Circulating autoimmune antibodies may be responsible for implantation failure in in vitro fertilization

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1 FERTILITY AND STERILITY Copyright 1994 The American Fertility Society Vol. 62, No.4, October 1994 Printed on acid-free paper in U. S. A Circulating autoimmune antibodies may be responsible for implantation failure in in vitro fertilization Eli Geva, M.D.* Yuval Yaron, M.D.* Joseph B. Lessing, M.D.* Israel Yovel, M.D.* Nurit Vardinon, Ph.D.t Michael Burke, M.B.B.S.t Ami Amit, M.D.*:j: Tel Aviv Sourasky Medical Center, Serlin Maternity Hospital, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To investigate the role of autoimmune factors as a possible cause for implantation failure as manifested by chemical pregnancy after IVF and ET. Design: Anticardiolipin, anti-double-stranded DNA (dsdna), antinuclear antibody, lupus anticoagulant, and rheumatoid factor serum levels were examined in patients with chemical pregnancies and in matched controls. Setting: An IVF unit, university-based IVF program. Patients: The study group included 21 patients who had had one or more chemical pregnancies and no deliveries. The control group consisted of 21 patients who had conceived and delivered after IVF-ET treatment, without any history of fetal wastage, matched for age, type and duration of infertility, and number of previous IVF cycles. Results: The incidence of circulating autoimmune antibodies in the study group was 33.3% (7 /21). Three patients (14.2%) were positive for anticardiolipin, two (9.5%) were positive for antidsdna, one (4.7%) for antinuclear factor, and one (4.7%) for rheumatoid factor. Autoimmune antibodies were not detected in any of the control group. Conclusion: Autoimmunity may play a role in implantation failure in IVF-ET. Circulating autoimmune antibody screening is therefore recommended after chemical pregnancy. Fertil Steril 1994;62:802-6 Key Words: IVF-ET, chemical pregnancy, autoimmune disease, autoantibodies, anticardiolipin Received December 23, 1993; revised and accepted April 28, * In Vitro Fertilization Unit. t Clinical Immunology Unit. :j: Reprint requests: Ami Amit, M.D., In Vitro Fertilization Unit, Serlin Maternity Hospital, Post Office Box 7079, Tel Aviv 61070, Israel (FAX: ). Autoimmunity has been implicated as being one of the possible causes for reproductive failure (1). Autoantibodies such as anticardiolipin and antinuclear antibodies have been shown to be associated with pregnancy wastage and poor outcome, namely, recurrent abortions, fetal growth retardation and stillbirths (1), all in the absence of clinical manifestations of any other autoimmune disease (2). Autoantibodies have also been implicated as one of the possible causes for IVF failure (3) and unexplained infertility (4). The antiphospholipid antibodies are of immunoglobulin (lg)g class and are directed against negatively charged phospholipids. The presence of raised serum levels of antiphospholipid antibodies has been found to be associated with a distinct clinical condition, the "antiphospholipid syndrome" (5). The immunopathological mechanisms that induce early pregnancy loss in patients with antiphospholipid syndrome may be uteroplacental thrombosis and vasoconstriction that result from binding of the lgg to both platelet and endothelial membrane phospholipids (1). This binding may in- 802 Geva et al. Autoimmunity and chemical pregnancy in IVF

2 duce platelet membrane instability and hyperaggregability (1) through mechanisms such as inhibition of protein C activation on endothelial prostacyclin synthetase activity (6). In addition, it may inhibit prekallikrein activity and endothelial plasminogen activator release (7). Chemical pregnancy in IVF represents implantation failure. The purpose of our study was to investigate the association of circulating autoantibodies in patients with chemical pregnancy after ET and IVF as a possible cause for implantation failure. Patients MATERIALS AND METHODS Over a 7 -year period, 2,357 IVF cycles were performed in our unit; 53 (2.2%) chemical pregnancies occurred. Chemical pregnancy was defined when the (3-subunit of hcg ((3-hCG) level had risen to at least twice that of 25 IU /L but declined before a gestational sac could be demonstrated by ultrasonography. Of the 53 patients with chemical pregnancies, 32 had either an antecedent successful pregnancy or subsequently had conceived with a normal pregnancy and delivery and were excluded from the study. The study group was comprised of 21 remaining patients with at least one chemical pregnancy and no deliveries. Patients with previous clinical abortions were not excluded. None of the patients had a history of rheumatic or autoimmune disease. The control group consisted of 21 patients who were computer matched for the following variables: age, type and duration of infertility, and the number of previous IVF cycles. All the patients in the control group conceived and delivered, and none of them aborted (Table 1). Superovulation Protocol Ovarian hyperstimulation was achieved using a combined regimen of GnRH agonist ( GnRH -a) and hmg. From the 1st day of the menstrual cycle GnRH-a (buserelin acetate nasal spray, Suprefact; Hoechst AG, Frankfurt am Main, Germany) was administered according to the "flare-up" regimen in a dose of 900 ttg/d until the stage of hcg injection. Three hmg ampules (Pergonal: 75 IU FSH and 75 IU LH; Teva Industries, Jerusalem, Israel) were administered from cycle day 3, using an indi- Table 1 Data Relating to the Study and Control Groups Age Duration of infertility Previous IVF cycles Primary /secondary infertility Etiology of infertility Mechanical Unexplained Male Study group (n = 21) 31.2 ± 4.0* 5.4 ± ± / Control group (n = 21) 30.2 ± ± ± /7 *Values are means ± SD. No statistically significant differences were noted for any of the variables. vidualized protocol. The hcg was administered when at least two follicles > 18 mm were detected and E 2 > 800 pg/ml (conversion factor to SI unit, 3.671). Ovum aspiration, ET, and laboratory techniques are detailed elsewhere (8). In all cases, the aspiration was ultrasonically guided (5 MHz, Combison 320; Kretz Technik, Zipf, Austria). Transfer of three to four embryos in all cases was performed 48 hours after ovum recovery. Pregnancy was defined as two rising serum (3-hCG levels. Autoantibodies Profile Sera was collected and stored at -20 C and examined together on the same day. Immunoglobulin G anticardiolipin antibodies were detected by ELISA, using cardiolipin (Reads Medical Products, Westminster, CO). Anti-double-stranded DNA (dsdna) was estimated using an RIA technique (Farr test). Fluorescent antinuclear antibody (FANA) was determined by indirect immunofluorescence, using heparin two-cell line substrate (Kallestad, Austin, TX). Screening for rheumatoid factor (RF) was performed by latex agglutination [PRL Diagnostics, Rishon-Le-Zion, Israel]. Positive results of RF were quantitated by indirect agglutination of coated latex particles (Fujirebiio, Tokyo, Japan). For activated partial thromboplastin time (PTT), prothrombin time (PT), and lupus anticoagulant estimation, the clotting time of citrated plasma was measured in a Coagulometer MLA 1000 (Electra 1000, Automatic Coagulation Timer; Medical Laboratory Automation, Pleasantville, NY). For PT and PTT, the added reagents were cerebral thromboplastin and kaolin-activated cephalin, respectively. Lupus anticoagulant was determined by Vol. 62, No.4, October 1994 Geva et al. Autoimmunity and chemical pregnancy in IVF 803

3 ~ ' measuring the plasma recalcification time in the presence of cephalin and a particulate activator, as described elsewhere (9). Quantitative complements C 3 and C 4 were measured by a turbidometric method, Turbox (Orion Diagnostics, Espoo, Finland). Plasma samples containing anticardiolipin antibodies < 23 GPL-U (lgg phospholipid unit)/ ml, anti-dsdna < 16%, FANA = 0, and RF < 1:160 were considered normal. Statistical Analysis Statistical analysis was made using Fisher's exact test and the paired Student's t-test as appropriate. A P value of <0.05 was regarded as significant. RESULTS The incidence of circulating autoimmune antibodies in the study group was 33% {7 /21), compared with no detectable antibodies in the control group (P = 0.04). Circulating autoimmune antibodies directed against cardiolipin (lgg isotype) were found in 14.2% (3/21) ofthe patients (Fig. 1). AntidsDNA antibodies were found in 9.5% (2/21) ofthe patients. Fluorescent antinuclear antibody was found in one ( 4.8%), and RF was found in one other patient (4.8%). The levels of PT, PTT, lupus anticoagulant, and complements c3 and c4 were normal (data not shown) in both the study and control groups. Of the 21 patients who comprised the study group, 6 had a history of at least one early spontaneous abortion 20 STL,D'r GROUP CONTROL GROUP 010 1i1 0 Bi1 0 L7 LJaJL7aJL7 ACA AntiDNA FANA RF AUTOIMMUNE ANTIBODIES Figure 1 The presence of circulating autoimmune antibodies in patients with chemical pregnancies and in the control group. (SAB), none of whom had circulating autoimmune antibodies. DISCUSSION In recent years, various types of reproductive failure have been considered as having an autoimmune component. Immunological effects are important at many levels of the reproductive process, including fertilization, implantation, and development of the placenta (10). The presence of raised serum levels of autoantibodies to cardiolipin have been found to be associated with a distinct clinical condition-the antiphospholipid syndrome (5), or more specifically, anticardiolipin syndrome (11). The prevalence of lgg anticardiolipin autoantibodies in an apparently healthy population has been reported as being 1.75% (12). In the present study, we found that 14.3% of the patients had lgg anticardiolipin autoantibodies, compared with none in the matched control group. Chemical pregnancy in an IVF -ET program represents implantation failure. Therefore, in view of the data presented here, it is tempting to assume that the implantation failure may be related to the high levels of anticardiolipin autoantibodies demonstrated in the sera of these patients. The study group ineluded six patients with a history of SABs. It should be noted that no circulating autoimmune antibodies were found in these women. Therefore, our hypothesis is that the presence of the anticardiolipin autoantibodies induced these chemical pregnancies and did not participate in the pathological mechanism of clinical abortion. However, further studies are still required to evaluate this hypothesis. Lupus anticoagulants are lgs (usually lgg, lgm, lga, or combined) that interfere with in vitro phospholipid-dependent coagulation tests (13). Based on population studies and definitions of normal assay ranges, lupus anticoagulants are found with an approximate frequency of <1% in the normal population ( 14). Lupus anticoagulants may represent antibodies produced in response to altered membrane phospholipid configuration secondary to injury (15). An association between fetal loss and lupus anticoagulant has been reported (16). The cause of the fetoplacental pathological features in patients with lupus anticoagulant is probably due to thrombosis of the decidual and placental blood vessels (17). In our investigation, lupus anticoagulants were not found in either the study or the control l I 804 Geva et al. Autoimmunity and chemical pregnancy in IVF

4 group. As reported previously, anticardiolipin antibodies were found to be a more specific and more sensitive predictor of early pregnancy loss (16). Also, our observations support the argument that there is poor correlation between lupus anticoagulant and specific autoimmune antibodies in reproductive failure. This point was also made in recent reports by several authors (1, 16, 18, 19). Rauch et al. (18) failed to detect any correlation between LA antibody activity and antiplatelet, anti-dsdna, and anticardiolipin activity. In contrast, antiplatelet activity was strongly correlated with anticardiolipin activity. The data presented here suggest that lupus anticoagulant screening is an inadequate method to detect abnormal B-cell function in women with implantation failure because of abnormal autoimmunity. Similar conclusions were reached by Cowchock et al. (19) who evaluated patients with repeated abortions primarily in reference to antiphospholipid antibodies. Anti-dsDNA has been found to be associated with unexplained infertility (4) in patients with repeated pregnancy loss (19) and premature ovarian failure (20). In our study, we found the antibodies to dsdna in 9.5% of the patients with chemical pregnancy. Women with FANA were prone to primary fetal loss in the first trimester (21). In contrast to the direct association of anticardiolipin antibodies with fetal loss, a positive F ANA suggests only an abnormal degree of autoimmunity and thus may serve as a marker for the presence of other circulating autoantibodies (19). According to our study, an isolated positive test for FANA may be associated with unexplained implantation failure. Rheumatoid arthritis (RA) has also been implicated in women with decreased fertility (22). This has been attributed to early pregnancy loss (21), antisperm antibodies (23), and ovarian antibodies (24). Moreover, women tending to develop RA suffer a higher fetal loss years before the onset of their disease (25). Our results may support those data; however, further examination of the reproductive performance in women seropositive for RF is necessary. Our findings of at least one circulating autoimmune antibody in 33% of the patients with chemical pregnancy could support the contention that autoimmunity may be one of the possible causes for implantation failure. However, the negative tests in these patients with very early pregnancy loss may indicate that the circulating autoimmune antibodies alone are insufficient to produce implantation failure. We concluded, therefore, that circulating autoimmune antibodies may play a role in very early postimplantation embryonic loss, and autoimmune antibodies screening is therefore recommended after chemical pregnancy in an IVF-ET program. Acknowledgment. We are indebted to Liat Lerner, M.D., for her valuable assistance. REFERENCES 1. Lockwood CJ, Reece EA, Romero R, Hobbins JC. Antiphospholipid antibody and pregnancy wastage. Lancet 1986;2: Alarcon-Segovia D. Pathogenic potential of antiphospholipid antibodies. J Rheumatol 1988;15: El-Roeiy A, Gleicher N, Confino E, Friberg J, Dudkiewicz AB. Correlation between peripheral blood and follicular fluid: autoantibodies and impact on in vitro fertilization. Obstet Gynecol 1987;70: Taylor PV, Campbell JM, Scott JS. Presence of autoantibodies in women with unexplained infertility. Am J Obstet Gynecol1989;161: Mackworth-Young CG. Anti -phospholipid antibodies: more than just a disease marker. lmmunol Today 1990;11: Carreras LO, Machin S, Demon R, Defregn G, Vermylen J, Spitz B, et al. Arterial thrombosis, intrauterine death and "lupus" anticoagulant. Detection of immunoglobulin interfering with prostacyclin formation. Lancet 1981;1: Angeles-Cano E, Sultan Y, Clauvel JP. Predisposing factors to thrombosis in systemic lupus erythematosus. Possible relation to endothelial cell damage. J Lab Clin Med 1979;92: Barak Y, Lessing JB, Amit A, Kogosowski A, Y ovel I, David MP, et al. The development of an efficient ambulatory in vitro fertilization (IVF) and embryo transfer (ET) program using ultrasonically guided retrieval. Acta Obstet Gynecol Scand 1988;67: Alving BM, Baldwin PE, Richards RL, Jackson BJ. The dilute phospholipid APTT: a sensitive assay for verification of lupus-17 -anticoagulants. Thromb Haemost 1985;54: Billingham RE, Head JR. Current trends in reproductive immunology: an overview. J Reprod Immunol 1981;3: Harris EN, Boey ML, Mackworth-Young CG, Gharavi AE, Patel BM, Loizou S, et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2: El-Roeiy A, Gleicher N. Definition of normal autoantibody levels in an apparently healthy population. Obstet Gynecol 1988;72: Vol. 62, No.4, October 1994 Geva et al. Autoimmunity and chemical pregnancy in IVF 805

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