Antisperm antibodies: origin, regulation, and sperm reactivity in human infertility*

Transcription

1 FERTILITY AND STERILITY Copyright" 1994 The American Fertility Society Vol. 61, No.6, June 1994 Printed on acid-free paper in U. S. A. Antisperm antibodies: origin, regulation, and sperm reactivity in human infertility* Rajesh K. Naz, Ph.D.tt Alan C. Menge, Ph.D. Albert Einstein College of Medicine, Bronx, New York, and the University of Michigan Medical School, Ann Arbor, Michigan Objective: To follow-up and expand discussion on the action mechanisms of antisperm antibodies in human infertility, the etiology and control of antisperm antibody induction, sperm antigens involved in immunoinfertility, and strategies for therapy. Design: A review of the recent literature with an emphasis on female immunoinfertility. Results: The role of antisperm antibodies in clinical infertility continues to be defined. Through assisted reproductive technologies, antisperm antibodies were shown to exert detrimental effects on different prefertilization and possibly postfertilization events. The female reproductive tract is part of the common mucosal immune system and is able to mount effective immune responses against infectious agents, foreign antigens, and, occasionally, sperm cells. Sperm membranes and constituents contain numerous antigenic components foreign to the human body, and yet antisperm antibodies become problematic in few women exposed to semen. Semen and sperm cells contain immunosuppressive factors capable of inhibiting different immune cells. Fertile women apparently produce antisperm antibodies but also possess neutralizing serum anti-idiotypic antibodies that are lacking in virgin and immunoinfertile women. Conclusions: Antisperm antibodies can affect adversely human fertility but normally may be controlled by anti-idiotypic antibodies, which along with immunosuppressor factors in semen prevent their induction to a significant degree. This balance between detrimental and "beneficial" immune response to sperm may be shifted toward an antisperm antibody response by stimulatory factors such as infection. Therapies may be devised to stimulate the anti-idiotypic antibody system, to induce immune tolerance to sperm antigens, and to use antigens to adsorb antisperm antibodies from spermatozoa. Fertil Steril1994;61: Key Words: Antisperm antibodies, sperm antigens, anti-idiotypic antibodies, immunoinfertility The saga continues as the significance of antisperm antibodies in infertility becomes clear; evidence ofthis significance includes sites and mecha- Received February 8, * Supported in part by the National Institutes of Health grant HD24425 (R.K.N.), Bethesda, Maryland. t Reproductive Immunology and Molecular Biology Laboratories, Department of Obstetrics and Gynecology, Albert Einstein College of Medicine. * Reprint requests: Rajesh K. Naz, Ph.D., Department ofobstetrics and Gynecology, Ullmann Building, Room 123, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York (FAX: ). Department of Obstetrics and Gynecology, University of Michigan Medical School. nisms of antibody action, the etiology of antisperm antibody formation, and possible modalities oftherapy. From a historical perspective, early claims of antisperm antibody incidence and involvement in involuntary human infertility probably were magnified because of unreliable assays and naivete concerning the complexities of the immune response and antigenic nature of the sperm cell. These factors, plus the dearth of well-designed and controlled experimental studies and the lack of effective therapy, resulted in confusion of the occurrence and importance of antisperm antibodies in human infertility. As a result, many clinicians rejected out of hand any evaluation of infertile couples for antisperm antibodies and the possible role that antisperm anti- Vol. 61, No.6, June 1994 N az and Menge Role of antis perm antibodies in infertility 1001

2 Table 1 Mechanisms and Sites of Action of Antisperm Antibodies in Infertile Women 1. Sites and isotypes of sperm antibody secretion Vagina: IgA and IgG; also IgM during inflammation; IgA of cervical origin, IgG from transudate Cervix: IgA and IgG; IgAl to IgA2 is 2:1, IgG4 > IgG3 Uterus: IgG and IgG + B cells predominate in human uterine fluids and endometrium, respectively Oviducts: IgA and IgG secreting B cells in mucosa Follicular fluids: IgA and IgG levels and antibody activities equivalent to serum; IgM is reduced. 2. Prefertilization effects Inhibits sperm migration through the cervical canal Sperm are motile but forward progression impeded primarily by IgA ASA Sperm death and complement activation primarily by IgG ASA Inhibit sperm capacitation-acrosome reaction Inhibit sperm attachment, binding, and penetration of zona pellucida of oocyte Inhibit sperm fusion with the vitelline membrane of the oocyte Inhibit sperm activation or pronuclear formation 3. Postfertilization effects Effects on sperm that are manifested by cleavage, pre- and postimplantation adverse outcomes of fertilized eggs Direct activity against developing embryo to decrease viability and pregnancy rates bodies could have in infertility. The development of more accurate assays and the discovery of mucosal immunity capable of responses independent of systemic immunity have caused inclusion of sperm cells and genital tract secretions in the analyses of antisperm antibodies. In addition, with progress in assisted reproductive technology, the role of antisperm antibodies in human infertility has become better defined, and effective forms of therapy also have evolved. In this article, we attempt to elucidate further modes of antisperm antibody action, the etiology of sperm antibody formation in infertile women, the role of characterized sperm antigens, and implications for future therapies. The article by Marshburn and Kutteh (1) updates the current status of antisperm antibody assays and classes of immunoglobulins involved and provides a survey of clinical infertility studies, some etiologic considerations, roles in infertility, and possible current treatments. Numerous investigations in human IVF, clinical studies, and animal models have demonstrated convincingly that antisperm antibodies are capable of inhibiting the fertility process at different steps (Table 1). Experimentally, monoclonal antibodies (mabs) against different human sperm antigens have shown cross-reactive binding with xenogeneic sperm, most often of the mouse (2). These interactions have identified antibodies capable of both prefertilization and postfertilization effects on fertility (3, 4). Although many mabs have been produced against human sperm, not all should be defined as antisperm antibodies but rather as sperm-reactive antibodies because they have no detectable effects on sperm function. Further elucidation of the inhibitory processes has occurred and will continue to occur with additional well-defined and characterized sperm antigens and complementary antibodies to enable fine dissection of the events preceding and including fertilization and early cleavage, as discussed later. The female reproductive tract is unique in that it needs to protect itself against invading pathogenic organisms while not mounting a significant destructive immune response against allogenic sperm cells and the developing conceptus. All the necessary components of mucosal immunity found in the respiratory and gastrointestinal tracts, including secretory component and immunoglobulin (Ig)A, are also found in the female reproductive tract (5, 6). It has become evident that the female reproductive tract is not an immunoprivileged site but that the immune system functions locally in a protective role against infectious agents and in the establishment and maintenance of pregnancy and adversely on fertility, with the generation of antisperm antibodies (7). It has been illustrated in animals by intragastric immunization with different types of antigens and the finding of secretory IgA antibodies in the reproductive tract secretions that the genital tract is a part of the common mucosal immune system (Fig. 1). Active immunization in the vagina of women against Candida albicans and inactivated polio virus resulted in localized IgA and IgG antibody responses (8,9). In addition, cervical secretory IgA to herpes simplex virus (HSV)-2 has been observed to increase with recrudescent cervical HSV, and its appearance is associated with the disappearance of detectable virus by culture (10). IMMUNE RESPONSES AGAINST SPERMATOZOA As described above, the components necessary for a mucosal immune response exist in the female genital tract. Antisperm antibodies found in genital tract secretions of infertile women are primarily of the IgA and IgG isotypes. Subclass identification of cervical mucus (em) antibodies revealed that approximately 70% of the IgA was IgAl, whereas the major IgG was IgG4, with some IgG3 present (11) N az and Menge Role of antisperm antibodies in infertility Fertility and Sterility

3 Humoral antibodies IgA IgG IgM stream SuppressIng Factors Female ReproducUva Treet Semfnol plasma: -prostaglandins -TGF-beto -large Mr molecules -cytoklnes (Il-e) Induce nonimmune disposal of sperm Sperm surface molecules -anti-complement activity -lga prot eases -unknown Idiotyplc ontlbody network Immune suppressor cells In tract Mucosal immune system GALT Blood stream Stimulating Factors IgA ond IgG committed end Ag-sensi t i2ed B-cells Ag-sensitlzed T-cells Infectious agents, Inflammatory cejls and cytokines In semen Active infection, inflommation or trauma In tract Deficiency In immunosuppressive factors In semen ASA-coated sperm activotlon of macrophoges and immune cells Deficient immune system -lack of idiotypic antibodies -lack of suppressor T-cell response Figure 1 Cartoon model of the systemic and mucosal immune responses of the female reproductive tract and some factors that suppress or stimulate the induction of antisperm antibodies. Sperm antigens (Ag) are processed locally in the epithelia or submucosal stroma by antigen-processing cells constituted from macrophages, Langerhans cells, and possibly HLA class II -positive epithelial cells or in draining local lymph nodes (LN). These accessory cells either activate and sensitize locally in the reproductive tract or more distally in the peritoneum and the gut-associated lymphoid tissues (GALT), the T and B lymphocytes. The sensitized immune cells undergo division and maturation in these latter sites before migrating to and populating the genital tract mucosal-associated tissues (primarily the cervix), where they undergo terminal differentiation to IgA and possibly IgG secreting plasma cells. Cytokines produced by distinct sets of T cells are responsible for the various steps of development and differentiation. Some immunosuppressing and stimulating factors could act at different sites, either in the tract or in the lymphoid tissues after absorption through the vaginal epithelium. Because IgA and IgG4 do not fix complement, the majority of CM samples with antisperm antibodies may not affect motility, but if IgG3 is present, complement activation and sperm damage could result. Complement-dependent sperm immobilizing activity was detected in CM samples of a significant number of infertile women (12). Antibodies against sperm secreted in the CM can interfere with fertility through multiple means. The foremost clinical manifestation is the inhibition of sperm migration into and through the cervical canal as often observed in postcoital tests (PCTs). This result of antisperm antibodies especially is due to secretory IgA binding sperm, with the complex being ensnared in the microfilament matrix of the CM gel, allowing continued sperm flagellation but without forward progression. Sperm exposed to antibodies in CM or in secretions from the upper parts of the reproductive tract also may become immobilized due to membrane damage from complement activation. The complement C3 component has been found in CM and other genital tract fluids in adequate quantities to react with antigen-antibody complexes with resultant membrane attack (13). The complement components are secreted by the epithelial cells of the endometrium and may be modulated by gonadal hormones and cytokines. Thus, it is reasonable to assume that either the cervical epithelium is capable of secreting complement components or that uterine secretions contributing to CM provide adequate complement to allow cytolytic reactions. Therefore, sperm bound with complement-fixing IgG antibodies upon reaching the uterine cavity would be liable to complement activation and deposition on the membrane, with resulting damage and possible death. Using the hemizona assay, a study recently found that complement-fixing antibodies against sperm from infertile women inhibited tight binding of sperm to the zona (14). These immune reactions could also initiate a heightened leukocytosis and phagocytosis of antibody-bound sperm by phagocytic cells with receptors for Fc of the IgA and IgG molecules. Semen and sperm cells are known to induce leukocytosis by the human cervix, possibly through interleukin (IL)-8, which is found in semen and has a strong chemotactic effect to attract and activate neutrophils (15). The IL-8 activity and leukocytosis likely is a natural mechanism for efficient elimination of excess sperm cells and, in conjunction with other seminal factors discussed below, prevent an immune response to sperm cells. In addition, sperm antigens could activate sensitized lymphocytes in women with antisperm antibodies to release cytokines in the reproductive tract that can exert adverse effects on sperm function, fertilization, or early embryo development (16). In another study carried out in our laboratory, we detected IL-6 in seminal plasma of fertile, infertile, and immunoinfertile men, and its levels were significantly lower in the seminal plasma of fertile men compared with those of infertile and immunoinfertile men (17). Similar trends also were noted in sera, with some immunoinfertile men having 4.3- to 5.8-fold higher values than the highest level observed in sera of fertile men. Interestingly, the levels in seminal plasma were higher than those in the sera, indicating a local production of IL-6. Higher Vol. 61, No.6, June 1994 N az and Menge Role of antisperm antibodies in infertility 1003

4 levels of IL-6 in seminal plasma of infertile and immunoinfertile men seem to have clinical significance as they correlated significantly with total sperm number, penetration rates, and some sperm motion parameters. Interleukin-6 is produced by a variety of cell types, including macrophages, endothelial cells, fibroblasts, and trophoblasts and has been demonstrated to influence the growth and differentiation of B cells. Because the male genital tract is an immunologically dynamic system and a number of studies have indicated an increased number of leukocytes in the ejaculates of infertile men compared with those of fertile men, the higher levels of IL-6 in seminal plasma of infertile and immunoinfertile men may be due to an increased number of leukocytes (secreting IL-6) in the semen of these patients. In addition, if sperm reach the site of fertilization, antibodies are capable of interfering with sperm interactions with the egg. Known observable effects include inhibition of sperm binding to and penetration of the zona pellucida and fusion with the egg vitelline membrane (18, 19). The presence of antisperm antibodies in maternal serum used for human IVF has been shown by many investigators to reduce success rates (20). Antisperm antibody activity in the CM of infertile women also effectively blocks sperm-hamster egg fusion (21). Because follicular fluid in some infertile women was shown to contain antisperm antibodies and complement, the oocyte-cumulus complex may well contain these immune components that could cause sperm damage and blockage of fertilization (22). Incubating sperm with follicular fluid containing antisperm antibodies indicated that the sensitized sperm have detectable levels of the complement complex deposited on plasma membranes (23). Sperm bound with antisperm antibodies over the head lose the ability to undergo capacitation and acrosome reaction, resulting in failure of the sperm to bind and penetrate the zona pellucida. A recent report suggests that antisperm antibodies prevent sperm membrane fluidity that blocks cholesterol loss and mannose ligand receptor formation on the cell surface (24). This is a putative ligand receptor for the zona pellucida. In addition, to these effects of inhibiting fertilization by sperm cells, antisperm antibodies especially of mucosal origin and against certain sperm antigens exert adverse fertility effects via two other means in experimental animals: reaction with sperm to affect embryo development and survival after fertilization and direct effects on cleaving embryos (4). Studies in humans also suggest that pregnant women with antisperm antibodies experience a higher incidence of spontaneous abortions than women without antisperm antibodies (25). Studies on women positive for antisperm antibodies undergoing IVF found reduced fertilization rates as expected, but the fertilized eggs had a lower assessed quality and cleavage rate and the women also had a somewhat lowered pregnancy rate (26, 27). Recently, antibodies against a sperm membrane antigen from antisperm antibody-positive IVF patients were characterized as exerting inhibitory effects on initial cleavage stages of pronuclear eggs (28). Other studies, however, suggest that once fertilization occurs, pregnancy rates between antisperm antibody-positive and antisperm antibody-negative groups do not differ (29, 30). This discrepancy may be due to the assay used, the sensitivity or parameters set, or just a chance occurrence. Also, the antisperm antibodies not showing effects on pregnancy rates may be reacting with different sperm antigens than the antisperm antibodies showing effects. ETIOLOGY OF ANTISPERM ANTIBODIES Role of Immunosuppressor Factors The origin of sperm antibodies in women still is unknown. Although the female genital tract as described above has the essential mucosal immune components to respond potentially to an antigenic challenge, the majority of the intraepitheliallymphocytes appear to be of the suppressor type (CD8+) (31). This suggests that an immune response probably requires a certain amount of antigenic and inflammatory challenge to activate T helper cells and a humoral immune response (Fig. 1). Seminal plasma and sperm containing multiple factors, however, that possess immunosuppressive activities identified in vitro would appear to reduce the probability of an immune response occurring against sperm cells (32). These substances range from large molecular weight unidentified compounds to smaller molecules, such as prostaglandins to transforming growth factor-fj (33, 34). Because seminal plasma effectively is eliminated from entering the upper reproductive tract of women by the cervix, it suggests these soluble factors must either act or be absorbed locally or both in the vagina. Immunosuppression could occur at multiple levels, that is, antigen processing as well as inhibition of lymphocyte activation. The actual role that these factors play in vivo is still largely 1004 N az and Menge Role of antisperm antibodies in infertility Fertility and Sterility

5 speculation. Likewise, the identification of spermbound components such as complement regulators and an IgA protease suggests that sperm cells may have defense mechanisms that impede immune reactions (35, 36). When examining the proliferative response of peripheral lymphocytes from women, their husbands' sperm were found to inhibit the response in 36% of women positive for antisperm antibodies, whereas the inhibition occurred in 81 % of antisperm antibody-negative women (37). There has been much speculation suggesting that antisperm antibodies may arise in women because of a deficiency of one or more of the immunosuppressive factors in her partner's semen, but, to date, convincing evidence is lacking to support the hypothesis. Also present even in "normal" semen but in greater numbers in ejaculates from men with genital tract infections are leukocytes, mostly neutrophils with some lymphocytes. A recent study (38) reported the presence of tumor necrosis factor (TNF) -a and IL-2 in seminal plasma of normal and infected men. They did not find any differences in the levels of TNF -a between normal and infected men. However, IL-2 levels were lower in bacteriainfected semen compared with normal semen. Role of Infectious Agents The major antigen-processing cells in the female genital tract are macrophages, which are found in the submucosal tissues and tract lumen in response to infection or antigens, and Langerhans cells, which are found in the vaginal epithelium. These dendritic cells express human lymphocyte antigen (HLA) class II (DR+) antigens that are associated with antigen-processing capabilities. In addition, epithelial cells of the glandular endometrium, and presumably cervix and oviducts, can be transformed into DR+ cells by inflammatory cytokines released by macrophages and other immune cells (39). DR+ epithelial cells from other tissues can process antigen and stimulate the immune system. Under normal conditions, the presence of sperm cells in the female genital tract or pelvis after coitus or placed in the uterine lumen after lui do not elicit a significant immune response. Sperm cells injected directly into the peritoneum for artificial insemination do appear to induce an immune response in some women (40,41). There is evidence, however, suggesting that such a response might occur with a concurrent infection in which sperm cells can become "innocent bystanders." Oral contraceptive users showed a high association between the incidence of antisperm antibodies and antibodies against chlamydia and candidiasis (42, 43). Women who experienced either clinical or "silent" pelvic inflammatory disease had a high incidence of antisperm antibodies in CM and serum as compared with sexually active but disease-free control women. Also, prostitutes who experience a high incidence of sexually transmitted diseases have a much higher incidence of antisperm antibodies than a control group (44). Microbes may be transported to the upper tract by attaching to the sperm cells. Culturing peritoneal fluids from women after cervical and uterine inseminations has confirmed the presence of seminal microorganisms reaching the pelvic cavity and thus the upper tract (45). Certain infectious agents and their products cause an intense local mucosal inflammation, resulting in an influx and activation of macrophages and lymphocytes. The endometrial stroma and epithelia have been shown to produce different cytokines and leukocyte adhesion molecules that may induce, activate, and distribute leukocytes throughout the reproductive tract. Inflammatory cytokines activate antigen processing cells and T-helper lymphocytes and stimulate division and maturation of sensitized B cells that then circulate through the lymphatic and circulatory systems, finally homing back to the site of the genital tract (46). Once in the lamina propria, local cytokines from monocytes, T cells, and epithelia transform B cells into plasma cells actively secreting antibodies that are transported across the epithelial lining into the tract lumen. Immunoglobulin G antibodies arise from transudation either from serum or plasma cell secretion, and IgA arises through active transport via the secretory component receptor mechanism, which is also upregulated by the inflammatory cytokines (7). A significant level of interferon gamma was found in the sera of 38% of infertile women with antisperm antibodies compared with 7% of antisperm antibody-negative women, suggesting that coitus may result in sperm-sensitized immune cells releasing cytokines, which in turn cause plasma cells to secrete antisperm antibodies (47). Role of Anti-Idiotypic Antibodies in Regulation of Antigenicity of Sperm Cell The immune responses are regulated by cells (including their soluble mediators) and antibodies that are specific for idiotypic as well as conventional antigenic determinants. Idiotype-based regulatory systems offer the possibility of extended chains of complementary members, that is, Vol. 61, No.6, June 1994 Naz and Menge Role of antisperm antibodies in infertility 1005

6 idiotype, anti-idiotype, anti-anti-idiotype, and so on. This theory of immune modulation regulated through the idiotype-anti-idiotype network was postulated by Jerne (48) and has been documented in various experimental and pathological conditions. Anti-idiotypic immunity has been suggested to play an important role in regulation of immune responses. Immune disease could result as a consequence of an alteration of the immunologic network, and anti-idiotypic immunity has been proposed as a treatment for individuals afflicted with disease (49). We have isolated and characterized a sperm -specific glycoprotein, designated fertilization antigen- 1 (FA-1), that has a role in human fertility from human and murine male germ cells (discussed below). Sera from infertile women react strongly with FA-1, indicating its involvement in infertility in humans. Recently, we conducted a study to investigate whether the sera from fertile and infertile women have anti-idiotype (ab-2) to antisperm antibodies (ab-1), especially to FA-1 antibodies (50). The study was conducted using sera from fertile women, infertile women, and virgin women who were not sexually exposed to sperm. Monovalent Fab' fragments (devoid to Fc portion) isolated from these sera were investigated for their reactions with the sperm-specific monoclonal Fab' antibody to FA-1 (discussed below) using the ELISA, Western blot procedure, immunoprecipitation procedure, and immunoaffinity purification procedure. Using these assays, ab-2 were detected in 71 % of sera from fertile women and in none of the sera from the virgin females (Fig. 2). Sera from infertile women that had antisperm antibodies showed a minimal presence of ab-2, with only 13% demonstrating the presence of low levels of ab-2. That ab-2 formation was a result of exposure to sperm cells was suggested by their absence in the sera of virgin women. Anti -F A- 1 Fab' reacted with the immunoglobulins from fertile women only when heavy and light chains were intact, as required by the idiotypic determinants for complete expression. The lack of detection of serum ab-2 in some of the fertile women could be due to the sensitivity of the methods or the amount of free ab-2 circulating, which is dependent upon the nature and kinetics of the immune response generated after exposure to sperm after intercourse. The ab-2 present in fertile women had biologic activity and were capable of neutralizing the fertilization-inhibitory activity of anti-fa-1 antibody in a concentration-dependent manner in a human sperm penetration assay (SPA) of zona-free hamster oocytes. Anti-idiotypic antibodies present in sera of fertile women were also capable of inhibiting the binding of antisperm antibodies present in sera of infertile women to the sperm by the immunobead binding technique (Table 2). There was a great variation in the neutralization capacity among Fabs from various fertile women. Even Fabs from the same fertile woman showed a varied degree of neutralization of the Fabs from different infertile women. This may be due to differences in antigenic specificities of the antisperm antibodies present in sera of infertile women and/or due to the differences in specificities of the ab-2 present in sera of fertile women. Indeed, the sera from various antibody-positive infertile women have been shown to react with a variety of different spermatozoal proteins on Western blots of detergent-solubilized human sperm preparations (51). In the present study, we investigated the presence of ab-2 in sera of fertile, infertile, and virgin women. To examine the in vivo relevance of these findings, studies need to be extended to investigate their presence in the genital tract secretions. However, the antibodies found in female genital tract secretions originate from both the mucosal immune system as well as the systemic immune system dependent on the organ, as discussed above. Thus, the immune induction of antisperm antibodies, either locally or systemically, by whatever etiology may be influenced by the anti-idiotypic antibodies, especially of the IgG isotype. The above results demonstrate for the first time that sera from fertile women who are sexually exposed to sperm have anti-idiotypic antibodies that are capable of neutralizing the antisperm antibody activity. These anti-idiotypic antibodies carried that internal image of the FA-1 antigen and thus were of the ab-2 {3 type. These data suggest that normally in fertile women exposure to semen elicits a systemic immune response with antibodies against sperm, as complementary anti-idiotypic antibodies were detected by several methods. These results support Jerne's network theory (48) of immune modulation regulated through idiotype-antiidiotype interaction. Based on these findings, one can speculate that the immune response to sperm is generated after exposure to sperm in both fertile and infertile women. However, in fertile women, ab-1 generated after exposure to sperm elicits the production of ab-2, which neutralizes the ab-1 response. In contrast, in infertile women, the ab-2 production is either weak or absent; thus it is insufficient to neutralize the ab-1 response, resulting in 1006 N az and Menge Role of antisperm antibodies in infertility Fertility and Sterility

7 CI) C) c II -e 0 fi).a c( AJ FA-1 Antigen BI Anti-FA-1 Fab... CJ Control Fab... I e3l !o _ ~7 ell e2s!.41 I- ~1' t t t e e' e e'..... AnII-!'MIle VIrgIn lib _II!'MIle InIert.. VIrgin lib _II!'MIle F... l (_) (11=12)... I-F... l (11=24) (11=23) F.td F.M (11=12).nll-F... 1 (n--24) Fib' 1nI_ (11=23) VIrgIn (11=12) Figure 2 Reaction of Fab' antibodies present in sera of fertile, infertile, and virgin females with FA-! antigen (A), anti-fa-! MCA Fab' (B), and control myeloma ascites fluid Fab' (C) in an ELISA. Absorbance values above the horizontal dotted line represent >2 SD units, indicating a positive reaction. The FA-! monoclonal antibody Fab' reacted strongly with the FA-! antigen (A). Rabbit anti-idiotypic antibodies (Rb ab-2) to anti FA-! Fab' also reacted strongly with anti-fa-! Fab' (B, lane a); the reaction diminished when FA-! antigen was added to compete with the anti-idiotypic antibody (B, lane a'). Control Fab' did not react with anti-idiotypic antibodies or Rb ab-2 whether tested before (C, lane a) or after addition offa-! antigen (C, lane a'). (Reproduced from Naz et al. [50] with permission) infertility. It can be hypothesized that immune infertility may be a consequence of derangement of the idiotype-anti-idiotype network. The derangement could be a result of either an inherent lack of ab-2 response to the primary ab-l production or hyperimmunization with sperm antigens occurring due to idiopathic reasons or as a concurrent event with infection in the female genital tract with elevated ab-l production ensuing. Besides the idiotype-anti-idiotype network, there may be additional mechanisms, such as cell-mediated immunity (CMI), including suppressor T cells, cytotoxic T cells, and various cytokines (lymphokines-monokines), as discussed above, involved in regulation of antigenicity of sperm cell in the female genital tract. Interactions between various components ofthe immune system in immunoregulation of sperm function require further study. SPERM ANTIGENS RELEVANT TO FERTILITY During the last decade, with the advent of hybridoma technology and Western blot procedure, it became possible to identify sperm-specific antigens that are relevant to fertilization and early embryonic development with potential implication in human infertility. Several sperm-specific antigens have been defined using bioeffective mabs (2). These mabs have been raised against sperm cells of various mammalian species, including mouse, rabbit, guinea pig, and human. Some of mabs have been investigated for cross-reaction with the human sperm cell, and it is envisaged that some of the reactive cognate antigens will be conserved evolutionarily among sperm of various mammalian species and others may be highly species specific. A few of these sperm antigens have been isolated and characterized biochemically and immunologically; cdnas encoding for some of these antigens have been cloned and sequenced. The antigens that have raised special interest recently are lactate dehydrogenase C. (LDH-C.), PH-20, rabbit sperm autoantigens, SP-IO, HSA-63, creatine phosphokinase (CPK), mannose-ligand receptor, FA-I, and CS-I; these antigens are described below. Lactate dehydrogenase-c. is a germ cell-specific Vol. 6!, No.6, June!994 Naz and Menge Role of antisperm antibodies in infertility 1007

8 Table 2 Effects of Anti-Idiotypic Antibodies Present in Sera of Fertile Women on Sperm-binding Activity of Antibodies Present in Sera of Infertile Women* Immunoadsorbed with Immunobead binding Infertile Serum Percent serum (patient) Amount sperm bound Localization p.g/30 p.l p.g/30 p.l Infertile no PBS-BSA Head and tail 15 Fertile (no. 1) Head and tail 15 Fertile (no. 6) Fertile (no. 7) Head and tail 15 Fertile (no. 8) Head and tail 15 Virgin (no. 49) Head and tail 15 Virgin (no. 50) Head and tail 15 Infertile (no. 29) Head and tail Infertile no PBS-BSA Head and tail 15 Fertile (no. 1) Head and tail 15 Fertile (no. 6) Head and tail 15 Fertile (no. 7) Head and tail 15 Fertile (no. 8) Head and tail 15 Infertile (no. 28) Head and tail Infertile no PBS-BSA Head 15 Fertile (no. 6) Head 15 Fertile (no. 7) Head 15 Fertile (no. 8) Head 15 Fertile (no. 28) Head * In these studies, affinity-purified Fab' antibodies showing a single band of 40 kd in sodium dodecyl sulphate-polyacrylamide gel were used. Experiments were performed using sera from eight different infertile women and using sperm from at least three different donors. Three representative experiments are shown here. All these Fabs from fertile women were negative for sperm binding (0% binding) in the immunobead binding test. (Reproduced from Naz et al. [50] with permission). but species cross-reactive enzyme that is present in the cytoplasm and midpiece of the spermatozoa (52). Antibodies to LDH-C 4 inhibit fertility by mechanisms that involve fertilization failure as well as postfertilization preimplantation embryonic morality. Although LDH-C 4 has been investigated extensively for its application in immunocontraception, it has no role in involuntary immunoinfertility in humans (53). Of several monoclones reported against guinea pig sperm, one has drawn considerable interest. This monoclone, directed against the PH-20 antigen, inhibits IVF in guinea pig by inhibiting sperm binding to the egg zona pellucida. PH-20 antigen raised a tissue-specific antibody response that completely blocked fertility in actively immunized male and female guinea pigs, and the contraceptive effect was long lasting and reversible (54). Recently, cdna encoding PH-20 has been cloned and sequenced from the guinea pig testes (55). PH-20 antigen is a highly species-specific molecule and may have limited, if any, crossreaction with the sperm of other species, including humans. Sera from immunoinfertile patients do not seem to have antibodies to PH-20 antigen. An autoantigen (RSA-l) has been isolated and characterized from rabbit sperm that functions as a sperm lectin-like molecule to bind the spermatozoon to the zona pellucida (56). Rabbit sperm autoantigen antibodies reduce fertilization rates in vitro and in vivo and also inhibit human sperm penetration of zona-free hamster eggs (57). It has not been investigated whether the sera from immunoinfertile patients have antibodies to rabbit sperm autoantigen. Even if an antigen shows an inhibition of fertilization in a variety of animal species, its role in human fertilization still has to be elucidated. Several monoclones have been reported that bind to human sperm and some of these also inhibit human sperm penetration of zona-free hamster oocytes (SPA). Many of these clones inhibit human sperm cell function by causing agglutination or complementmediated immobilization of sperm. Agglutination and immobilization can be specific as well as nonspecific and can, at best, partially reduce rather than completely block fertility. Wright et al. (58) have reported a tissue-specific monoclonal antibody (MHS-10) against a sperm protein (SP-10) that impairs human sperm penetration of zona-free hamster oocytes by causing agglutination of sperm. SP-lO is an intra-acrosomal protein, and cdnas encoding SP-10 protein have been cloned and sequenced from human cdna expression library (58). At the present time, it is not known whether active immunization with SP-10 causes a reduction of fertility. It also has not been determined if the sera from immunoinfertile patients have antibodies reactive with SP-lO. Another mab, monoclone HS- 63, that reacts with acrosome of spermatozoa of mouse and human has been shown to inhibit IVF in mice and SPA in humans. The species cross-reactive cognate antigen (HSA-63) recognized by HS- 63 monoclonal antibody has been purified partially and characterized from murine and rabbit testes. The HSA-63 is immunogenic in female mice and rabbits, and polyclonal antisera block murine IVF and human SPA. However, active immunization with HSA -63 does not reduce fertility in vivo. The HSA-63 antiserum reacted with four clones from murine cdna testis library constructed in Agtll expression vector. One of these clones has been sequenced (59). Sera from immunoinfertile patients do not seem to have antibodies to HSA-63. Additionally, two other proteins, namely the CPK and mannose-ligand receptor, have been 1008 N az and Menge Role of antisperm antibodies in infertility Fertility and Sterility

9 reported to be involved directly or indirectly in human sperm maturation, and in human spermhuman zona pellucida binding and acrosomal exocytosis, respectively. Creatine phosphokinase, involved in the creatine-creatine phosphate shuttle, a key enzyme of sperm energy transport, was found to have inverse correlation between sperm CPK activity and sperm concentration, with significant elevation in sperm of infertile versus fertile men (60). The level of mannose-ligand receptor on sperm surface was found to have significant relation to capacitation, acrosome status, and content of nonesterified cholesterol in sperm (24). In context of immunofertility, it was found that antisperm antibodies present in the sera of immunoinfertile patients are not directed against the mannose-ligand receptor of 48 and 61kd. However, antisperm antibodies inhibit sperm surface expression of mannose-ligand receptors indirectly by altering the membrane fluidity (24). The CPK and mannose-ligand receptors are not specific to sperm and are present in a variety of somatic cells. We have isolated a glycoprotein, FA-1, from human and murine testes using germ cell-specific but species cross-reactive mab (MA-24) raised against human spermatozoa that inhibits murine IVF and human SPA (61, 62). Fertilization antigen-1 is a glycoprotein comprised of a monomer of 23 kd and/ or a dimer of 51 ± 2 (mean ± SD) kd; the forces of dimerization are ionic in nature rather than disulfide bonds. A significant reduction of fertility occurred in female rabbits after immunization with FA-1, with a majority of animals showing a complete block (63). Antisera collected after immunization were tissue specific and inhibited fertilization by mechanism(s) other than agglutination and immobilization of sperm. In the mouse, FA-1 activated presensitized lymphocytes to secrete soluble mediators that activated macrophages and significantly inhibited sperm motility and embryonic development (64). The results indicate that sperm antigens specifically can induce CMI factors in vitro that have detrimental effects on sperm motility and preimplantation embryos. Likewise, active immunization with FA-1 and other sperm antigens might affect fertility by inducing humoral and cellular immune responses, both of which are detrimental to sperm and embryos. These results also suggest that CMI factors (cytokines), besides antisperm antibodies, are involved in the etiology of immunoinfertility. Fertilization antigen-1 reacts with sera from immunoinfertile males and females (65) and vasectomized men (66) and not with sera from fertile men and women. There was a strong correlation between the presence of antibodies and failure of IVF in humans (65). The couples that had FA -1 antibodies in sera and seminal plasma of male partners of the infertile couples showed a block of fertilization. It was further observed that immunoinfertile sera that showed reduced penetration rates in human SP A demonstrated an increase after adsorption with FA-I. Additional evidence that antibodies to FA-1 may playa role in human infertility was obtained in a series of collaborative studies. In a collaborative study with Bronson et al. (67), it was found that the addition of purified FA-1 to the immunobead-positive immunoinfertile sera render them negative in the immunobead technique. In another study with Naz et al. (68), it was found that FA -1 specifically induced lymphocytes from infertile men and women (not from fertile men and women) to proliferate in vitro, indicating that the lymphocytes from immunoinfertile patients are sensitized to FA-I. In another recent study with Naz et al. (19), it was found that the antibodies to FA-1 inhibit human sperm-human zona interaction, reinforcing the fact that FA -1 may be involved in sperm-zona pellucida ligand interaction. However, it also was found that the immunoaffinitypurified anti-fa-1 mab inhibits acrosome reaction of human sperm cells in solution (but not on the zona pellucida surface) (69), thus indicating that FA-1 antigen is also involved in human sperm capacitation and acrosomal exocytosis, besides its involvement in sperm -zona pellucida interaction. Presently, we are cloning and sequencing the FA-1 antigen from human testes-xgtll expression library and delineating its bioeffective epitope involved in biologic action. Mammalian developmental biologists are searching for a signal that is required for the fertilized oocyte to cleave. Transcription starts at four- to eight-cell stage in human embryos and between one- to two-cell stage in murine embryos (70). Thus, the signal for the first cleavage has to be provided by an extranuclear preformed message-molecule. The research carried out in our laboratory indicates for the first time that a sperm surface antigen, the cleavage signal (CS-1) protein, may provide the first signal to the oocyte to cleave. The CS-1 is present on the sperm surface of various mammalian species, including humans and mice (28), and is different from GA-1 (71). Interestingly, the immunoinfertile patients have antibodies to CS-1 and, once the antibodies are present, they exercise their Vol. 61, No.6, June 1994 N az and Menge Role of antisperm antibodies in infertility 1009

10 effects by inhibiting first cleavage of the pronuclear stage zygotes (28). We have cloned and sequenced the CS-1 from the human testes, and the extensive search in Genbank and protein library databases did not indicate any homology with any known sequence (72). Presently, we are studying the effects of recombinant CS-1 on oocyte cleavage by microinjecting into the oocyte. We also are investigating the potential application of recombinant CS-1 in immunoinfertility in humans. Recently, there has been an enhanced interest in the signal transduction pathway(s) leading to sperm cell maturation and differentiation, especially capacitation and acrosome reaction, which have remained an enigma since their discovery in There are three different groups of molecules-processes, though not mutually exclusive, namely the proto-oncogene products, guanine nucleotide-binding regulatory proteins (G-proteins), and membrane phosphotyrosine proteins, which have been investigated extensively as participants in the signal transduction pathway(s) relevant to sperm cell function (73-78). Antibodies present in sera of immunoinfertile patients reactive with these components directly or indirectly can affect the fertilization process and fertility. It will be interesting to investigate whether some of the infertile men that have abnormal sperm cell function, especially in capacitation and/or acrosome reaction, have a defect in expression-structure-activity of proto-oncogenes products-g-proteins-tyrosine phosphorylation. CONCLUSIONS Research activities as related to the reproductive immunology and molecular biologic aspects of antisperm antibodies are going in four main directions. The first major emphasis is on delineating sperm antigens that are relevant to fertility (fertilization and embryonic development) and against which the immune response is generated in immunoinfertile men and women. As discussed earlier, the sera from immunoinfertile patients react with various sperm antigens in the Western blot and immunoprecipitation procedures; however, no definite pattern has been found. At this time only two sperm antigens, FA-1 and CS-1, that have been characterizedextensively and/or cloned and sequenced have been implicated in immunoinfertility in humans, and the involvement of FA -1 has been confirmed by various leading investigators working in the field of immunoinfertility. It is envisaged that additional antigens are also involved because anti-fa-1 antibodies have been detected in only 70% of sera from immunoinfertile patients and immunoadsorption of sera from immunoinfertile patients with purified human FA-1 causes a complete neutralization of fertilization-inhibitory activity in majority of the sera, and only a partial neutralization in the others, indicating the presence of antibodies to others antigens that are relevant to fertility. Recently, research in the area of sperm antigens has received a strong impetus due to renewed interest in immunocontraception and immunoinfertility. These antigens will be used for specific diagnosis and treatment of immunoinfertility. The second major emphasis is on delineating the cells, molecules, and mechanisms involved in regulation of antigenicity of sperm cell in the female genital tract. Again, the area of local genital tract immunity (versus systemic immunity) has regained impetus due to advancement of hybridoma and recombinant technologies and enhanced interest in immunocontraception. The class and subclass of antibodies' especially to well-characterized fertilityrelated sperm antigens (79), their local synthesis, relevance to in vivo fertility, and kinetics of the immune response generated are being extensively investigated. Also, the role of CMI involving welldefined cytokines is being examined extensively. The third major emphasis is on the role of signal transduction pathways (proto-oncogenes, G-proteins, and phosphotyrosine protein-kinases) in sperm cell function and fertilization and use of latest molecular biologic techniques such as polymerase-chain reaction (PCR) and Northern and Southern blot analyses in investigating the presence and expression of relevant genes involved in sperm cell function. Recently, using PCR and in situ hybridization, we found that the sperm cell has mrna in the residual cytoplasm of mature sperm cell (80). The role of this stored mrna in sperm cell function and subsequent oocyte cleavage and in regulation of immunogenicity needs to be investigated. The fourth major emphasis is on the immunotherapy of immunoinfertile patients. Immunoinfertile patients are otherwise healthy individuals without any disease concomitant with infertility, and antisperm antibody production does not induce immune complex formation (81, 82), so that the only treatment needed is to stop or neutralize the antisperm antibody production. Based on the idiotype-anti-idiotype network, it may be possible by providing gamma globulin to turn off the immune 1010 Naz and Menge Role of antisperm antibodies in infertility Fertility and Sterility

11 mechanism responsible for continued production of antisperm antibodies. This concept is based on the success that has been achieved in controlling antibody formation in Rh-negative pregnant women carrying Rh-positive fetuses. Another approach that is gaining credence is the oral administration of peptide preparations of some antigens, which will induce tolerance to the native antigenic molecule when administered or reduce levels of existing antibodies in cases of prior immunization. This approach would be limited to a single or few sperm antigens, such as FA-I, that are relevant to human immunoinfertility. The competitive binding or blocking of antisperm antibody binding sites by epitopes of relevant antigens also may be a method of in vitro therapy, freeing sperm from fertility-inhibiting antibodies. In conclusion, the field of sperm antigens and antisperm antibodies is growing rapidly. Advances in technologies and renewed interest in immunocontraception and immunoinfertility are increasing our understanding of the molecules and mechanisms involved in immunoregulation of sperm function in the female genital tract. This information will provide a basis for development of better methods for specific diagnosis and treatment of immunoinfertility in humans. Acknowledgments. We sincerely thank Ms. Sheree Salas and Ms. Margaret O'Connell for excellent typing assistance. 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Characterization of the fertilization antigen (FA-I) for the development of a 1012 N az and Menge Role of antisperm antibodies in infertility Fertility and Sterility