2-Glycoprotein 1 as a marker of antiphospholipid syndrome in women with recurrent pregnancy loss

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1 FERTILITY AND STERILITY VOL. 73, NO. 3, MARCH 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. 2-Glycoprotein 1 as a marker of antiphospholipid syndrome in women with recurrent pregnancy loss Rodney D. Franklin, B.S., Nicole Hollier, M.D., and William H. Kutteh, M.D., Ph.D. Division of Reproductive Endocrinology and Immunology, Department of Obstetrics & Gynecology, University of Tennessee, Memphis, Tennessee Received July 13, 1999; revised and accepted September 23, Presented at the 55th Annual Meeting of the American Society for Reproductive Medicine, Toronto, Ontario, Canada, September 15 30, Reprint requests: William H. Kutteh, M.D., Ph.D., Director of Reproductive Endocrinology, Director of Reproductive Immunology, Department of Obstetrics and Gynecology, The University of Tennessee, Memphis, 956 Court Avenue, Room D324, Memphis, Tennessee (FAX: ; wkutteh@utmem.edu) /00/$20.00 PII S (99) Objective: To determine if 2-glycoprotein 1 ( 2-GP1) antibodies are a better marker of the antiphospholipid antibody syndrome (APS) in women with recurrent pregnancy loss (RPL). Design: Evaluation and testing of sera from women with RPL. Setting: A university-affiliated reproductive endocrinology practice. Patient(s): 90 women with RPL; 45 women met criteria for APS and 45 women met criteria for RPL without antiphospholipid antibodies (APA). Both groups were of similar age and had a similar history of RPL. Intervention(s): Patient sera were obtained from women with RPL and were tested for APA and 2-GP1. Main Outcome Measure(s): A standard antiphospholipid antibody assay was employed to detect the presence of immunoglobulin (Ig)G, IgM, and IgA antibodies in serum against cardiolipin, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl serine, and phosphatidyl ethanolamine. Samples were also assayed with a commercial 2-GP1 assay for IgG antibodies. Result(s): Among the 45 women with APS, 10 (22.2%) had positive IgG antibodies for 2-GP1. Only 1 woman (2.2%) of 45 was positive for 2-GP1 among the control group of women with RPL but negative APA. There was no correlation noted among the 2-GP1 positive patients for a specific phospholipid antibody or isotype. Conclusion(s): These data suggest that IgG 2-GP1 antibodies are less sensitive than antiphospholipid antibodies for the diagnosis of APS. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: Antiphospholipid antibodies, antiphospholipid syndrome, 2-glycoprotein 1, recurrent pregnancy loss Antiphospholipid antibodies (APA) are acquired antibodies against phospholipids that have been associated with placental thrombosis and can lead to fetal death. Clinical features, such as venous or arterial thrombosis, recurrent pregnancy loss (RPL), or thrombocytopenia in conjunction with positive laboratory finding (positive immunoglobulin [Ig]G or IgM anticardiolipin antibodies or positive lupus anticoagulant tests), will satisfy criteria for diagnosis of the antiphospholipid antibody syndrome (1, 2). Antiphospholipid antibodies are thought to induce thrombosis by binding to phospholipids on the surface of platelets and the vascular endothelium. This binding in turn is characterized by decreased prostacyclin production by the endothelial cells, increased thromboxane production by the platelets, and decreased protein C activation, resulting in vasoconstriction (3). The APA that have been investigated in association with RPL include antibodies to cardiolipin, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl serine, and phosphatidyl ethanolamine (4). A recent review summarized APA prevalence rates in different patient populations: 5.3% of normal obstetric patients (n 7,278), 24% of women undergoing IVF (n 3,343), 37% of women with systemic lupus erythematosus (n 1,579), and 20% of women with RPL (n 2,226 women) (5). Phospholipid-dependent clotting tests, which detect lupus anticoagulant, and the enzyme-linked immunosorbent assay (ELISA), which uses phospholipids as the coating antigens, are used to detect APA (6). False-positive tests for anticardiolipin antibodies have been 531

2 reported secondary to certain infectious diseases (syphilis) and some autoantibodies (double-stranded DNA) (7, 8). Studies suggest that some antibodies in APS actually bind to a cofactor known as 2-glycoprotein 1 ( 2-GP1) and that the phospholipid merely serves as a link to the solid phase (9, 10). 2-Glycoprotein 1, also known as apolipoprotein H, is a 50-kD serum cofactor required for anticardiolipin antibodies to bind to cardiolipin coated plates (9 11). The physiologic function of 2-GP1 is uncertain; however, it has known activity as an in vitro inhibitor of the intrinsic blood coagulation pathway (12), adenosine diphosphate dependent aggregation of platelets (13), and prothrombinase activity of activated platelets (14). Some investigators have suggested that 2-GP1 antibodies may be a better prognostic marker for APS and potential coagulation problems. It has been proposed that 2-GP1 ELISA would be more specific and would eliminate the false-positive results of the traditional anticardiolipin test (9, 10). However, the use of 2-GP1 antibodies as a tool in diagnosing APS in women with RPL is controversial. Therefore, we sought to determine the value of the IgG 2-GP1 assay as a screening test to diagnose APS in women with RPL. We used APA ELISA and 2-GP1 ELISA to ascertain the more relevant screening method. MATERIALS AND METHODS Patients Women were diagnosed with RPL on the basis of a history of three or more spontaneous losses fathered by the same partner. Women were diagnosed with APS on the basis of a clinical history of three or more pregnancy losses at any gestational age. Forty-five women who satisfied the above clinical criteria for APS were selected on the basis of laboratory criteria of positive IgG antibodies to one or more of the phospholipids or a lupus anticoagulant (or both). Some women also had positive IgM and IgA antiphospholipid antibodies. All women had positive APA tests confirmed at least 6 weeks after initial testing. Forty-five age-matched women with RPL who were negative for APA were tested as controls. Patients were excluded if they had systemic lupus erythematosus, a history of thrombosis, or ectopic pregnancies. Women were considered to be positive for the presence of lupus anticoagulant if they had a prolonged dilute Russell viper venom time, confirmed with a negative mixing study, and correction of the clotting time with a platelet neutralization test. APA ELISA All serum samples were evaluated for IgG, IgM, and IgA antibodies against cardiolipin, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl serine, and phosphatidyl ethanolamine by using the ELISA method as described by Harris (6). Briefly, individual 96-well microtiter plates (Immulon-2; Dynatech Labs, Chantilly, VA) were coated with 30 L of either purified phospholipids (Sigma Chemical Co., St. Louis, MO) at a concentration of 45 g/ml (cardiolipin) in ethanol or 50 g/ml (phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl serine, and phosphatidyl ethanolamine) in methanol. The plates were air-dried overnight at 4 C, then blocked with 200 L of 10% fetal calf serum (GIBCO, Long Island, NY) in 1 phosphate-buffered saline (GIBCO), washed, and incubated at 37 C for 2 hours with 50 L of patients sera diluted 1:50 in 10% fetal calf serum in phosphate-buffered saline. Each unknown sample was run in duplicate. The plates were then washed to remove unbound antibody and proteins, and a secondary antibody, alkaline phosphatase conjugated antihuman IgG (Caltag Labs, San Francisco, CA) or IgM (Biosource, Tago Immunologicals, Camarillo, CA) was added to the plate. After incubation and washing, p-nitrophenyl phosphate substrate (Sigma 104) was added and used to indirectly measure the level of APA in a patient s serum. The optical density of the samples, caused by the cleavage of the substrate by the enzyme, was determined at 405 nm by a Bio-Tek Microplate Reader Model EL 340 (Bio-Tek Instruments, Winooski, VT) and was used to quantitate the amount of APA in the sera. Every assay plate also included a known high-positive anticardiolipin sample ( 100 GPL), run in duplicate. Plates were incubated until the high-positive wells achieved an optical density 1.0; typically, this required an incubation of 20 to 30 minutes. Referenced standard sets for cardiolipin (Louisville APL Diagnostics, Louisville, KY) and known negative sera were used on every plate. All results were defined in phospholipid units for IgG (GPL) and IgM (MPL) as follows: 10 units, negative; units, borderline; units, positive; and 80 units, high positive. Phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl serine, and phosphatidyl ethanolamine values were interpreted on the basis of the multiples of the median method, as described previously (15). Briefly, phospholipid units for IgG and IgM were calculated for each serum sample, and the median value was determined from the nongaussian distribution. The cutoff value in phospholipid units of each APA was determined by using the 99th percentile of the normal population, approximately 3.0 times the median value. All values reported as positive were the mean of duplicated determinations, with background absorbance obtained from wells prepared without the coating phospholipid subtracted. Any values with an SE 10% were discarded and reassayed. Interassay variation was 8%, and intraassay variation was 6%. 2-GP1 ELISA Serum samples were assayed for 2-GP1 in duplicates with commercially obtained ELISA kits from INOVA (San Diego, CA). The accuracy and reproducibility of each assay was determined by running a negative, high, and moderate reactive sample six times within a run morning and afternoon on three consecutive days. The interassay and intraas- 532 Franklin et al. 2-glycoprotein 1 and pregnancy loss Vol. 73, No. 3, March 2000

3 TABLE 1 Comparison of women with recurrent pregnancy loss based on APA status. Variable Antiphospholipid syndrome (n 45) Recurrent pregnancy loss (n 45) Mean SD 95% CI Mean SD 95% CI P value Age at entry (y) ( ) ( ).65 No. of prior pregnancies per patient ( ) ( ).43 No. of prior live births per patient ( ) ( ).18 No. of prior miscarriages per patient ( ) ( ).17 Gestational age at loss (%)* 12 weeks 185/188 (98.4) (95 100) 146/164 (89.0) (83 93) weeks 2/188 (1.1) ( ) 13/164 (7.9) (4.0 13) weeks 1/188 (0.5) ( ) 5/164 (3.0) ( ).10 Positive for 2-GP1 (%) 10/45 (22.2) (11 37) 1/45 (2.2) ( ).01 Note: P values.05 were considered statistically significant. APA antiphospholipid antibody; CI confidence interval; 2-GP1 2-glycoprotein 1. * Values represent no. of losses at specified gestational week/total no. of losses at all gestational weeks (percentage of losses). Values represent no. of patients with 2-GP1 antibodies/total no. of patients in the group. say coefficients of variation for the negative, moderate, and high samples were 4.3%, 5.4%, and 3.5% and 4.0%, 5.3%, and 3.2%, respectively. Briefly, samples and calibrators were added to the precoated microtiter strips. Plates were incubated at 37 C for 30 minutes. After washing, streptavidin horseradish peroxidase was added and plates incubated at 37 C for 30 minutes. The plates were washed again. The color was developed in the presence of the conjugated enzyme with a stabilizing chromogen in the dark at room temperature. After 30 minutes, the reaction was stopped and read at 450 nm with a Bio-Tek plate reader. Samples were calculated from the mean absorbance of a five-point calibration curve against their concentration. A 2-GP1 value of at least 8 standard IgG anti 2-GP1 units was considered positive. Statistical Analysis Statistical analysis was performed with the two-tailed Fisher s exact test. Levels were considered significant when P.05. The potential value of the IgG 2-GP1 assay as a diagnostic and screening assay was determined as described previously (16). RESULTS The study population consisted of 90 women with RPL. The APA-positive and APA-negative patients were tested for 2-GP1. One patient was positive for lupus anticoagulant. No significant differences were found among the patients for age, number of pregnancies, or number of deliveries (Table 1). In the 45 women with APS, there were a total of 188 losses; of these losses, 185 (98.4%) occurred before the 12th week of gestation. Similarly, in women with RPL but without APA, 146 of 164 losses (89.0%) occurred before the twelfth week of gestation; these differences were not significant (P.54). Significantly more losses occurred at 13 or more weeks of gestation in women with RPL and without APA than in women with RPL and APA. The sera of the women with APA positive and the APA negative women with RPL were assayed to detect antibodies to 2-GP1. All 45 women with APS were selected on the basis of one or more positive test for IgG antiphospholipids. Among 45 women with APS, 10 (22.2%) had positive IgG antibodies to 2-GP1. Only 1 woman (2.2%) in the control group of women with RPL but no APA was positive (Table 1). The frequency of positive 2-GP1 antibodies between the APA-positive and APA-negative groups was significantly different (P.01). No correlation among the 10 APA-positive women who were also positive for 2-GP1 was found for a specific phospholipid antibody. The distribution of APA among women with RPL was evaluated. All 45 patients selected were positive for at least one IgG APA to be included in the APS group. Some women were also positive for IgM and IgA phospholipids. Positive results were found in all of the Ig isotypes and APA except for IgM phosphatidyl ethanolamine. The majority of patients had IgG anticardiolipin (48.8%) or antiphosphatidyl serine (33.3%) or both. The remaining IgG APA detected were phosphatidyl inositol (26.7%), phosphatidyl ethanolamine (24.4%), and phosphatidyl glycerol (20.0%) (Table 2). Some patients were positive for more than one phospholipid antibody. The IgG 2-GP1 assay was analyzed to determine its possible usefulness as a diagnostic and screening test (Table 3). The positive and negative predictive values were.909 and.557, respectively. The assay was highly specific (.978) but was less sensitive (.222) for classifying diseased and nondiseased patients. FERTILITY & STERILITY 533

4 TABLE 2 Distribution of positive APA based on immunoglobulin isotypes (IgG, IgM, and IgA). Phospholipid antibody IgG (%) IgM (%) IgA (%) Cardiolipin 22/45 (48.8) 4/45 (8.8) 3/45 (6.6) Inositol 12/45 (26.7) 2/45 (4.4) 2/45 (4.4) Glycerol 9/45 (20.0) 2/45 (4.4) 1/45 (2.2) Serine 14/45 (33.3) 1/45 (2.2) 2/45 (4.4) Ethanolamine 11/45 (24.4) 0/45 (0.0) 2/45 (4.4) Note: APA antiphospholipid antibody. DISCUSSION These data suggest that IgG 2-GP1 is less sensitive than APA for the diagnosis of APS in women with RPL. The prevalence of 2-GP1 was 22.2% in women with APS and 2.2% in APA-negative women with RPL. These data are in agreement with other reports that do not support the routine use of 2-GP1 assay in the evaluation of women with APS (17, 18). Some patients with RPL have positive APA specific to epitopes other than cardiolipin (19, 20). However, in the present study, 48.8% of the patients were positive for IgG anticardiolipin, supporting the original association between anticardiolipin antibodies and RPL. 2-Glycoprotein 1 has high specificity for APA but low sensitivity; thus, testing in conjunction with cardiolipin has been suggested (21, 22). Cardiolipin has been described as having two subtypes: 2-GP1 dependent and 2-GP1 independent (23). 2-Glycoprotein 1 independent antibodies were not associated with adverse pregnancy outcomes (24, 25); in contrast, the 2- GP1 dependent subtype had a significant association with adverse pregnancy outcome in a prospective study (26). Conversely, Lynch et al. (27) prospectively demonstrated that the odds ratio for pregnancy loss increased with levels of IgG anticardiolipin and antiphosphatidyl serine; however, the same association was not present for women with RPL positive for antibodies to 2-GP1. TABLE 3 Statistical value of 2-glycoprotein 1 as a diagnostic and screening test for APS. Sensitivity of test.222 Specificity of test.978 False positive rate.022 False negative rate.778 Predictive value of positive test.909 Predictive value of negative test.557 Arterial and venous thrombosis are complications that have been described in patients with APS. Some women with systemic lupus erythematosus who have anticardiolipin antibodies (or produce low affinity anticardiolipin antibodies) may not exhibit thrombotic events. However, in patients with lupus, the presence of IgG 2-GP1 has been associated with a history of thrombosis (22, 28). The absence of IgG 2-GP1 antibodies in women with lupus correlated with a subgroup that was not at risk for associated thrombosis (29). Thus, IgG 2-GP1 may be clinically useful in identifying patients with lupus who are at risk for thrombotic events. In a recent study by Stern et al. (30), IgM 2-GP1 was associated with implantation failure in IVF patients and with pregnancy loss in RPL patients. These investigators reported a prevalence of IgM and IgG 2-GP1 in women with RPL (16% and 6%, respectively) compared with IgM and IgG 2-GP1 in a control group of women without RPL (0% and 6%, respectively). Moreover, in a study of 72 women with RPL, it was reported that the prevalence of IgG 2-GP1 was the same as that of normal women (31). Our study identified IgG 2-GP1 antibodies in 10 of 45 (22.2%) patients with APS compared with only 1 of 45 (2.2%) women who had RPL without APA. Our findings of a lack of correlation between IgG 2-GP1 antibodies and APA in women with RPL are in agreement with those of Stern et al. (30) and Maejima et al. (31). Further testing is necessary to confirm the potential of IgM 2-GP1 as a diagnostic marker for APS in women with RPL. Statistical analysis does not support the use of the IgG 2-GP1 assay as a diagnostic or screening test in assessing APS in patients with pregnancy loss. Immunoglobulin G 2-GP1 demonstrated high specificity but very low sensitivity in detecting APA in patients with APS. In this study, the predictive value of a positive result for 2-GP1 IgG antibody would correctly identify APS patients who are positive for APA in over 90% of cases; however, the predictive value of a negative IgG 2-GP1 test was just slightly better than chance in correctly identifying RPL patients without APA. We emphasize a standard measurement of APA correlated with the obstetric history as the preferred method to diagnose APS. References 1. Pattison NS, Chamley LW, McKay EJ, Liggins GC, Butler WS. Antiphospholipid antibodies in pregnancy: prevalence and clinical associations. Br J Obstet Gynecol 1993;100: Lockwood CJ, Romero R, Feinberg RF. 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