Prevalence and heterogeneity of antiphosphatidylethanolamine antibodies in patients with recurrent early pregnancy losses

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1 FERTILITY AND STERILITY VOL. 71, NO. 6, JUNE 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Prevalence and heterogeneity of antiphosphatidylethanolamine antibodies in patients with recurrent early pregnancy losses Toshitaka Sugi, M.D., Ph.D.,* Junko Katsunuma, M.D.,* Shun-ichiro Izumi, M.D., Ph.D.,* John A. McIntyre, Ph.D., and Tsunehisa Makino, M.D., Ph.D.* Tokai University School of Medicine, Kanagawa, Japan, and Methodist Hospital of Indiana, Indianapolis, Indiana Received September 3, 1998; revised and accepted January 5, Supported in part by a Grant of the Science Frontier Program by Ministry of Education, Science and Culture, Japan, a grant-in-aid for scientific research ( ) from the Ministry of Education, Science and Culture, and 1997 Tokai University School of Medicine Research Aid, Knagawa, Japan. Reprint requests: Toshitaka Sugi, M.D., Ph.D., Department of Obstetrics and Gynecology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, , Japan (FAX: ; sugi@is.icc.u-tokai.ac.jp). * Department of Obstetrics and Gynecology, Tokai University School of Medicine. Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana /99/$20.00 PII S (99) Objective: To describe the prevalence of antiphospholipid antibodies to both anionic and zwitterionic phospholipids in women with early recurrent pregnancy losses (RPLs). Design: Retrospective data analysis. Setting: Tokai University Hospital, Kanagawa, Japan. Patient(s): One hundred thirty-nine patients with unexplained early RPLs. Intervention(s): None. Main Outcome Measure(s): Enzyme-linked immunosorbent assays were used to measure autoantibodies to phosphatidylethanolamine, cardiolipin, and phosphatidylserine. Result(s): Twenty-eight (20.1%), 17 (12.2%), and 2 (1.4%) patients of the 139 total patients were positive for immunoglobulin (Ig) G, IgM, and IgA antiphosphatidylethanolamine antibodies, respectively. Because 3 patients had two isotypes, 44 (31.7%) of the patients were positive for antiphosphatidylethanolamine antibodies. Six patients (4.3%) and 1 patient (0.7%) were positive for IgG and IgM antiphosphatidylserine antibodies, respectively. Seven patients (5%) were positive for 2 -glycoprotein I independent anticardiolipin IgG, and 1 patient was positive for 2 -glycoprotein I dependent anticardiolipin IgG. Two patients (1.4%) had lupus anticoagulant. Conclusion(s): Our data show a statistically stronger association between RPLs and antiphosphatidylethanolamine antibodies than between RPLs and antibodies to anionic phospholipids for early gestational losses. Our data suggest that antiphosphatidylethanolamine antibodies may be a risk factor in patients with early RPLs. (Fertil Steril 1999;71: by American Society for Reproductive Medicine.) Key Words: Antiphosphatidylethanolamine antibodies, antiphospholipid antibodies, kininogen, recurrent pregnancy loss, lupus anticoagulant Antiphospholipid antibodies (apas) to anionic phospholipids such as cardiolipin and phosphatidylserine have been described in patients with thrombosis, thrombocytopenia, and recurrent fetal loss (1 3). Similar but fewer reports have focused on autoantibodies to the zwitterionic phospholipid phosphatidylethanolamine (PE) (4 8). Because PE is a major component of both the outer and inner leaflets of cell plasma membranes, the production of autoantibodies to PE should not be viewed as without consequence. Recent evidence shows that many apas to negatively charged phospholipids do not target anionic phospholipids per se but are specific for anionic phospholipid-binding plasma proteins. At present, the most common and best characterized plasma protein apas antigenic targets are 2 -glycoprotein I ( 2 GPI) and prothrombin (9). We recently reported that certain antiphosphatidylethanolamine antibodies (apes) are not specific for PE per se but are directed to PE-binding plasma proteins, such as high-molecular-weight kininogen (HK), low-molecu- 1060

2 lar-weight kininogen (LK), and proteins in complex with HK, factor XI, or prekallikrein (8). We also demonstrated that kininogen-dependent apes can augment thrombin-induced platelet aggregation in vitro (10, 11). Recurrent pregnancy loss (RPL), occurring mostly in the second and third trimesters of pregnancy in apa-positive mothers, is one of the hallmark clinical manifestations of the antiphospholipid syndrome (12 14). Recurrent pregnancy loss in the first-trimester fetal period ( 10 weeks gestation) also has been associated with the antiphospholipid syndrome (15). However, the association between apas and recurrent embryonic losses ( 10 weeks gestation) are not well documented (15). In fact, several studies have questioned whether an association exists between apas to negatively charged phospholipids and early RPL (15 17). The female reproductive tract is the second richest site for kininogen and its metabolic products in the body (18 21). Adam et al. (21) measured 12.2, 10.9, 0.4, and 1.2 g/mg of T-kininogen in rat plasma, uterus, liver, and kidney, respectively. The kininogen concentration in reproductive tissues and plasma was reported to fluctuate during ovulation, pregnancy, and parturition (18, 21). Why the female reproductive system is so rich in kininogen and what governs the fluctuation of kininogen concentrations at the local level remains to be elucidated. Because numerous studies (12 14) have concluded that RPLs are associated with apas to anionic phospholipids, and in view of the conspicuous presence of kininogen in reproductive tissues, we tested patients with RPLs for apes, especially those patients with RPLs during the embryonic period. We showed a strong association between RPLs and apes, the latter of which require the presence of kininogen or other plasma proteins. Our data suggest that apes may represent a significant risk factor for early RPLs. MATERIALS AND METHODS Patients and Controls Plasma samples were obtained from 139 nonpregnant patients with a history of RPLs during the embryonic period (15). The patients met our study entry criteria, which were as follows: [1] two or more pregnancy losses before 10 weeks gestation, exclusive of ectopic pregnancy and elective abortion; [2] no presumptive cause found for RPLs after routine evaluation for detection of uterine factors (i.e., normal hysterosalpingography and ultrasound examinations); [3] absence of chromosomal abnormalities; [4] absence of endocrine factors (normal prolactin and progesterone levels and normal thyroid function); [5] absence of infectious factors (no group B streptococcal or Chlamydia trachomatis infection); and [6] absence of diabetes mellitus. The mean age of the patients was 31 years (range, years), and the mean number of pregnancy losses was 2.8. All plasma samples were stored at 70 C until use. Two hundred age-matched, healthy, nonpregnant female volunteers with no history of miscarriage were tested as controls. Positive apa samples from patients with systemic lupus erythematosus were used at 1:100 dilutions to establish standard curves. This study was approved by the Institutional Review Board of the Tokai University School of Medicine. Methods The ELISA for apas followed a previously described procedure (8, 10, 23), with slight modifications. Immulon 3 microtiter plate wells (Dynatech Laboratories, Chantilly, VA) were coated with 30 L of50- g/ml of PE (Avanti Polar Lipids, Birmingham, AL) or phosphatidylserine (Sigma Chemical Co., St. Louis, MO), diluted in chloroform and methanol at a ratio of 1:3, and dried under nitrogen. Each well was blocked for 1 hour with 10% bovine serum albumin (Sigma Chemical Co.) in tris(hydroxymethyl)aminomethane (Tris) buffered saline (0.02 M of Tris hydrochloride and 0.15 M of NaCl, ph 7.3). To detect phospholipid-binding plasma protein dependent and independent apa ELISA reactivity, 50 L of patient plasma diluted 1:100 in Tris-buffered saline containing either 10% adult bovine plasma (Sigma Chemical Co.) or 1% bovine serum albumin was incubated in triplicate for 1 hour. Antiphospholipid antibodies were assessed with the use of alkaline phosphatase conjugated monoclonal antibody to human immunoglobulin (Ig) G, IgM, and IgA (Sigma Chemical Co.). The plates were washed three times with Tris-buffered saline after phospholipid coating, blocking, primary antibody incubation, and conjugate incubation. Color development, produced by paranitrophenyl phosphate substrate, was measured by optical density at 405 nm. Color development was stopped with 75 L of 3N NaOH when the positive controls reached an optical density of 1.0 at 405 nm. Nonspecific binding control wells (without phospholipid coating) were processed in parallel with the ELISA for apas, and the background values were subtracted. Positive values were determined as previously described (7, 8, 22, 23). Data were reported as multiples of the mean of the optical density values of 200 nonpregnant women. Results were defined as positive if they were equal to or greater than the multiples of the mean that encompassed the optical density determinations for 95% of the control values. This approach was used because of the nonparametric distribution of ELISA data for apas obtained from 200 control samples studied for each antibody isotype and phospholipid antigen combination. Positive samples were retested to confirm the results. Samples positive for phospholipid-binding plasma protein dependent IgG apes were investigated for kininogen dependence in an ELISA according to previously described procedures (7, 8). Patient plasma positive for plasma protein dependent IgG apes was diluted (1:100) in either a FERTILITY & STERILITY 1061

3 TABLE 1 Antiphosphatidylethanolamine antibodies in 139 patients with recurrent early (embryonic stage) pregnancy losses and 200 normal controls. Independent apes* Dependent apes Independent and dependent apes Total Isotype RPL Controls RPL Controls RPL Controls RPL Controls P value IgG 7 (5.0) 8 (4.0) 16 (11.5) 7 (3.5) 5 (3.6) 1 (0.5) 28 (20.1) 16 (8.0).001 IgM 8 (5.8) 10 (5.0) 8 (5.8) 6 (3.0) 1 (0.7) 0 (0) 17 (12.2) 16 (8.0) NS IgA 2 (1.4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (1.4) 0 (0) NS Note: Values represent no. of patients (%). apes antiphosphatidylethanolamine antibodies; Ig immunoglobulin; RPLs recurrent pregnancy losses; NS not significant. * Phosphatidylethanolamine binding protein independent apes (patient sample diluted 1:100 in 1% bovine serum albumin). Phosphatidylethanolamine binding protein dependent apes (patient sample diluted 1:100 in 10% adult bovine plasma). partially purified kininogen preparation eluted from a carboxymethyl-papain-sepharose affinity column or 1% bovine serum albumin, and the assay was continued as described for the apas ELISA. The partially purified kininogen preparation from the carboxymethyl-papain column contained HK, LK, and the HK-binding proteins factor XI and prekallikrein (8). Plasma IgG to the 2 GPI-cardiolipin complex was measured with a commercially available ELISA (Yamasa Corporation, Tokyo, Japan) according to the manufacturer s recommendations (14, 16, 24, 25). This kit contains human recombinant 2 GPI in the dilution buffer. Immunoglobulin G to cardiolipin also was measured in the absence of 2 GPI (24, 25). Positive values were determined by previously described methods (14, 16). Lupus anticoagulant activity was detected with screening assays and confirmed with neutralization procedures. Detection was performed with the activated partial thromboplastin time and dilute Russell s viper venom time according to the method of Thiagarajan et al. (26) (LA-Screen; MBL, Nagoya, Japan). These assays were performed on the control pooled plasma and on each control individual plasma that was used to obtain the control pooled plasma. For each assay, control clotting time ratios (control individual plasma clotting time/control pooled plasma clotting time) were calculated and normal values of clotting time ratios were assumed to be less than or equal to the 99th percentile of the obtained control clotting time ratios. Thus, a clotting time ratio between that of the patient s sample and the control pooled plasma of 1.2 for the activated partial thromboplastin time and/or 1.2 for the dilute Russell s viper venom time indicated a prolonged time and was scored as a positive result. Each time a prolonged activated partial thromboplastin time or dilute Russell s viper venom time was found, a mixing study was performed. The corresponding clotting time of a 1:1 mixture of sample and control pooled plasma was performed. The clotting time ratio of the mixture (mixture of sample and control pooled plasma clotting time/ control pooled plasma clotting time) was calculated. The clotting times of each 1:1 mixture of control individual plasma and control pooled plasma were evaluated, and the control mixture clotting time ratio was calculated. Normal results of mixing studies performed on a patient s plasma sample were assumed if the corresponding mixture clotting time ratio was less than or equal to the 99th percentile of the corresponding control mixture clotting time ratio. Thus, samples with a mixture clotting time ratio of 1.2 for the activated partial thromboplastin time and/or 1.2 for the dilute Russell s viper venom time indicated the presence of inhibitor activity. Lupus-like anticoagulant activity was confirmed by high phospholipid concentrations (LA-Confirm, MBL) according to the manufacturer s recommendations. Antinuclear antibodies were detected by indirect immunofluorescence on Hep-2 slides (FANAwell; Nihon DPC Corp., Tokyo, Japan) (27). Patient sera were diluted (1:80) in the dilution buffer. Statistical Analysis Differences between the two groups were analyzed for statistical significance (P.05) with the 2 test. RESULTS Detection of apes in Patients with RPLs Patients with RPLs (n 139) were screened for phospholipid-binding plasma protein dependent versus independent IgG apes with the use of 10% adult bovine plasma versus 1% bovine serum albumin in the patient plasma diluent. As shown in Table 1, 28 (20.1%) of the patients with RPLs were positive for IgG apes. A positive test result for IgG apes was more frequent in the patients with RPLs than in the control group (P.001). The patients with RPLs also were screened for phospholipid-binding plasma protein dependent versus independent IgM and IgA apes. Seventeen 1062 Sugi et al. Antiphosphatidylethanolamine antibodies Vol. 71, No. 6, June 1999

4 TABLE 2 Antiphosphatidylserine antibodies, anticardiolipin antibodies, and lupus anticoagulant in 139 patients with recurrent early (embryonic stage) pregnancy losses. Antiphospholipid antibody Negative Positive Independent* Dependent Independent and dependent Total Phosphatidylserine IgG 133 (95.7) 6 (4.3) IgM 138 (99.3) 1 (0.7) IgA 139 (100.0) 0 Cardiolipin IgG 6 (4.3) 0 1 (0.7) 7 (5.0) Lupus anticoagulant 137 (98.6) 2 (1.4) Note: Values represent no. of patients (%). Ig immunoglobulin. * 2 GPI-independent anticardiolipin antibodies (patient sample diluted 1:200 in 0.3% bovine serum albumin). 2 GPI-dependent anticardiolipin antibodies (patient sample diluted 1:200 in 0.3% bovine serum albumin containing recombinant 2 GPI). Patient sample diluted 1:100 in 10% adult bovine plasma. (12.2%) of the 139 patients with RPLs were positive for IgM apes (Table 1). Two (1.4%) of the 139 patients with RPLs had IgA apes (Table 1). A positive test result for IgM or IgA apes was not statistically more frequent in the patients with RPLs than in the members of the reference group. Three patients with apes had two isotypes: 1 patient had IgG and IgM apes, and 2 patients had IgG and IgA apes. No patient had three apa isotypes. To summarize, 44 (31.7%) of the 139 patients with RPLs were positive for apes. A positive test result for apes was more frequent in the patients with RPL than in the members of the control group (P.0002). Anticardiolipin Antibodies, Antiphosphatidylserine Antibodies, and Lupus Anticoagulant in Patients With RPLs The same 139 patients with RPLs also were tested for anticardiolipin antibodies, antiphosphatidylserine antibodies, and the lupus anticoagulant. As shown in Table 2, 6 patients (4.3%) and 1 patient (0.7%) were positive for IgG and IgM antiphosphatidylserine antibodies, respectively. Seven patients (5%) were positive for 2 GPI-independent IgG anticardiolipin antibodies, and 1 patient (0.7%) was positive for 2 GPI-dependent IgG anticardiolipin antibodies. No patient had IgA antiphosphatidylserine antibodies. Five of the 7 patients who were positive for antiphosphatidylserine antibodies also were positive for apes. Two patients (1.4%) had lupus anticoagulant detected by dilute Russell s viper venom time. No patient was positive for two or more antibodies to phosphatidylserine, cardiolipin, or lupus anticoagulant. There was no statistically significant difference in the incidence of positive test results for antiphosphatidylserine antibodies and/or lupus anticoagulant between the RPL group and the control group. Antinuclear Antibodies in Patients With RPLs Antinuclear antibodies in the 100 healthy women, the 139 patients with RPLs, and the 28 patients with RPLs who were positive for IgG apes were measured. In the control group, 13 patients (13%) were positive for antinuclear antibodies. Among the patients with RPLs, 31 (22.3%) were positive for antinuclear antibodies. Among the patients with RPLs who were positive for IgG apes, 10 (35.7%) were positive for both IgG apes and antinuclear antibodies. The incidence of antinuclear antibodies in the last two groups was higher than that in the control group (P.02). Kininogen Dependence of apes The requirement of kininogens for plasma protein dependent IgG apes detection was determined with the use of partially purified kininogens as the patient plasma diluent. In this study, 21 patients were positive for plasma protein dependent IgG apes. Nineteen (90.5%) of these 21 patients were kininogen-dependent and 2 (9.5%) were kininogenindependent. The kininogen-independent IgG apes must recognize other PE-binding plasma proteins because they were not positive when bovine serum albumin was used as the patient plasma diluent but were positive when adult bovine plasma was used. The plasma protein involved in these two apes-positive sera will be the subject of a separate study. DISCUSSION Associations have been reported between apas, mainly anticardiolipin antibodies and/or the lupus anticoagulant, and RPLs (1 3). Relatively few studies describing apes have been published (4 8). We recently reported that certain apes are not specific for PE per se but are directed to PE-binding plasma proteins, such as HK, LK, and the HKbinding plasma proteins factor XI and prekallikrein (8). Because kininogens appear in mammalian reproductive tissues, we screened patients with RPLs for kininogen-dependent apes by ELISA using adult bovine plasma and/or kininogens partially purified from adult bovine plasma. We now report a stronger association between RPLs and apes FERTILITY & STERILITY 1063

5 than between RPLs and apas to the anionic phospholipids during early gestational losses. Many patients with RPLs who fit a clinical profile compatible with antiphospholipid syndrome are negative for apas when tested by ELISA. Explanations for the negative findings are manyfold but often can be attributed to assay variations. For example, some commercial detection kits for apas are designed to use purified or recombinant 2 GPI as the patient sample diluent. In this situation, patients with apas that are dependent on the presence of prothrombin would appear to have false-negative results. Other kits combine anionic and zwitterionic phospholipids in the ELISA plates. This can lead to false-negative findings because of the differential affinities of phospholipid-binding plasma proteins for the respective phospholipids. Moreover, binding to a mixture of phospholipids may result in false-negative results because of steric hindrance or a change in the physical phospholipid separation in the mixtures that alters their protein-binding properties. False-negative results in the ELISA for apes also can result from the use of a patient sample diluent that is low or deficient in the kininogens; fetal calf serum and newborn calf serum contain notably low concentrations of HK and LK (28, 29) and should be avoided. A few studies implicated apes in patients with RPLs (30 32). In these studies, fetal calf serum or newborn calf serum was used as the patient sample diluent. This suggests that many kininogen-dependent apes may not be detected in these studies. Finally, prolonged storage of adult bovine serum and adult bovine plasma at 4 C can result in decreased kininogen activity (Sugi T, unpublished observations). Midgestation pregnancy losses in patients with the primary antiphospholipid syndrome often are associated with anticardiolipin antibodies (12 14). This may reflect appearance more than reality because most published studies have limited their analyses to cardiolipin and to IgG and IgM isotypes. It may be an oversimplification to assume that midgestation to late gestation pregnancy losses in these patients are mediated by placental thrombosis; although placental thrombosis can occur, it often is insufficient to explain the pregnancy loss (33). Nevertheless, low-dose aspirin and/or subcutaneous heparin therapy often are effective and result in successful subsequent pregnancies (34). Recurrent pregnancy losses in the first trimester also may be associated with apas; however, a relatively low incidence of anticardiolipin antibodies and/or lupus anticoagulant has been reported. Gris et al. (35) reported that the prevalence of anticardiolipin antibodies and lupus anticoagulant in patients with RPLs before the end of the 16th week of amenorrhea was 2.2% and 4%, respectively. Ozawa et al. (24) reported that the prevalence of 2 GPI-dependent and -independent IgG anticardiolipin antibodies in patients with RPLs in the first trimester was 1.1% and 4.3%, respectively. Data from these investigators are similar to our RPL data. When we could find no association between anticardiolipin antibodies or lupus anticoagulant and early pregnancy loss, we extended our studies to include apes and discovered a significant number of positive patients. Because antinuclear antibodies have been accepted as serologic markers for the presence of autoimmune disease, patients with RPLs were screened for them. Many patients with RPLs who were positive for IgG apes also were positive for antinuclear antibodies (35.7%). In some instances, clinicians treat unexplained patients with RPLs who have antinuclear antibodies as if they had the antiphospholipid syndrome. We found that 44% of these patients also were positive for apes. However, we emphasize that antinuclear antibodies are not apas, and their pathogenesis remains to be elucidated (36). We suggest that all patients with unexplained RPLs who are positive for antinuclear antibodies be screened for apes to avoid unnecessary medical treatment. Patients with RPLs who are positive for antinuclear antibodies but have no evidence of apas do not require medication because the presence of antinuclear antibodies does not predict subsequent pregnancy loss (37). We recently reported that kininogen-dependent IgG apes augmented thrombin-induced platelet aggregation in vitro (11). Kininogens bind to platelets and inhibit thrombininduced platelet aggregation. Our data support the hypothesis that kininogen-dependent apes may cause thrombosis in vivo as a result of disruption of the normal antithrombotic effects of kininogen (11, 38, 39). As shown by our data, most ape-positive patients with RPLs were dependent on kininogens. In the future, antiplatelet therapy such as lowdose aspirin may be beneficial for kininogen-dependent apepositive patients with RPLs. This possibility awaits confirmation in appropriately designed clinical trials. Acknowledgments: The authors thank Atsuko Ohnishi, M.T. and Motoko Sato, M.T. for technical assistance. References 1. McNeil HP, Chesterman CN, Krilis SA. Immunology and clinical importance of antiphospholipid antibodies. Adv Immunol 1991;49: Hughes GRV, Harris EN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol 1986;13: Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-sle disorders. 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