4 %) | 2 (0.9 %) | 16 (2.0 %) | 9 (4.2 %) | NS | |
|
IgG | 5 (2.3 %) | 20 (9.0 %) | 39 (5.2 %) | 29 (13.6 %) | 0.01 |
| IgA | 0 (0 %) | 1 (0.4 %) | 10 (1.3 %) | 3 (1.4 %) | NS | |
|
IgG | 16 (7.2 %) | 8 (3.6 %) | 60 (8.0 %) | 23 (10.8 %) | NS |
| IgM | 0 (0 %) | 0 (0 %) | 0 (0 %) | 3 (1.4 %) | NS | |
|
IgG | 5 (2.3 %) | 9 (4 %) | 37 (4.9 %) | 32 (15.0 %) | 0.01 |
Discussion
In a normal pregnancy, the mother is exposed to a great number of autoimmune and alloimmune antigens which modulate her immune activities. The immunologic mechanisms of pregnancy loss have been the subject of several reviews e.g. 7, 8, 9, 12, 13, 16, 17, 18, 20, 23. This paper summarizes our experience in a long-termed study of women who used HC for more than ten years and displayed significantly increased levels of IgG aPLs, predominantly against ph-inositol, ph-serine, ph-ethanolamine, β2-GPI, and annexin V. These autoantigens contribute to the pathogenesis of pregnancy failure through multiple mechanisms in which the central feature could be thrombosis and/or microthrombosis 23. We did not find information on these findings in the literature. However, the effects of estrogen and progesterone in autoimmune diseases such as systemic lupus erythematosus (SLE) have been previously described 24, 25, 26, 27, 28, 29, 30. Therefore, we aimed to determine whether HC played a role in the elevation of autoimmune factors such as aPLs which contribute to fertility failure. It is well known 29, 30 that estrogens have both pro-inflammatory and anti-inflammatory effects, with certain immunomodulatory and immunosuppressive activities enhancing the proliferation and activation of B-cells. Estrogen administration has the potential effect to exacerbate autoimmune processes and can also increase the risk of thrombosis, especially in women positive for aPLs. Our patients had taken oral combined hormonal contraceptives with different concentrations of the biologically active synthetic estrogen (ethinyl estradiol) and one of the 17-alpha ethinyl analogs of 19-nortestosterone as the progesterone component. However, it is not known which drug combination led to the induction of aPLs. Thromboembolism is a major complication of HC, the risk being further increased by the presence of positive autoimmune factors such as lupus coagulant and aPLs 15. Could HC be the reason for the long-term effects of exogenous estrogen and progesterone through the impact on female autoimmunity? Our study focused on screening for aPLs positivity and determination of aPLs profiles in women with varying lengths of HC use. Although we also considered other causes of fertility problems (through the evaluation of gynecological and endocrinological information, reproductive immunology and genetics), the results of the aPLs panels were clear. A minority of women who did not use HC but had infertility problems also displayed certain immunological abnormalities such as the presence of aPLs antibodies. Although the current classification of APS is based only on positive identification using one or more of three standardized laboratory assays (anti-cardiolipin antibodies, lupus anti-coagulant, and anti-β2-GPI according to the report of aPLs congresses 7, 31), it is clear that other antiphospholipid antibodies may also play an important role in pregnancy failure. To determine the immunological cause of infertility this definition is insufficient. This is the reason why our laboratory, which specializes in reproductive immunology, uses a wider range of aPLs. Results generated suggest that women with previous long-term use of HC have significantly increased levels of aPLs, especially of the IgG isotype, against ph-inositol and ph-serine, and that these levels are higher than those against β2-GPI, and annexin V. Based on our results and extensive experience with human infertility and reproductive immunology, we stress that prolonged use of HC is likely to affect female autoimmunity through the induction of various autoantibodies such as aPLs. Immune response could be also associated with aging, particularly in older patients 32, 33, 34, but none of our patients were elderly; in all groups, only a minority of patients with proven anovulation and higher number of positives for aPLs were aged 40 or older. We speculate that the long-term intake of a combination of artificial hormones such as those present in HC and patient age of more than 35 years could influence hormonal and immunological responses.
Conclusion
We suggest that prolonged HC use represents a new and significant risk factor for immunological reproductive failure 33, 34. The presence of high levels of aPLs could alert clinicians, prompting them to initiate timely anticoagulant treatment to prevent APS.
Acknowledgements
Supported by grant MSM 002 162 0812 and by the Charles University Research Fund (project number P36).
Footnotes
Conflict of Interest None declared.
References
- 1.Cameron S T, Glasier A. London: Wiley-Blackwell; 2012. Chapter 40; pp. 495–512. [Google Scholar]
- 2.Sulak P J, Scow R D, Preece C. et al. Hormone withdrawal symptoms in oral contraceptive users. Obstet Gynecol. 2000;95:261–266. doi: 10.1016/s0029-7844(99)00524-4. [DOI] [PubMed] [Google Scholar]
- 3.Müller-Jahncke W D. [Ludwig Haberlandt (1885–1932) and the development of hormonal contraception] Z Gesamte Inn Med. 1988;43:420–422. [PubMed] [Google Scholar]
- 4.Perone N. The history of steroidal contraceptive development: the progestins. Perspect Biol Med. 1993;36:347–362. doi: 10.1353/pbm.1993.0054. [DOI] [PubMed] [Google Scholar]
- 5.Wasserman A. Über die Entwicklung und den gegenwärtigen Stand der Serodiagnostik gegenüber Syphilis. Berlin: Klin Wochenschr. 1907;44:1599–1605. [Google Scholar]
- 6.Pangborn M C. A new serologically active phospholipids from beef heart. Proc Soc Exp Biol Med. 1941;48:484–486. [Google Scholar]
- 7.Cervera R, Piette J C, Font J. et al. Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients. Arthr Rheumat. 2002;46:1019–1027. doi: 10.1002/art.10187. [DOI] [PubMed] [Google Scholar]
- 8.Ruffatti A, Del Ross T, Ciprian M. et al. Risk factors for a first thrombotic event in antiphospholipid antibody carriers: a prospective multicentre follow-up study. Ann Rheum Dis. 2011;70:1083–1086. doi: 10.1136/ard.2010.142042. [DOI] [PubMed] [Google Scholar]
- 9.Shoenfeld Y, Blank M. Autoantibodies associated with reproductive failure. Lupus. 2004;9:643–648. doi: 10.1191/0961203304lu2009oa. [DOI] [PubMed] [Google Scholar]
- 10.Roubey R A. Immunology of the antiphospholipid antibody syndrome. Arthritis Rheum. 1996;39:1444–1454. doi: 10.1002/art.1780390903. [DOI] [PubMed] [Google Scholar]
- 11.Gleicher N, El-Roeity A, Confino E. et al. Reproductive failure because of autoantibodies unexplained infertility and pregnancy wastage. Am J Obstet Gynecol. 1989;160:1376–1380. doi: 10.1016/0002-9378(89)90858-2. [DOI] [PubMed] [Google Scholar]
- 12.Chaouat G. Regulation of cellular immune response from the mother to the paternal antigens of the conceptus. Prog Clin Biol Res. 1981;70:137–144. [PubMed] [Google Scholar]
- 13.Hemberger M. Immune balance at the foeto-maternal interface as the fulcrum of reproductive success. J Reprod Immunol. 2013;97:36–42. doi: 10.1016/j.jri.2012.10.006. [DOI] [PubMed] [Google Scholar]
- 14.McNeil H P, Chesterman C N, Krilis S A. Immunology and clinical importance of antiphospholipid antibodies. Adv Immunol. 1991;49:193–280. doi: 10.1016/s0065-2776(08)60777-4. [DOI] [PubMed] [Google Scholar]
- 15.Cervera R, Balasch J. Bidirectional effects on autoimmunity and reproduction. Human Reprod Update. 2008;14:359–366. doi: 10.1093/humupd/dmn013. [DOI] [PubMed] [Google Scholar]
- 16.Carp J A, Selmi C, Shoenfeld Y. The autoimmune bases of infertility and pregnancy loss. J Autoimmun. 2012;38:266–274. doi: 10.1016/j.jaut.2011.11.016. [DOI] [PubMed] [Google Scholar]
- 17.Ulcova-Gallova Z. Repeated miscarriages in patients with antiphospholipid syndrome and subjected to in vitro fertilization: the importance of preimplantation genetic diagnosis. Lupus. 2012;21:744–746. doi: 10.1177/0961203312442581. [DOI] [PubMed] [Google Scholar]
- 18.Ulcova-Gallova Z, Krauz V, Novakova P. et al. Anti-phospholipid antibodies against phosphatidylinositol, and phosphatidylserine are more significant in reproductive failure than antibodies against cardiolipin only. Am J Reprod Immunol. 2005;54:112–117. doi: 10.1111/j.1600-0897.2005.00294.x. [DOI] [PubMed] [Google Scholar]
- 19.Ulcova-Gallova Z, Mukensnabl P, Hadravska S. et al. Antibodies against annexin V and prothrombin, their correlation with other anti-phospholipid antibodies in recurrent pregnancy loss. J Reprod Contraception. 2005;16:35–46. [Google Scholar]
- 20.Tincani A, Spatola M, Cinquioni M. et al. Animal models of antiphospholipid syndrome. Rev Rheumat. 1998;65:614–618. [PubMed] [Google Scholar]
- 21.World Health Organization . Geneva, Switzerland: WHO Press; 2010. WHO Laboratory Manual for the Examination and Processing of human Semen. 5th ed; pp. 1–286. [Google Scholar]
- 22.Mackworth-Young C G. Antiphospholipid syndrome: multiple mechanisms. Clin Exp Immunol. 2004;136:393–401. doi: 10.1111/j.1365-2249.2004.02497.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Buyon J P, Petri M A, Kim M Y. et al. The effect of combined estrogen and progesterone hormone replacement therapy on disease activity in systemic lupus erythematosus: a randomized trial. Ann Intern Med. 2005;142:953–962. doi: 10.7326/0003-4819-142-12_part_1-200506210-00004. [DOI] [PubMed] [Google Scholar]
- 24.Girolami A, Zanon E, Zanardi S. et al. Thromboembolic disease developing during oral contraceptive therapy in young females with antiphospholipid antibodies. Blood Coagul Fibrinolysis. 1996;7:497–501. doi: 10.1097/00001721-199606000-00011. [DOI] [PubMed] [Google Scholar]
- 25.Julkunen H, Kaaja R, Jouhikainen T. et al. Malignant hypertension and antiphospholipid antibodies as presenting features of SLE in a young woman using oral contraceptives. Br J Rheumatol. 1991;30:471–482. doi: 10.1093/rheumatology/30.6.471. [DOI] [PubMed] [Google Scholar]
- 26.Duarte C, Inês L. Oral contraceptives and systemic lupus erythematosus: what should we advise to our patients. Acta Reumatol. 2010;35:133–140. [PubMed] [Google Scholar]
- 27.Lakasing L, Khamashta M J. Contraceptive practices in women with systemic lupus erythematosus and/or antiphospholipid syndrome: what advice should we be giving? Fam Plann Reprod Health Care. 2001;27:7–12. doi: 10.1783/147118901101195029. [DOI] [PubMed] [Google Scholar]
- 28.Yazdany J, Trupin L, Kaiser R. et al. Contraceptive counseling and use among women with systemic lupus erythematosus: a gap in health care quality? Arthr Care Res. 2011;63:358–365. doi: 10.1002/acr.20402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Lateef A, Petri M. Hormone replacement and contraceptive therapy in autoimmune disease. J Autoimmun. 2012;38:J170–J176. doi: 10.1016/j.jaut.2011.11.002. [DOI] [PubMed] [Google Scholar]
- 30.Bernier M O, Mikaetoff Y, Hudson M. et al. Combined oral contraceptive use and the risk of systemic lupus erythematosus. Arthr Rheumat. 2009;61:476–481. doi: 10.1002/art.24398. [DOI] [PubMed] [Google Scholar]
- 31.Devreese K M. Antiphospholipid antibody testing and standardization. Int J Lab Hematol. 2014;36:352–363. doi: 10.1111/ijlh.12234. [DOI] [PubMed] [Google Scholar]
- 32.Globerson A, Effros R B. Ageing of lymphocytes and lymphocytes in the aged. Immunol Today. 2000;21:515–521. doi: 10.1016/s0167-5699(00)01714-x. [DOI] [PubMed] [Google Scholar]
- 33.Hradecky L, Subrt I, Ulcova-Gallova Z. Urgent termination of pregnancy in pre-eclampsia and panel of antiphospholipid antibodies. Am J Reprod Immunol. 2009;62:412–417. doi: 10.1111/j.1600-0897.2009.00759.x. [DOI] [PubMed] [Google Scholar]
- 34.Kwak-Kim J, Agcaoili M S, Aleta L. et al. Management of women with recurrent pregnancy losses and antiphospholipid antibody syndrome. Am J Reprod Immunol. 2013;69:596–607. doi: 10.1111/aji.12114. [DOI] [PubMed] [Google Scholar]