Alan C. Menge, Ph.D.:!: Rajesh K. Naz, Ph.D.

Transcription

1 FERTILITY AND STERILITY Vol. 60, No.4, October 1993 Copyright 1993 The American Fertility Society Printed on acid-free paper in U. S. A. Immunoglobulin (Ig) G, IgA, and IgA subclass antibodies against fertilization antigen-l in cervical secretions and sera of women of infertile couples*t Alan C. Menge, Ph.D.:!: Rajesh K. Naz, Ph.D. The University of Michigan, Ann Arbor, Michigan, and The Albert Einstein College of Medicine, Bronx, New York Objectives: To assess the occurrence of immunoglobulin (Ig) G, IgA, and IgA subclass antibodies against human sperm fertilization antigen-1 (FA-I) in cervical mucus (CM) and serum of women of infertile couples. Design: Enzyme-linked immunosorbent assay methodology was used to detect anti-fa-1 antibodies. Antisperm antibodies were detected by agglutinating, immobilizing, and indirect immunobead (IB) methods. Control samples for the ELISA were from 10 women negative in the antisperm antibody assays. Participants: Samples were from women of 32 infertile couples undergoing antisperm antibody analysis. Results: One of 10 control CM samples was slightly positive for IgG anti-fa-1 and none for IgA. Of the 22 CM samples from antisperm antibody-positive women, 9 were positive for IgG antibodies, 9 for IgA, 7 for IgA1, and 6 for IgA2. Cervical mucus samples from eight women were positive for both IgA and IgG antibodies. Assay of 19 serum samples, including 8 controls, by ELISA, indicated 9 of 11 from antisperm antibody-positive women and none from controls were positive for IgA and IgG (7 of 9 identical women). In addition, of the nine IgA-positive sera, seven were of the Al subclass and five were of the A2 subclass. Positive IB assays occurred more frequently in CM and serum samples positive for anti-fa-1 antibodies than in negative samples. Conclusion: The results suggest that cervical secretions and sera of antisperm antibody-positive women contain IgA and IgG antibodies against sperm antigen FA-1 that may be involved in antifertility effects. Fertil Steril 1993;60: Key Words: Anti-FA-1 antibodies, cervical mucus, serum, IgA, IgG Involuntary human infertility has been associated with the occurrence of antisperm antibodies in the circulation as well as in genital tract secretions of both women and men (1, 2). Antisperm antibodies are detected primarily by assays using motile sperm cells, namely agglutinating, immobilizing, Received April 12, 1993; revised and accepted June 23, * Supported in part by grant HD from The National Institutes of Health, Bethesda, Maryland (R.K.N.). t Reprints are not available. :\: Department of Obstetrics and Gynecology, The University of Michigan. Department of Obstetrics and Gynecology, The Albert Einstein College of Medicine. and immunobead techniques (3). Sperm bound with antisperm antibodies, in addition to lowered motility characteristics, have exhibited reduced capacity to migrate through cervical mucus (CM), penetrate zona-free hamster eggs, bind to and penetrate the human zona pellucida, and fuse with the vitelline membrane of human oocytes (1). Cervical mucus containing antisperm antibodies also presents a barrier to sperm migration to the upper reproductive tract (4, 5). Antibodies found in genital tract secretions are primarily of the immunoglobulin (Ig) A and IgG classes (6). There are four subclasses of IgG, three of which (IgGl, 2, and 3) are capable of activating complement. IgA has two sub- 658 Menge and Naz Cervical antibody to sperm antigen FA-J Fertility and Sterility

2 classes, IgA1 and IgA2; neither are able to bind complement components. Sperm antibodies of both IgA subclasses have been detected on sperm cells and in the seminal plasma of infertile men (7) and in the CM of infertile women (8). The predominant subclass detected in the majority of genital tract samples examined in both studies, however, was IgAl. The majority of the reports on antisperm antibodies have used whole sperm cells (intact or solubilized preparations) as the antigen target in the assays. Further studies to understand the immune mechanisms responsible for inhibition of fertility require the use of defined sperm antigens. A spermspecific glycoprotein, fertilization antigen -1 (FA- 1), has been purified from human and mouse male germ cell plasma membranes (9). Tissue-specific but species-cross-reactive monoclonal antibodies against FA -1 inhibited both human sperm of zona-free hamster eggs and mouse sperm fertilization of mouse eggs (10). Anti-FA-1 antibodies have been demonstrated to be involved in human immunologic infertility (11-13). Antibodies against FA -1 were found in the sera of infertile patients only and in the sera and seminal plasma of men that failed fertilization in vitro. The purpose of our study was to assess the occurrence of IgG, IgA, IgA1, and IgA2 anti-fa-1 antibodies in CM samples and sera from women of infertile couples undergoing analysis for antisperm antibodies. MATERIALS AND METHODS Human Serum and em Samples Thirty-two infertile couples referred to the University of Michigan Laboratory of Assisted Reproductive Technologies for antisperm antibody analysis underwent phlebotomy and the women CM sampling at midcycle. Mucus was collected from the endocervical canal using sterile Tygon tubing (Norton Performance Plastics, Akron, OH) (3 mm inner diameter) attached to a lo-ml syringe. The CM sample was weighed, a portion used for a CMsperm assay and the remainder diluted with phosphate-buffered saline (PBS). The diluted sample was sonicated with 10-second bursts of 30% full power until solubilized (Bronson Sonic Power Co., Danbury, CT). The sample was centrifuged and stored frozen (-20 C) as was the serum until used in the assays. The procedures for specimen collection and study followed the guidelines of the University of Michigan Internal Review Board. Antisperm Antibody Assays The serum assays were the tray agglutination technique (TAT); the sperm immobilization test (SIT), as previously described (14); and the indirect immunobead (IB) technique (15). The IB and SIT were used to assay the CM samples. The CM -sperm assay consisted of CM drawn into a microslide tube (0.2 mm path length; Vitro Dynamics, Rockaway, NJ) (16). The distance that a vanguard of five motile sperm migrated was measured at 30 minutes at 37 C. The criterion established was poor migration, ::;;20 mm; good migration> 20 mm. FA-l Antigen Preparation The FA-1 antigen preparation was purified from lithium diiodosalicylate-solubilized human sperm by immunoaffinity chromatography using monoclonal antibodies that inhibit fertilization (9). Each batch of FA -1 antigen was examined for homogeneity by polyacrylamide gel electrophoresis. Only sampies showing a specific band of 47 to 50 kd after high-sensitivity silver staining were used in the present study. Anti-FA-l ELISA Methodology Anti-FA-1 antibodies were assayed in CM samples of all 32 women and in sera of 19 of the women (samples available at the time of assay) using the solid-phase ELISA (12, 17). Microtiter plates were coated overnight at 5 C with FA -1 antigen at a concentration of 5 mg/ml in 100 ML PBS/well. The washing solution consisted of PBS at ph 7.4 or PBS with 0.5% Tween 20. After washing, the plates were blocked with 5% fetal calf serum (FCS) in PBS for 60 minutes at 37 C and then treated with sera and CM samples obtained from the infertile women at midcycle. Serum samples were diluted 1:20 and 1:40 and CM samples 1:10 and 1:20, respectively, with PBS containing 1 % FCS and each was run in duplicate. After 3 hours incubation at 37 C, the plates were washed and second antibodies were added. Murine monoclonal antibodies (IgG isotype) against human IgA1 and IgA2 subclasses were used at concentrations of 5 mg/ml and polyclonal-affinity purified biotinylated goat antihuman IgA and IgG antibodies (TAGO, Inc, Burlingame, CA) were used at 1:1,000 dilution. The plates were incubated 2 hours, washed, and, in the case of the IgA sub- Vol. 60, No.4, October 1993 Menge and Naz Cervical antibody to sperm antigen FA-J 659

3 classes, a third antibody, biotinylated goat antimurine IgG antibody (Southern Biotech Corp., Birmingham, AL) was added to the wells for 60 minutes at 37 C. Avidin-peroxidase enzyme conjugate (Sigma Chemical Co., St. Louis, MO) diluted 1:2,000 was added to the wells for 60 minutes at room temperature. After a final wash with PBS, the substrate 2,2'-azinobis(3-ethylbenzthiazoline- 6-sulfonic acid) (Sigma Chemical Co.) containing % H was added to each well and the plates were read at 414 nm wavelength. The absorbance readings were converted to SD units using the formula: SD units = mean (test) - mean (control)/sd of control group. Samples ::?: 2 SD units from the control mean were considered positive. The Fisher Exact Test was run on frequency distributions using the University of Michigan Interactive Data Analysis System. RESULTS Of the 32 women assayed for antisperm antibodies, 10 were negative by TAT, SIT, and IB in both CM and sera. In addition, 4 of the 10 antisperm antibody-negative women became pregnant by assisted reproductive technology shortly after their antisperm antibody analyses. Three women conceived at the first GIFT cycle and one after intrauterine insemination. The CM of these 10 antisperm antibody-negative women and sera from 8 of these 10 women served as negative controls for the ELISA analysis of antibodies against FA -1 antigen. One control CM sample exceeded (2.1 SD units) the 2 SD unit limit for the IgG anti -F A -1 response with the range for the control values being from -1.6 to 2.1. Of the 22 antisperm antibody-positive women, 9 (41 %) exhibited positive IgG, 9 (41 %) IgA, 7 (32%) IgA1, and 6 (27%) IgA2 antibodies against FA-1 antigen in the CM samples (Fig. 1). The mean SD units for the ELISA results on the CM samples from antisperm antibody-positive women were 3.4, 4.8,2.9, and 1.3 for IgG, IgA, IgA1, and IgA2, respectively. Eight of nine CM samples positive for IgG antibodies were also positive for IgA. The SD units for IgA1 were greater than IgA2 in six of seven cases by 131 % to 750% in the positive samples. IgA2 was greater (133%) in only one case. Of the antisperm antibody assays performed on CM, only IB appeared to be associated with anti FA-1 results. Cervical mucus samples with positive IgA anti -FA -1 responses were mostly positive in the IB (7 of9 samples) whereas in ELISA-negative CM the IB positive response was less (3 of 13, P < 0.05) (Table 1). The IB response with antisperm antibody-positive CM was not different between samples negative and positive for anti-fa-1 IgG antibodies (6 of 13 versus 5 of 9) (Table 2). The presence of anti -FA -1 antibodies of either immunoglobulin class was not associated with sperm of CM. Also, serum antisperm antibody as detected by TAT or SIT, as well as the anti-fa-1 ELISA results, did not appear related to CM antisperm antibody levels. Nineteen serum samples from the 32 women were run in the ELISA for the detection of antibodies against FA-I. Sera from eight women negative for antisperm antibodies served as ELISA control samples. Nine of the 11 antisperm antibody-positive sera were positive for IgA and IgG antibodies against FA-1, eight for IgA1, and five for IgA2 (Fig. 1). Seven sera were positive for both Ig classes. The mean SD units for the ELISA results on sera of antisperm antibody-positive women were 15.9, 11.3,8.6, and 2.6 for IgG, IgA, IgA1, and IgA2, respectively. The IB was Ig-class positive in eight of nine sera with IgA and seven of nine for IgG anti FA-1 antibodies compared with none of two for anti-fa-1 negative samples (P < 0.10) (Tables 1 and 2). DISCUSSION Immune responses against the human spermspecific antigen FA-1 have been implicated in the etiology of involuntary human infertility (11). Anti-FA-1 antibodies have been detected in the sera of infertile men and women and associated with infertility. Also, IVF was significantly impaired in couples with antibodies against FA -1 and in those sperm samples bound with anti-fa-1 antibodies. The present study suggests that sera and cervical secretions of women positive for antisperm antibodies are likely to possess antibodies against FA-1 of different Ig isotypes and IgA subclasses because 50% of antisperm antibody-positive CM samples and 100% of sera positive for antisperm antibodies demonstrated anti-fa-1 antibodies. In contrast, only one CM sample from an antisperm antibody-negative women possessed a slightly elevated IgG anti -FA -1 antibody. In general, there was an positive association between the occurrence of IgG and IgA antibodies and the occurrence of IgA and IgA1 and IgA2 antibodies. The mode of action of anti-fa-1 antibodies in inhibiting fertility may be via interfering with the egg-sperm receptorbinding mechanism. A recent study demonstrated 660 Menge and Naz Cervical antibody to sperm antigen FA-J Fertility and Sterility

4 60 60 III 'c Figure 1 Distribution of :::II 20 ELISA results expressed as c 10 SD units of IgG, IgA, IgA1, 0 and IgA2 antibodies against... CI 6 sperm antigen FA-1 as mea- > sured in the serum and CM GI '1:1 samples of women assayed for 6 '1:1 antisperm antibody. Values... CI represented by open circles 4 '1:1 (0) and by closed circles (.) c CI are' respective samples from... 2 antisperm antibody-negative women and antisperm anti- 0 If) ~_o oil. o ~I :hl g8 :1 ~ol body-positive women. Control J>o o 0 o. o CM samples were from 10-2 antisperm antibody-negative women and control sera were IgG IgA IgAI IgA2 IgG IgA IgA 1 IgA2 from 8 of the antisperm antibody-negative women. Cervical mucus Serum 00 that FA-l has a lectin-binding property that binds and neutralizes the sperm ligand activity of purified ZP3 from zonae pellucidae of oocytes (18). In addition, anti -FA -1 antibodies prevented sperm from interacting with and penetrating the zona pellucida of oocytes by possibly blocking capacitation and acrosome reaction (19, 20). Secretory IgA (S-IgA) found in mucosal secretions is largely polymeric and quite effective in binding to the target antigen. The presence of S-IgA is hypothesized to protect mucosal epithelia from immunologic damage that would occur from inflammatory immune reactions. This is thought to occur by two mechanisms: lack of complement activation by IgA by either the classical or alternative pathway in humans and an IgA inhibition of C activation by IgG or IgM (21). Of the two IgA subclasses, IgAI is predominant (80% to 90%) in serum, whereas in mucosal secretions the ratio may vary (6). The numbers of IgAI to IgA2 plasma cells found in the genital tract tissues were in equal proportions (22). Assaying the actual quantities of IgA subclasses in CM from women undergoing infertility workups gave a distribution of 2:1 of IgAI to IgA2 (6). In general, the occurrence of the different subclasses appears to be dependent on the nature of the antigen, with proteins inducing IgAI and lipopolysaccharides and most carbohydrates stimulating IgA2 production (23, 24). Our limited results suggest that IgA antibodies against FA -1 were of both subclasses, although IgAI tended to give a greater ELISA response. This might be expected because the FA-l antigen is a glycoprotein possessing epitopes of Table 1 Incidence of Positive Antisperm Antibodies, Poor CM-Sperm Penetration Assay, and IgAl and IgA2 Antibodies in Women ofinfertile Couples Assayed for IgA Anti-FA-1 Antibodies by ELISA Cervical mucus samples Serum samples CM-sperm CM-sperm ELISA SD units No. SIT IBT* assay IgAl IgA2 No. TAT IBTt IBT* assay IgAl IgA2 Control < Antisperm antibodies positive < > Total * CM assay. t Serum assay. Vol. 60, No.4, October 1993 Menge and N az Cervical antibody to sperm antigen FA-l 661

5 Table 2 Incidence of Positive Antisperm Antibodies and Poor CM-Sperm Penetration Assay in Women of Infertile Couples Assayed for IgG Anti-FA-1 Antibodies by ELISA Cervical mucus samples CM-Sperm ELISA SD units No. SIT IBT* assay Control < Antisperm antibodies positive < > Total * CM assay. Serum samples CM-Sperm TATt No. TAT IBTt IBT* assay t Serum assay. both peptide and carbohydrate natures (9). An advantage of IgA2 antibodies compared with IgA1 for local immunity is the relative resistance to the proteases of microbes commonly found in the female genital tract (25). Immunologic-contraceptive vaccines could be so constructed by molecular biology to stimulate S-IgA2 production preferentially at the level of the reproductive tract. The potential of FA -1 sperm antigen as a contraceptive vaccine is enhanced by the demonstration of the presence of IgA and IgG antibodies against FA-1 in the genital tract secretions of infertile women. The results suggest that anti-fa-1 antibodies induced by natural means or possibly by vaccination could be secreted locally in the female reproductive tract and therefore be available to prevent sperm cells from initiating pregnancy. In conclusion, the results suggest that cervical secretions and sera of antisperm antibody-positive women contain IgA and IgG antibodies against sperm antigen FA-1, which may be involved in antifertility effects. The ELISA method using the sperm-specific antigen F A-1 should aid in elucidating the role of discrete sperm antibodies in human infertility. Acknowledgment. We thank Jiri Mestecky, M.D., University of Alabama at Birmingham, for generously providing the monoclonal antibodies against IgAl and IgA2. REFERENCES 1. Bronson RA. Immunology. In: Seibel MM, editor. Infertility: a comprehensive text. Norwalk, CN: Appleton and Lange, 1990: Menge AC. Clinical immunological infertility: diagnostic measures, incidence of antisperm antibodies, fertility and mechanisms. In: Dhindas DS, Schumacher GFB, editors. Immunological aspects of infertility and fertility regulation. New York: Elsevier-North Holland, 1980: Bronson RA, Cooper GW, Rosenfeld DL. Sperm antibodies: their role in infertility. Fertil Steril1984;42: Menge AC, Medley NE, Mangione CM, Dietrich JW. The incidence and influence of antisperm antibodies in infertile human couples on sperm-cervical mucus interactions and subsequent fertility. Fertil Steril 1982; 38: Alexander NJ. Antibodies to human spermatozoa impede sperm of cervical mucus or hamster eggs. Fertil Steril1984;41: Menge AC, Edwards RP. Mucosal immunity of the reproductive tract and infertility. In: Naz RK, editor. Immunology of reproduction. Boca Raton, FL: CRC Press, 1993: Bronson RA, Cooper GW. Documentation oflgal and IgA2 antisperm antibodies within seminal fluid. Am J Reprod Immunol1988; 18: Haas GG,Jr, D'Cruz OJ. A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies. Fertil Steril1989;52: Naz RK, Phillips TM, Rosenblum BB. The characterization of fertilization antigen 1 for the development of contraceptive vaccine. Proc Natl Acad Sci USA 1986;83: Naz RK, Alexander NJ, Isahakia M, Hamilton MS. Monoclonal antibodies to a human sperm cell membrane glycoprotein that inhibits fertilization. Science 1984; 225: Naz RK. Involvement of fertilization antigen (FA-I) in involuntary immunoinfertility in humans. J Clin Invest 1987;80: Bronson RA, Cooper GW, Margalioth EJ, Naz RK, Hamilton MS. The detection in human sera of antisperm antibodies reactive with FA-I, an evolutionarily conserved antigen, and with murine spermatozoa. Fertil Steril1989;52: Naz RK, Chaudhry A, Witkin SS. Lymphocyte proliferative response to fertilization antigen in patients with antisperm antibodies. Am J Obstet Gynecol 1990; 163: Korte MK, Menge AC. Detection of agglutinating and immobilizing antisperm antibodies. In: Keel BA, Webster, BW, editors. CRC handbook ofthe laboratory diagnosis and treatment of infertility. Boca Raton, FL: CRC Press, 1990: Menge and Naz Cervical antibody to sperm antigen FA-l Fertility and Sterility

6 15. Clarke GN. Detection of antisperm antibodies using immunobeads. In: Keel BA, Webster, BW, editors. CRC handbook of the laboratory diagnosis and treatment of infertility. Boca Raton, FL: CRC Press, 1990: Menge AC, Beitner O. Interrelationships among semen characteristics, antisperm antibodies, and cervical mucus assays in infertile human couples. Fertil Steril 1989;51: Czerkinsky C, Russell MW, Lycke N, Lindblad M, Holmgren J. Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes a strong antibody response in salivary glands and extramucosal tissues. Infect Immun 1989;57: Naz RK, Sacco AG, Yurewicz EC. Human spermatozoal FA-1 binds with ZP3 of porcine zona pellucida. J Reprod ImmunoI1991;20: Naz RK, Brazil C, Overstreet JW. Effects of antibodies to sperm surface fertilization antigen-ion human sperm-human zona interaction. Fertil SteriI1992;57: Kaplan P, Naz RK. The fertilization antigen-1 does not have proteolytic/acrosin activity, but its monoclonal antibody inhibits human sperm capacitation and acrosome reaction. Fertil Steril 1992; 58: Russell MW, Mansa B, Kilian M. Inhibition of complement activation by human IgA antibodies and their Fab fragments. In: McDonald TT, Challacombe SJ, Bland PW, Stokes CR, Heatley RV, editors. Advances in mucosal immunology. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1990: Kutteh WH, Hatch KD, Blackwell RE, Mestecky J. Secretory immune system of the female reproductive tract: 1. Immunoglobulin and secretory component-containing cells. Obstet GynecoI1988;71: Brown TA, Mestecky J. Subclass distribution of IgA antibodies to microbial and viral antigens. In: Strober W, Lamm ME, McGhee JR, James SP, editors. Mucosal immunity and infections at mucosal surfaces. New York: Oxford University Press, 1988: Tarkowski A, Lue C, Moldoveanu Z, Kiyono H, McGhee JR, Mestecky J. Immunization of humans with polysaccharide vaccines induces systemic, predominantly polymeric IgA2-subclass antibody responses. J Immunol 1990; 144: Kutteh WH, Edwards RP, Menge AC, Mestecky J. IgA Immunity in female reproductive tract secretions. In: Talwar GP, editor. Proceedings of the symposium on local immunity in reproductive tract tissues. Cambridge, U.K.: Cambridge University Press, U92: Vol. 60, No.4, October 1993 Menge and N az Cervical antibody to sperm antigen FA-1 663