Catharyn Stern, M.D.,* Lawrence Chamley, Ph.D., Lyndon Hale, M.D.,* Michael Kloss, M.D., Andrew Speirs, M.D.,* and H. W. Gordon Baker, Ph.D.

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1 FERTILITY AND STERILITY VOL. 70, NO. 5, NOVEMBER 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Received April 29, 1998; revised and accepted June 26, This work was supported by Melbourne IVF Research Fund, East Melbourne, Victoria, Australia, the Bonnie Babes Foundation, Knoxfield, Victoria, Australia, and the New Zealand Lottery Grants Board, Wellington, New Zealand, whose contribution is gratefully acknowledged. Lawrence Chamley is an Overseas/ Repatriation Research Fellow of the Health Research Council of New Zealand. Part of this work was presented at the British Fertility Society meeting, April 1998, Sheffield, United Kingdom. Address for correspondence and reprints: Catharyn Stern, M.D., Reproductive Biology Unit, Royal Women s Hospital, Grattan Street, Carlton, 3053, Victoria, Australia (FAX: ; sternc@cryptic.rch. unimelb.edu.au). * Reproductive Biology Unit, Royal Women s Hospital, Melbourne, Australia. Department of Obstetrics and Gynaecology, University of Auckland, National Women s Hospital, Auckland, New Zealand. Recurrent Miscarriage Clinic, Royal Women s Hospital, Melbourne, Australia. University Department of Obstetrics and Gynaecology, Royal Women s Hospital, Melbourne, Australia /98/$19.00 PII S (98) Antibodies to 2 glycoprotein I are associated with in vitro fertilization implantation failure as well as recurrent miscarriage: results of a prevalence study Catharyn Stern, M.D.,* Lawrence Chamley, Ph.D., Lyndon Hale, M.D.,* Michael Kloss, M.D., Andrew Speirs, M.D.,* and H. W. Gordon Baker, Ph.D. Royal Women s Hospital, Melbourne, Victoria, Australia, and University of Auckland, National Women s Hospital, Auckland, New Zealand Objective: To investigate whether antiphospholipid and related autoantibodies are associated with IVF implantation failure as well as with recurrent spontaneous miscarriage. Design: Prevalence study. Setting: University teaching hospital and associated IVF unit. Patient(s): Patients with at least three consecutive first-trimester miscarriages (n 97), undergoing IVF who had at least 10 embryos transferred without any resulting clinical pregnancy (n 105), fertile women (n 106), and newly referred for IVF treatment (n 52). Intervention(s): Antibodies tested included lupus anticoagulant; immunoglobulin (Ig) G and IgM isotypes of each of anticardiolipin antibody, antiphosphatidylserine, antiphosphatidylethanolamine, and antiphosphatidylinositol; 2 glycoprotein I antibodies; and antinuclear antibodies. Statistical analysis included 2 and Fisher s exact tests for differences between groups, and multiple linear regression analysis and Spearman s nonparametric tests for relations between results. Main Outcome Measure(s): Seropositivity for autoantibodies tested. Result(s): Overall, 84 (23%) of the 360 samples tested positive for at least one autoantibody. 2 Glycoprotein I IgM antibody and antinuclear antibody were significantly associated with both IVF implantation failure and recurrent miscarriage. Conclusion(s): Autoantibodies, particularly 2 glycoprotein I antibodies and antinuclear antibodies, are associated with IVF implantation failure as well as with recurrent spontaneous abortion, although the mechanism is still unclear. The high seroprevalence of antibodies to 2 glycoprotein I suggests that it may have an important role in autoimmune reproductive failure that needs to be explored further. (Fertil Steril 1998;70: by American Society for Reproductive Medicine.) Key Words: Antiphospholipid antibodies, 2 glycoprotein I, antinuclear antibodies, implantation failure, recurrent miscarriage Antiphospholipid antibodies (apls) are a group of autoantibodies that react with negatively charged phospholipids. Lupus anticoagulant and anticardiolipin antibodies are the two most commonly encountered apls. Tests also have been introduced for antibodies to other phospholipids, including phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine (1, 2). Although the pathogenetic mechanisms by which apls affect reproductive performance are poorly understood, this group of autoantibodies is associated with both thrombotic disorders and adverse pregnancy outcomes (3 7). The term reproductive autoimmune failure syndrome has been coined to designate the entire spectrum of the reproductive process that is susceptible to immunologic disruption (8). It is possible that similar mechanisms may underlie both implantation failure and recurrent spontaneous abortion. In this study, we examined autoimmune mechanisms associated with unsuccessful pregnancy. The putative role of antiphospholipid antibodies in with infertility is uncertain (9, 10), particularly in undergoing IVF 938

2 who have implantation failure in whom repeated ET has been unsuccessful (11 13). Although most groups report an increased prevalence of autoantibodies in with infertility (12, 14), there is conflicting evidence regarding IVF outcome (7, 15 18). Part of this confusion relates to differences in the antibodies tested. Some groups have tested primarily for anticardiolipin antibodies, lupus anticoagulant, and/or antinuclear antibodies (12), whereas others have evaluated a more comprehensive range, including antibodies to the negatively charged phosphatidylserine and phosphatidylinositol, and to the zwitterionic phosphatidylethanolamine (1, 2, 14, 15, 18). With the exception of anticardiolipin (19), there is no universally accepted standard for the determination of apl levels, and this lack of standardization contributes to the confusion (18). Since the discovery that autoimmune apls do not bind directly to phospholipid, but rather to a complex antigen comprised of phospholipid and cofactor proteins, attention has shifted to these proteins (20 23). 2 Glycoprotein I ( 2 GPI), also known as apolipoprotein H, has been identified as the cofactor for anticardiolipin, and it is possible that testing for antibodies to cofactor proteins such as 2 GPI may be more clinically discriminating than traditional apl assays. The possibility that autoimmune mechanisms are involved in implantation failure is of great importance because IVF is hampered by relatively low implantation rates. Many undergo repeated transfers without success; 15% of embryos transferred actually implant in most IVF cycles, whereas with natural conception, implantation rates are at least 20%. Given the conflicting evidence relating to the importance of apls in with failed IVF cycles, we performed a prospective prevalence study to determine whether with IVF implantation failure exhibit an increased prevalence of antinuclear antibodies, apls, and, particularly, antibodies to 2 GPI. Control groups included fertile women, newly referred for IVF treatment and women with recurrent spontaneous miscarriage. MATERIALS AND METHODS This prospective prevalence study was approved by the Royal Women s Hospital institutional review board. Patients were recruited from the hospital clinics and associated IVF units and gave informed consent. Patient population Patients with IVF implantation failure We evaluated 105 aged 24 to 47 years (mean age, 35 years) who enrolled in our IVF program between January 6, 1996, and July 8, 1997, and who had undergone multiple ETs and previously had at least 10 embryos transferred (mean total transferred, 16; range, 10 42) without achieving a clinical pregnancy. For the purpose of this study, a clinical pregnancy was defined as a pregnancy diagnosed initially by biochemical means at 17 days after ET (serum hcg level of 100 IU) with consequent evidence of a gestational sac with or without a fetal heart observed on transvaginal ultrasound 28 days after ET. Major infertility diagnoses for these were occlusive tubal disease diagnosed by laparoscopy or radiologic examination in 28 (27%); significant endometriosis involving the ovaries, classified as revised American Fertility Society stage III or IV (24) diagnosed by laparoscopy in 8 (8%); isolated male factor infertility (as per World Health Organization guidelines for subfertility) in 37 (35%); combined tubal disease and male factor infertility in 9 (8%); and unexplained infertility in 23 (22%). New IVF Fifty-two women newly referred to our clinic for IVF who had not yet commenced treatment were tested to ascertain whether infertility per se may contribute to an increased frequency of autoantibodies in women in whom treatment with IVF fails. These women had a mean age of 32.8 years (range, years). Their major infertility diagnoses were occlusive tubal disease in 9 (17%), significant endometriosis in 6 (12%), isolated male factor infertility in 25 (48%), and unexplained infertility in 12 (23%). Patients with recurrent spontaneous abortion We also evaluated 97 women with a mean age of 32.7 years (range, years) who were attending the Recurrent Miscarriage Clinic. All these women had had at least three sequential first-trimester clinical pregnancy losses (mean, four, range, 3 16) and were classified into the recurrent spontaneous abortion group. Thirty-seven women in this group (38%) were pregnant at the time of the study. Patients were investigated for obvious causes of their implantation failure or recurrent miscarriage with hysteroscopy and karyotypic analysis of both partners. Patients in whom a major uterine or karyotypic abnormality was found to contribute to their reproductive failure were excluded from analysis in this study. Fertile controls The fertile control group consisted of 106 women who had at least one child born at term without any major pregnancy complications and without any period of subfertility (i.e., 6 months of trying) before conception. These women ranged in age from years (mean age, 33.6 years). Fifty-six of these women (55%) were pregnant at the time of the study. FERTILITY & STERILITY 939

3 Laboratory evaluation Lupus anticoagulant Testing was performed according to established recommendations (25) and involved a panel of coagulation tests, including activated partial thromboplastin time (normal, seconds), kaolin clotting time ratio (normal, 1.2), kaolin clotting time mixing test ratio (normal, 1.2), and dilute Russell viper venom ratio (normal, ). Two percent of specimens were reported as unsatisfactory for assessment because of activation. Fresh samples were obtained from these and the results were included in the analysis. Antinuclear antibodies Antinuclear antibodies were detected by immunofluorescence using Hep 2 cells as the antigen (Hep-2000ANA Test System; Immunoconcepts, Sacramento, CA). A titer of 1/80 was classified as positive for the purpose of this study. Solid-phase antiphospholipid antibody ELISAs The phospholipids (phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol) and cardiolipin were obtained from Sigma (Sydney, New South Wales, Australia). Antibody screening was conducted as previously described (26). Briefly, the relevant phospholipid was diluted to 50 g/ml in ethanol, and 50 L was used to coat a 96-well ELISA plate (Corning High Binding, Corning, Corning, NY), by evaporation at 4 C overnight. Plates were exposed to blocking solution (10% newborn calf serum in phosphatebuffered saline [PBS], ph 7.4) for 1 hour at room temperature. The blocking solution was discarded and the plates were washed three times with PBS (ph 7.4). Serum samples, diluted 1:100 in blocking solution, then were incubated on the plates for 1 hour at room temperature. The plates then were washed three times with PBS (ph 7.4) and horseradish peroxidase conjugated goat antihuman -chain or -chain antiserum (Tago, Burlingame, CA), diluted 1:8,500 in blocking solution, was added for 1 hour at room temperature. The plates again were washed three times with PBS (ph 7.4) and the assay was developed by the addition of 1 mg/ml of -phenylamine diamine dihydrochloride (Sigma) in 0.1M citrate buffer (ph 5.5) containing freshly prepared.005% H 2 O 2. The reaction was stopped by the addition of 10% HCl, and the optical density at 490 nm was determined. Multiples of the median were used to determine which samples were positive. Samples were considered to be positive when the optical density of a sample exceeded the multiple of the median of the 95th percentile of 284 normal serum samples (188 men and 104 fertile women). All assays included serum from normal samples. The intra-assay coefficients of variation ranged from 5% to 13%, and the interassay coefficients of variation ranged from 7% to 17%. Solid-phase 2 GPI antibody ELISAs 2 Glycoprotein I was purified from normal human plasma as previously described (27). Enzyme-linked immunosorbent assay plates were coated overnight at 4 C with 50 L of purified 2 GPI (10 g/ml in 0.1M carbonate buffer, ph 9.0). The antigen solution was discarded and the plates were blocked with 5% nonfat milk powder dissolved in PBS containing 0.05% Tween 20 (ph 7.4) for 1 hour at room temperature. The plates were washed three times with PBS- Tween before serum samples, diluted 1:200 in the blocking solution, were added for 1 hour at room temperature. Thereafter, the method was the same as for the phospholipid antibody tests. The intra-assay and interassay coefficients of variation were 10% and 6%, respectively, for the immunoglobulin (Ig) G assay and 6% and 12%, respectively, for the IgM assay. Statistical Analysis Differences between the frequencies of positive results were tested by the 2 or exact test. Antibody levels were related to patient characteristics by Spearman s nonparametric correlation test or regression analysis. RESULTS A significantly higher frequency of antinuclear antibodies and 2 GPI IgM antibodies was found in the with recurrent miscarriage and IVF implantation failure than in the fertile controls (Table 1 and Fig. 1). There was no statistically significant association between any other autoantibodies and any particular patient group. No in any of the study groups tested positive for lupus anticoagulant. The incidence of individual antibodies was low in all groups, although some tended to be higher in the recurrent miscarriage and IVF implantation failure groups without reaching statistical significance. Overall, 84 of the 360 samples (23%) tested positive for at least one antiphospholipid antibody or 2 GPI antibody (Table 2): 16% of in the control group, 21% in the new IVF group, 26% in the recurrent miscarriage group, and 30% in the IVF implantation failure group. The last finding was significant compared with the fertile control group (P.019). Six in the recurrent miscarriage group and 6 in the IVF implantation failure group were positive for 2 antiphospholipid antibodies compared with 1 patient in the fertile control group. These results approached statistical significance (P.054). In addition to a strong association between the two isotypes of anticardiolipin (P.001), there was a tendency for both anticardiolipin IgG and IgM to be found in association with the other apls, particularly antiphosphatidylethanolamine IgM, antiphosphatidylserine IgG, and antiphosphati- 940 Stern et al. Antibodies and IVF Vol. 70, No. 5, November 1998

4 TABLE 1 Prevalence of different autoantibodies in control and patient groups. No. (%) of different autoantibodies in indicated patient group TABLE 2 Presence of multiple antiphospholipid and/or 2 glycoprotein I antibodies in control and patient groups. No. of (%) in each indicated group who tested positive for one or more autoantibodies* Test Fertile controls (n 106) New IVF (n 52) RSA (n 97) IVF-IF (n 105) No. of positive antibodies Controls (n 106) New IVF (n 52) RSA (n 97) IVF-IF (n 105) acl IgG 2 (1.9) 0 4 (4.1) 3 (2.9) acl IgM 1 (1.0) 0 4 (4.1) 6 (5.7) LAC ape IgG 6 (5.6) 7 (13.5) 3 (3.1) 5 (4.8) ape IgM 1 (1.0) 1 (1.9) 2 (2.1) 5 (4.8) aps IgG 2 (1.8) 0 4 (4.1) 4 (3.8) aps IgM 4 (3.8) 0 2 (2.1) 6 (5.7) api IgG 1 (1.0) 1 (1.9) 3 (3.1) 4 (3.8) api IgM 2 (1.9) 1 (1.9) 4 (4.1) 4 (3.8) a 2 GPI IgG 6 (5.6) 1 (1.9) 6 (6.2) 7 (6.7) a 2 GPI IgM 0 2 (3.8) 15 (15.5)* 9 (8.6)* ANA 10 (9.4) 5 (9.6) 22 (22.7)* 22 (21.0)* * P.05 (patient groups vs. fertile controls). Note: a 2 GPI antibody to 2 glycoprotein I; acl anticardiolipin antibody; ANA antinuclear antibody; ape antibody to phosphatidylethanolamine; api antibody to phosphatidylinositol; aps antibody to phosphatidylserine; IVF-IF IVF implantation failure; LAC lupus anticoagulant; RSA recurrent spontaneous abortion. dylinositol IgG, as well as 2 GPI IgM antibodies (P.005). Antinuclear antibody was associated with apls, particularly anticardiolipin IgG (P.023) and 2 GPI IgM antibodies (P.042). Multiple linear regression analysis suggested a strong 1 10 (9) 9 (17) 16 (17) 18 (17) 2 6 (6) 2 (4) 3 (3) 7 (7) 3 1 (1) 2 (2) 4 (4) 4 1 (1) 1 (1) 5 3 (3) 1 (1) * Includes the following antibodies: a 2 GPI, acl, ape, api, aps, and LAC. Note: a 2 GPI antibody to 2 glycoprotein I; acl anticardiolipin antibody; ape antibody to phosphatidylethanolamine; api antibody to phosphatidylinositol; aps antibody to phosphatidylserine; IVF-IF IVF implantation failure; LAC lupus anticoagulant; RSA recurrent spontaneous abortion. association between antinuclear antibodies and tubal infertility (P.005). No statistically significant relation was found between antibody status and age, race, number of previous miscarriages, or number of embryos transferred. DISCUSSION Our results demonstrate a new finding: that IgM antibody to 2 GPI, the anticardiolipin cofactor, is significantly associated with IVF implantation failure as well as with recurrent spontaneous abortion. The prevalence of 2 GPI antibody FIGURE 1 Prevalence of IgM antibodies to 2 glycoprotein I in control and patient groups. IVF-IF IVF implantation failure; RSA recurrent spontaneous abortion. FERTILITY & STERILITY 941

5 was 16% in the recurrent miscarriage group and 9% in the IVF implantation failure group compared with 0% in a control group of fertile women. Women with infertility who had not yet commenced treatment also demonstrated a greater frequency of positivity than controls but a lower frequency than women with implantation failure. Although follow-up of these new IVF is required to allow further insight into the significance of these results, it is possible that testing for this antibody may provide prognostic information with respect to the future success of IVF treatment. There also was a tendency toward higher frequencies of antiphospholipid antibodies tested in the groups of women with recurrent miscarriage and IVF implantation failure, particularly the combination of three or more positive tests. Tubal disease was the only infertility diagnosis that correlated with antibody positivity. The increased prevalence of antinuclear antibodies in women with recurrent miscarriage or IVF implantation failure found in our study concurs with results reported elsewhere (12, 28), and is thought to be suggestive of an underlying autoimmune disturbance rather than a specific phospholipid-related thrombogenic mechanism. Antiphospholipid antibodies are found in low levels in many normal individuals, and the level of antibodies does not appear to vary with current pregnancy status (28, 29). The reported prevalence of apls ranges from 1% 17%, depending on the cutoff point used, the isotype, and the range of the antigenic specificities investigated (28 30). In our study, 16% of healthy fertile women were positive for at least one isotype of a panel of five antiphospholipid antibodies, which is significantly higher than the 5% seroprevalence recently reported by Coulam et al. (15). The role of apls in with infertility who are undergoing treatment with IVF is controversial. Most studies suggest that presenting for IVF have higher levels of autoantibodies, but the relation with IVF outcome is less clear (7, 14, 18). Some groups have found an association between the presence of apls and unsuccessful IVF treatment (11, 12, 15), whereas others have failed to confirm this (7, 31 33). In this study, the high incidence of apls suggests an association of these antibodies with implantation failure. The strongest association was with 2 GPI IgM antibody. It is conceivable that apls can arise in undergoing IVF as a result of the treatment process. The high prevalence of apls in infertile women who had not commenced IVF treatment in our study argues against this hypothesis, and is in accord with the results of Fisch et al. (34) and of Birdsall et al. (7). No clear-cut factors (e.g., previous IVF procedures, pregnancy, hormonal states, specific infertility diagnoses) have been identified as a source of autoantibody inducement or activation, and to date, no autoantibody markers have been identified that can reliably predict the outcome of individual IVF cycles. The term antiphospholipid antibody syndrome has been used most commonly to describe clinical problems such as venous or arterial thrombosis in the presence of lupus anticoagulant and/or anticardiolipin IgG/IgM (35). However, there are many women in whom the only clinical presentation of this syndrome is recurrent pregnancy failure. Although the association between autoantibodies, thrombosis, and pregnancy failure is not clear, it appears that autoantibodies exert their actions either by interfering with cell membrane surface hemostatic reactions or by reacting with antigens on cell surfaces, resulting in altered cell activity (36). The importance of 2 GPI became apparent when it was discovered that autoimmune anticardiolipin antibodies do not bind directly to phospholipid, but rather to a complex antigen comprised of phospholipid and a cofactor, identified as 2 GPI (20 22). In one of the earliest studies of the clinical significance of 2 GPI, Aoki et al. (37) investigated the association between 2 GPI-dependent and 2 GPI-independent anticardiolipin antibodies and pregnancy outcome. These investigators did not test for antibodies to 2 GPI itself. Whereas 3.3% of healthy pregnant women were positive for 2 GPI-independent anticardiolipin antibodies, none were positive for 2 GPI-dependent anticardiolipin antibodies. Thus, they concluded that 2 GPI-independent anticardiolipin antibodies were less useful markers of miscarriage risk (37). In vitro, 2 GPI is a phospholipid-dependent anticoagulant that inhibits the prothrombinase activity of platelets and adenosine diphosphate P induced platelet aggregation (38, 39). This anticoagulant activity results from binding to negatively charged phospholipid surfaces such as activated platelets. Although it is appealing to postulate that the pathogenic mechanisms of apls relate to neutralization of the anticoagulant functions of 2 GPI, a deficiency of 2 GPI does not appear to correlate with a predisposition to thrombosis (40). However, mechanisms by which 2 GPI antibodies may inhibit phospholipid-dependent reactions recently have been described (41), and involve inhibition of binding of other phospholipid-binding proteins and/or anticoagulants. The accurate comparison of studies of apls is extremely difficult because of the differing methods used to determine the positive cutoff point, and because of the range of phospholipid antigens tested. However, we are unaware of any other studies that have tested for 2 GPI antibodies in with implantation failure. A particularly disquieting development is the proliferation of tests for different phospholipid-reactive autoantibodies that may be used for diagnosis before the prevalence of these antibodies in the normal population has been estab- 942 Stern et al. Antibodies and IVF Vol. 70, No. 5, November 1998

6 lished. It is surprising that this lack of standardization of prevalence testing has not inhibited the development of treatment protocols for presumed to have autoimmune-related reproductive failure. Although the pathogenic mechanisms of apls in reproductive failure are still unknown, their presumed thrombotic effects have led to the use of heparin and aspirin for women with apls and recurrent spontaneous abortion or IVF implantation failure (13, 42 44). In conclusion, we demonstrated that with IVF implantation failure as well as with recurrent spontaneous abortion have an increased seroprevalence of IgM antibodies reactive with 2 GPI, a protein implicated in aplmediated reproductive failure. Testing for this antibody thus may provide useful prognostic information with respect to the success or failure of IVF treatment. However, it is important to acknowledge Gleicher s (17) timely reminder that the mere presence of autoantibodies only denotes the risk of a disease or clinical problem and not necessarily the presence of the disease itself. We also must remember that the links found between apls and unsuccessful pregnancy outcomes, although clinically intriguing, do not provide definite evidence that these antibodies cause the disease phenomena, but merely that they are associated with these circumstances. It is possible that an underlying immunologic disturbance may be associated both with infertility (and IVF failure) and with the presence of these antiphospholipid antibodies (17). Further research is required before the disappointingly low implantation rates that are found in some who undergo IVF, and that are presumed to be mediated by autoimmune dysfunction, can be treated in accordance with the principles of evidence-based medicine (45). Acknowledgments: The authors thank Andrea Duncalf, BSC, for technical assistance, Kevin Townend, BSC, for statistical advice, Helen Norris, RM, for data collection and trial coordination, and Lisa Dunlop, RM, and Sonia Grover, MD, for patient recruitment. References 1. Matzner W, Chong P, Xu G, Ching W. Characterisation of antiphospholipid antibodies in women with recurrent spontaneous abortions. J Reprod Med 1994;39: Yetman DL, Kutteh WH. Antiphospholipid antibody panels and recurrent pregnancy loss: prevalence of anticardiolipin antibodies compared with other antiphospholipid antibodies. Fertil Steril 1996;66: Harris EN, Gharavi AE, Boey ML, Portel BM, Mackworth-Young CC, Loizou S, et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2: Cowchock S, Smith JB, Gocial S. Antibodies to phospholipids and nuclear antigens in with related abortions. 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7 34. Fisch B, Rikover Y, Shohat L, Zurgil N, Tadir Y, Ovadia J, et al. The relationship between in vitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid autoantibodies. Fertil Steril 1991;56: Triplett D. Antiphospholipid antibodies and recurrent pregnancy loss. Am J Reprod Immunol 1989;20: Roubey RA, Hoffman M. From antiphospholipid syndrome to antibody-mediated thrombosis. Lancet 1997;350: Aoki K, Matsuura E, Sasa H, Yagami Y, Dudkiewicz A, Gleicher N. 2 Glycoprotein I-dependent and independent anticardiolipin antibodies in healthy pregnant women. Hum Reprod 1994; Nimpf J, Bevers E, Bomans P, Til U, Wurm H, Kostner G, et al. Prothrombinase activity of human platelets is inhibited by 2 GPI. Biochim Biophys Acta 1986;884: Nimpf J, Wurm H, Kostner G. 2 GPI inhibits the release reaction of human platelets during ADP-induced aggregation. Atherosclerosis 1987;63: Laszlo F, Bancsi M, van der Linden I, Bertina R. 2 Glycoprotein I deficiency and the risk of thrombosis. Thromb Haemost 1992;67: Takeya H, Mori T, Gabazza EC, Kuroda K, Deguchi H, Matsuura E, et al. Anti- 2 glycoprotein I monoclonal antibodies with lupus anticoagulant-like activity enhance the 2 GPI binding to phospholipids. J Clin Invest 1997;99: Kutteh W. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low dose aspirin is superior to low dose aspirin alone. Am J Obstet Gynecol 1996;174: Kutteh W, Yetman D, Chantilis S, Crain J. Effect of antiphospholipid antibodies in women undergoing in vitro fertilization: role of heparin and aspirin. Hum Reprod 1997;12: Rai R, Cohen H, Davey M, Regan L. Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with antiphospholipid antibodies. Br Med J 1997;314: Grimes D. Introducing evidence-based medicine into a department of obstetrics and gynecology. Obstet Gynecol 1995;86: Stern et al. Antibodies and IVF Vol. 70, No. 5, November 1998