Antisperm antibodies: etiology, pathogenesis, diagnosis, and treatment

FERTILITY AND STERILITYt VOL. 70, NO. 5, NOVEMBER 1998

MODERN TRENDS

Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Edward E. Wallach, M.D. Associate Editor

Antisperm antibodies: etiology, pathogenesis, diagnosis, and treatment Setu Mazumdar, M.D.,* and Adam S. Levine, M.D.*† Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, Maryland and Tampa Obstetrics, Fertility and Gynecology, Tampa, Florida

Objective: To critically review the English-language literature and describe the current diagnosis, prevalence, etiology, and treatment of antisperm antibodies (ASA). Design: A comprehensive literature search of the English-language literature published between 1966 and December 1997 was performed on MEDLINE. Articles were also located via bibliographies of published works. Result(s): Data were excerpted from articles identified by MEDLINE search. The diagnosis, prevalence, etiology, and treatment of ASA are described. Conclusion(s): There is sufficient evidence that ASA impair fertility in couples with unexplained infertility. A number of different methodologies are available, which may be used in their detection. However, in many cases, test interpretation is subjective. Although there is not enough evidence to support systemic treatment for ASA, application of a variety of assisted reproductive technologies improves outcome. (Fertil Sterilt 1998;70:799 – 810. ©1998 by American Society for Reproductive Medicine.) Key Words: Antisperm antibodies, andrology, infertility, immunology, unexplained infertility

Received January 7, 1998; revised and accepted March 13, 1998. Reprint requests and present address: Adam S. Levine, M.D., Tampa Obstetrics, Fertility, and Gynecology, 888 South Parsons Avenue, Brandon, Florida 33511 (FAX: 813654-1384; E-mail: [email protected]). * Division of Reproductive Endocrinology and Infertility, Johns Hopkins University School of Medicine. † Tampa Obstetrics, Fertility and Gynecology. 0015-0282/98/$19.00 PII S0015-0282(98)00302-1

One in five reproductive age couples in the United States is infertile. Despite an adequate medical evaluation, approximately 15% of them will have unexplained infertility (1). There is now mounting evidence to support the immunomodulation of fertility in many of these couples. One major aspect of this immunomodulation may be the presence or absence of antisperm antibodies (ASA). Antisperm antibodies research began in 1899 when Landsteiner initially reported that sperm could be antigenic if injected into a foreign species. After this, Metalnikoff found that sperm were also antigenic when injected into the same species (2). Those early investigations led to the possibility that naturally occurring male and female ASA could serve as one mechanism for human infertility. In 1954, Wilson reported two cases of infertile men exhibiting spontaneous sperm agglutination. These men had natural spermagglutinating autoantibodies in their seminal plasma and serum (2). Later, Franklin and Dukes used a microscopic sperm agglutinization assay with the sera of women with unexplained infertility to demonstrate that as many

as 80% (15 of 19) of infertile women had ASA. However, the assay they used was subject to a high false-positive rate. Since their pioneering work, a wide range of ASA prevalence has been reported (2). A precise estimate of ASA prevalence is limited by the methods used for detection and their subsequent interpretation. There are numerous physical locations in which ASA may be located, including male or female serum, semen, ovarian follicular fluid, vaginal or cervical secretions, or as antigenic epitopes bound directly to the outer sperm plasma membrane. In addition, different isotypes of ASA, including the immunoglobulins A, G, and M (IgA, IgG, IgM), have been identified. Finally, the populations previously studied were limited by small sample sizes. This makes the conclusions subject to error from lack of sufficient power. This review examines the methods used to detect ASA, their prevalence, the etiology of their formation, the pathogenesis by which they may lead to infertility, and, finally, possible treatments for ASA-mediated infertility. 799

ANTISPERM ANTIBODY DIAGNOSIS Numerous methodologies are used to detect ASA. Each has distinct advantages and disadvantages. Cunningham et al. (3) summarize our experience to date with ASA, stating that “neither a specific antigen(s) nor a superior antibody detection assay exists, although both are requisite to an understanding of the significance of antisperm antibody production and the potential manipulation of the female immune response to spermatozoa for the purpose of infertility reduction and potential immuno-contraception.” The ideal assay should detect the presence of ASA, their location, and their isotype with high sensitivity and specificity. Commercially available ASA assays either directly measure ASA bound to sperm or indirectly measure ASA in solution (serum, semen, vaginal or cervical secretions, or follicular fluid). These assays include immunobead assays (IBD), mixed antiglobulin reaction (MAR) tests, ELISA, tray agglutination tests (TAT), sperm immobilization assay tests flow cytometry, and radiolabeled agglutinin assays. An IBD assay (4, 5) is composed of polyacrilimide beads that are coated with a specific antiimmunoglobulin. The coated beads then are mixed with fresh, viable, washed or unwashed sperm samples and ultimately bind to sperm bound ASA. This test is evaluated by light microscopic visual appearance. A qualitative score is based on the percent of sperm-bead binding and location of bound beads. It is notable that there is a degree of nonspecific cross-reactivity between normal sperm plasma membrane epitopes and IBD binding. This may cause difficulty interpreting between positive and negative tests. Advantages of an IBD assay include its semiquantitative nature, the ability to detect the isotype and physical location of the ASA, good assay sensitivity and specificity, and the ability to use the test on viable sperm. The disadvantages of IBD testing include a requirement for a skilled staff, expense, time, and occasional difficulty in test interpretation. The MAR (6, 7) assay is similar to IBD testing. Blood group O Rh-positive erythrocytes are coated with human IgG or IgA and, subsequently, mixed with washed or unwashed, viable sperm. Antiserum specific to the immunoglobulin used to coat the erythrocytes is added, and spermerythrocyte agglutination occurs in the presence of ASA. This agglutination then may be determined semiquantitatively by light microscopy. The advantages of MAR testing include a rapid assay time, good specificity, and the ability to use viable sperm. The MAR testing is limited by its inability to provide quantitative information about ASA binding or location, an unknown sensitivity, expense, and the need for skilled technical assistance. Another option for ASA detection is ELISA. Antibodies to isotype specific immunoglobulins are covalently linked to an enzyme and added to a sample (fixed sperm, sperm extracts, cervical mucus extracts, or sera). Antibody-enzyme800

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immunoglobulin complexes are detected by addition of a specific enzyme substrate, usually resulting in a color change that may be quantified. The advantage of this method is that it is both specific and quantitative. The major disadvantage of this assay is that samples are generally subjected to fixation, which may disrupt the outer plasma membrane. This disruption of the plasma membrane may result in the detection of internal antigens. Another disadvantage of this assay is that the antibody used may bind to nonspecific antigens, resulting in false-positive identification. ELISA also is limited by the time involved, its cost, poor sensitivity, and inability to determine ASA location and isotype. TAT (8) is used to detect ASA in patient serum or semen. Fluid samples are diluted serially after heat inactivation of native complement activity. Washed, motile sperm from a healthy donor are added to the dilute patient samples and to known positive and negative control samples. The percent of sperm agglutination then is determined by light microscopic evaluation. Sperm immobilization assay tests (9, 10) use heat-fixed patient serum mixed with motile sperm and a measured external source of complement (such as rabbit serum) to detect loss of sperm motility. Patient serum is compared with known positive and negative sera. Both of these methods are limited by their reliance on skilled assistance, and neither provides quantitative data. Two other methods of ASA testing include flow cytometry and radiolabeled agglutinin assays. For both, an isotype specific antiimmunoglobulin is labeled with a marker (fluorescent, magnetic, radioactive) and mixed with a sperm sample. A flow cytometer uses an appropriate method of detection (laser or magnet) to process a continuous stream of the sample being tested. Labeled cells are evaluated and, in some cases, sorted. The major advantage to using flow cytometry is a quantitative analysis and specific isotype determination. However, nonmotile sperm must be used for this assay, and false positives may result after immobilization or fixation. The disadvantages of using flow cytometry include the time involved, expense, and requirement for skilled labor. Radiolabeled agglutinin assays (11) uses radiolabeled antibodies to detect and quantify ASA. This method is limited by an inability to determine specific ASA location, expense, and reliance on skilled labor. There is a close correlation between Radiolabeled agglutinin assay test results and IBD (12). The conflicting data reported by various investigators may be a result of many different factors. In addition to actual differences between ASA testing modalities, specimen preparation and subsequent test interpretation vary by laboratory. Furthermore, sperm specimens are dynamic, undergoing maturational changes including capacitation and the acrosome reaction. This results in changing ASA epitopes as the outer acrosomal membrane and its associated Vol. 70, No. 5, November 1998

proteins are lost and the antigens present on the inner acrosomal membrane are exposed. Haas et al. (13) noted that there are several pitfalls associated with comparisons between the different methods of ASA detection. Many methods rely on subjective determinations and variable specimen preparations. False-positive results may occur with agglutination tests in the presence of nonantibody factors. Furthermore, sensitivity and specificity vary for each of the testing modalities. Immunoglobulins bound to the sperm surface may be either different or absent from semen or serum. They compared flow cytometry, MAR, and IBD in 36 patients (18 IBD-positive serum samples and 18 IBD-negative serum samples). In this study, IBD was considered positive if .20% of motile sperm demonstrated immunobead binding. Radiolabeled agglutinin assay was considered positive in patient sera when sperm-associated radioactivity was .3 SDs above that of the 18 IBD-negative patients. Flow cytometry and MAR were both negative in IBDnegative patients. Patients (77%) with IBD-positive serum samples were also positive by flow cytometry compared with 27% who were MAR positive, suggesting that flow cytometry is more sensitive than MAR. They concluded that IBD testing provides investigators with a highly sensitive diagnostic test. Rajah et al. (14) found a significant correlation between IBD, MAR, and TAT testing in a comparison of semen samples from 109 infertile men. For this study, the MAR was considered positive if .10% of motile sperm adhered to agglutinated erythrocytes, and the IBD was considered positive if .20% of the motile sperm were attached to one or more beads. They noted that there were five men with a positive TAT (serum test) but negative MAR or IBD tests (sperm surface bound ASA). Furthermore, five additional men had positive IBD testing but negative TAT assays. They concluded that, given similar results, MAR should be used as a screening test because it is more rapid than IBD (3 minutes versus 30 minutes) and less expensive (one-third the cost). If ASA are detected by MAR, further IBD testing could be used to determine immunoglobulin isotype. Andreou et al. (15) support this view, concluding that MAR testing for IgG and IgA is both sensitive and specific as well as easier to perform and more accurate than respective IBD testing. Eggert-Kruse et al. (16) examined the usefulness of serum ASA evaluation by ELISA in 95 couples undergoing infertility investigation. They compared ELISA results with MAR testing, semen analyses, postcoital testing, spermcervical mucus penetration tests, and subsequent fertility. In this study, MAR was considered positive if .30% of the motile sperm were agglutinated, and ELISA was considered positive when the sperm-antibody concentration was $95 ng/mL. No patient who was MAR positive was ELISA positive. Furthermore, serum evaluation by ELISA revealed FERTILITY & STERILITYt

no statistically significant differences between infertile women, virgin women, pregnant women, or prostitutes. They concluded that ELISA should not be used during an infertility evaluation.

ANTISPERM ANTIBODY PREVALENCE It is difficult to estimate the actual prevalence of ASA given the vast array of diagnostic testing modalities available and their subsequent interpretations. Haas et al. (17) used radiolabeled agglutinin assays to evaluate sera from 614 men and women with unexplained infertility. They determined that 7% of men and 13% of women were ASA-positive. Nip et al. (18) used ELISA to report that ASA were present in the sera of 77% of women with unexplained infertility, 75% of women with endometriosis, and 60% of women with tubal infertility. In this study, only 5% of control women had sera positive for ASA. Furthermore, they suggested that ASA could potentially affect IVF rates because ASA were not demonstrated in the follicular fluid of control patients, but were demonstrated in the follicular fluid of 13% of women with unexplained infertility, 30% of women with endometriosis, and 20% of women with tubal infertility. Pattinson and Mortimer (19) used IBD to examine seminal fluid from 300 infertile men undergoing evaluation and reported an ASA prevalence of 10.7%. Witkin (20) reported a 15% incidence of sperm bound by ELISA. Finally, Mandelbaum et al. (21) used IBD testing of sera, semen, and follicular fluid in 36 couples undergoing IVF to determine that ASA to the sperm head were present in 10% of men and in 15% of women.

ETIOLOGY OF ASA FORMATION The basic function of the immune system is to provide a mechanism by which individual organisms are able to recognize self from nonself or foreign. This identification of self usually depends on protein moieties present on the outer cellular plasma membrane, which serve as antigens. Lipids, polysaccharides, and nucleic acids also may function as antigens. Recognition that an antigen is self or foreign with subsequent antibody formation depends on Burnet’s clonal selection theory (22). This theory suggests that the immune system develops during early fetal life when tolerance to self occurs after presentation and subsequent recognition of self by the thymus gland. Antigens not present during fetal life are recognized subsequently as foreign; antibodies are produced, and the cells bearing these antigens ultimately are destroyed. Immunoglobulins are antibodies produced in response to a specific antigen. There are five known immunoglobulin isotypes: IgA, IgG, IgM, IgE, and IgD. Each is composed of two isotype-specific heavy chains and two light chains (k or l). Immunoglobulins are divided into two regions, the Fab 801

(amino-terminal) portion binds to antibodies, and the Fc (carboxy-terminal) portion binds to other immunoreceptors. Three immunoglobulin isotypes are intimately involved in ASA recognition. Immunoglobulin M is a pentamer weighing approximately 900 kD, with a serum concentration of between 50 and 400 mg/dL, and a biologic half-life of 5 days. Immunoglobulin G is a monomer weighing approximately 150 kD, with a serum concentration of between 600 and 1,500 mg/dL, and a biologic half-life of between 21 and 23 days. Immunoglobulin M is generally the first immunoglobulin synthesized in response to a novel antigen followed in several weeks by IgG synthesis. Immunoglobulin A may be either a monomer or a dimer, weighing between 150 and 400 kD, with a serum concentration between 85 and 380 mg/dL, and a biologic half-life of 6 days. Immunoglobulin A is a secretory immunoglobulin present in a variety of bodily secretions, including semen and cervical mucus (23). Sperm have foreign antigens because they are not present until after puberty and most likely develop in an immunoprivileged site. Sperm production takes place as a developmental syncitium protected from recognition by the bloodtestis barrier, which is located at the basement membrane of the seminiferous tubules and formed by tight intercellular junctions between Sertoli cells. Active spermatogenesis is a requisite for the development of ASA in men. Bronson et al. (24) demonstrated that ASA were detected only after the onset of puberty in a cohort of men with cystic fibrosis (CF). Importantly, the prevalence of ASA in men with CF is normally greater than in men without CF. The time-course of ASA development is not clear. Data from a rat model suggest that IgM ASA develop within 2 weeks of a vasectomy (25). Immunoglobulin M titers subsequently diminish over 4 – 8 weeks followed by increasing titers of ASA IgG between 8 and 12 weeks. There are several hypotheses for ASA formation in men. Theoretically, the blood-testis barrier may be breached by a variety of mechanisms resulting in exposure of immunogenic sperm antigens to the immune system. Ultimately, this exposure could initiate an immune response, resulting in an inflammatory reaction and ASA formation. Mechanical obstruction of the genital tract may occur as a result of congenital anomaly, vasectomy, or trauma (26, 27). Extravasation of sperm is common in men after vasectomy. Several reports suggest that between 50% and 70% of these men subsequently have sera positive for ASA (2, 28). Occlusion of the vas deferens is common in men with cystic fibrosis. Vazquez-Levin et al. (29) examined the association between vas obstruction secondary to CF and ASA formation using IBD testing. The IBD testing was defined as positive when .10% of the beads bound to motile sperm. Immunoglobulin M was the only antibody isotype identified in CF patients who were peripubertal. This finding suggests that ASA formation in these men resulted from fairly recent 802

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exposure. They further reported a 75% ASA incidence in adult males with CF and a 71% incidence of ASA in patients with congenital bilateral absence of the vas deferens. Matsuda et al. (30) initially reported that some male infertility patients have either a unilateral or bilateral occlusion of the vas deferens following childhood inguinal herniorrhaphy. They subsequently (31) used indirect IBD testing to examine the sera of 13 of these men. Immunoglobulin G was identified in 54% of the patient’s sera, and IgA was identified in 15% of the patient’s sera. Histologic evaluation after vasovasostomy revealed the presence of epididymal disruption in 5 of these men. They concluded that, although epididymal disruption was not present in most men, the incidence of ASA after childhood inguinal herniorrhaphy is similar to the incidence of ASA in vasectomy patients. The impact of varicoceles on male infertility is controversial. Varicoceles are present in 5%–15% of the general population (32) and in 30%– 40% of infertile men (33, 34). Theoretically, impaired venous drainage of the testis may result in damage to the seminiferous tubules and lead to ASA production. Gilbert et al. (35) used ELISA to demonstrate the presence of sperm-bound immunoglobulins in 32% (27 of 84) of infertile men with palpable varicoceles. They found IgA in 85% of the men, IgG in 67% of the men with ASA, and IgM in 74% of the men with ASA. They also noted that men with sperm-bound immunoglobulins demonstrated significant decreases in sperm concentration and motility as well as an increase in abnormal morphology. These changes were not evident in men with sera positive for ASA, but without sperm-bound immunoglobulins. They speculated that greater epididymal damage was present in those men with sperm-bound immunoglobulins, resulting in the impaired semen analyses. Notably, Witkin (20) previously reported an overall 15% ASA incidence by ELISA in infertile men without palpable varicoceles. Inflammation may lead potentially to genital tract disruption and ASA formation. Kortebani et al. (36) examined leukocyte populations in semen samples from 279 infertile men. Eighty-five (30%) of the semen samples were leukocytospermic (.1 3 106 white blood cells/mL). Leukocytospermic samples demonstrated a decrease in sperm motility, poor sperm morphology, and a lower semen fructose concentration than nonleukocytospermic samples. The IBD testing was used, and .20% immunobead binding to motile sperm was considered positive. Antisperm antibodies were more common in the highconcentration granulocyte group with low-fructose levels than in the granulocyte group with normal fructose levels. There was a 38.3% prevalence of ASA in men with dysfunctional seminal vesicles compared with a 13% ASA prevalence in men with normal glandular function. There were also increases in macrophage and granulocyte concentrations Vol. 70, No. 5, November 1998

in men with hypofunctional glands compared with men with normal glandular function. They concluded that there was a link between leukocytospermia, seminal gland dysfunction, and occurrence of ASA. Wolfe et al. (37) confirmed an association between leukocytospermia and a reduction in semen analysis parameters. They found that leukocytospermia (.1 3 106/mL) was correlated with significant decreases in total sperm number (41% reduction), percent motile sperm (22% reduction), sperm velocity (12% reduction), sperm motility index (40% reduction), and total number of motile sperm (66%). However, with the use of direct immunobead testing, they could not demonstrate an association between the presence of ASA and leukocytospermia. Organisms that cause sexually transmitted diseases may serve as initiators of ASA formation through both inflammatory processes and autoimmune mechanisms. Numerous studies have demonstrated that various bacterial, viral, and fungal particles may attach themselves to the outer sperm membrane. These particles themselves may serve as antigens or haptens, inciting an immune response. A prospective study of 197 randomly selected asymptomatic, infertile men demonstrated an 18.8% prevalence of IgA antibodies to Chlamydia in semen (38). Witkin et al. (39) compared the seroprevalence of antibodies to Chlamydia trachomatis in 227 asymptomatic male partners of infertile couples with ASA testing by IBD. A positive IBD was defined as .20% immunobead binding to motile sperm. Immunoglobulin A antibodies to Chlamydia were present in 25% of semen samples and in 15% of serum samples. It is of interest that 75% of the semen samples that were positive had negative serum samples. Immunoglobulin G antibodies to Chlamydia were present in 22% of semen samples and in 11% of serum samples. Antisperm antibodies were demonstrated in 16.3% of all samples. It is notable that antichlamydial antibody was present in 51.4% of men with positive ASA compared with 16.8% of men with negative ASA. Munoz and Witkin (40) used IBD testing to examine the relationship between asymptomatic male genital tract infection with C. trachomatis, gd and ab T-lymphocyte populations in semen, and ASA. For this study, .30% bead binding to motile sperm was considered positive. They concluded that there was a positive association between asymptomatic chlamydial infection in men and autoimmunity to sperm. In their study of 48 infertile men, 29.2% had antichlamydial IgA in semen, and 27.1% had ASA in semen. The men with positive seminal antichlamydial IgA demonstrated an increased gd T-cell concentration in semen, but not an associated increase in serum IgG or IgA. They believed that their data supported the hypothesis that an immune reaction could occur in response to a local phenomena in the genital tract. Finally, Greskovich et al. (41) used a rat model to demonstrate first that ASA may be FERTILITY & STERILITYt

formed after an induced genital tract infection (epididmyitis) and second, that the ASA response could be blunted by prompt antibiotic treatment. Immunosuppression is another potential mechanism by which ASA may be produced. Liu et al. (42) compared the lymphocyte subpopulations in peripheral blood from 28 men with ASA with men without ASA. Antisperm antibodies were detected by either sperm immobilization assay tests or ELISA. They found that men with ASA had an increased percentage of B cells, an increase in the CD4-CD8 ratio, and a decrease in total CD4 and CD8 cells. They concluded that ASA result in greater B-cell function as a result of an increase in mitogenic B-cell activity. They also concluded that T-cell function was decreased in men with ASA after a decrease in mitogenic T cell activity. The greater CD4-CD8 ratio may represent a decline in suppressor T-cell function, which would suggest that T-cell suppressor function in the testis prevents an immune response to sperm and that loss of suppressor T cell function may lead ultimately to ASA formation. Theoretically, suppressor T cells regulate B-cell antibody production. If suppressor T-cell activity decreases, B-cell antibody production could increase. Production of ASA in women may occur in a variety of ways. Mechanical or chemical disruption of the mucosal layer of the female genital tract may permit exposure to foreign sperm antigens and, ultimately, ASA formation. However, the reason most women do not develop an immune response after repeated sperm exposure is not clear. Furthermore, sperm within the peritoneal cavity after transtubal passage also could induce serum ASA formation through macrophage phogocytosis and presentation to T cells leading to an immune response. However, Mashu et al. (43) used sperm immobilization assay tests to compare ASA prevalence between 49 infertile women after artificial insemination by donor (AID) with 151 infertile women undergoing some other form of assisted reproductive technology (ART) with their husband’s sperm. These 49 women had a mean of 14.5 AIDs (range, 1– 44), each of which was usually from a different donor. No differences in ASA prevalence were noted between the two groups. They concluded that semen exposure from multiple donors does not necessarily increase the chance of inducing sperm-immobilizing antibodies. Using an indirect MAR for IgG, Eggert-Kruse et al. (44) reported a 1.6% incidence of ASA in the cervical mucus of 192 infertile women with a median of 5 years of infertility (range, 1–12 years). They were not able to demonstrate a relationship between MAR-positive cervical mucus and the presence of other pathogenic microorganisms. All positive MAR women had poor postcoital tests, and no pregnancies occurred in the 13 months after testing. This study was also unable to demonstrate a relationship between ASA in cervi803

cal mucus and T-cell populations in the cervical mucus. They concluded that although ASA prevalence in cervical mucus is infrequent, it may result in significantly impaired fertility.

tility. This study supported previous work by Upadhyaya et al. (50) and by Mathur et al. (55) in which men with ASA had similar decreases in linearity, velocity, and percent motility.

Antisperm antibody formation also may occur as a consequence of local inflammation after genital infection in women (45). Cunningham et al. (46) confirmed this in an examination of the prevalence of ASA in reproductive age nulligravid women with various gynecologic infectious processes. They used an indirect sperm MAR test to detect IgG in patient sera and cervical mucus.

Sperm with abnormal motility may not penetrate the cervical mucus. Steen et al. (56) found that men with ASA against the equatorial segment of the sperm cell present in either serum or semen have less cervical mucus penetration than men without ASA. Eggert-Kruse et al. (57) used MAR to evaluate the effect of ASA on sperm penetration into cervical mucus in 209 infertile couples. Mixed antiglobulin reaction was considered positive when .30% of the motile sperm became agglutinated.

Forty-six percent of women diagnosed with upper genital tract disease (pelvic inflammatory disease [PID], n 5 81 women) had sera and cervical mucus positive for ASA compared with an ASA prevalence of 20% in women who had lower genital tract infection (fungal, chlamydial, or bacterial vaginitis, n 5 86 women). Antisperm antibodies also were detected in 69% of women with laparoscopically confirmed pelvic adhesive disease or hydrosalpinx (n 5 45) but with no prior history of PID.

PATHOGENESIS OF ASA-MEDIATED INFERTILITY The precise mechanism for ASA-mediated fertility impairment is unclear. In either the male or female reproductive tract, ASA may have an adverse impact on sperm maturation and function or overall semen quality (47). Fertilization requires that enough sperm undergo capacitation or other maturational changes resulting in the development of fertilizing potential. In addition, sperm must maintain some degree of forward motility to reach the oocyte. There, sperm must bind and penetrate the zona pellucida. Finally, after penetration, sperm nuclei must decondense, and embryo cleavage must begin. The relationship between ASA and sperm concentration is controversial (48, 49, 50). A decrease in sperm concentration potentially could decrease the chance for fertilization to occur. Antisperm antibodies may disrupt normal sperm function by damaging sperm motility. This may occur after exposure to ASA in the presence of heterologous complement (51, 52). Computer-aided semen analysis (CASA) is used to evaluate and quantify various parameters of sperm motility, including sperm head amplitude, linearity, straight and curvilinear velocity, and percent motility. The effect of ASA on CASA results are controversial (49, 53). Check et al. (49) used a direct immunobead test (54) to compare the effect of ASA on CASA in 239 men. They reported that 10% of the men had positive ASA (.50% immunobead binding [n 5 24]), 4.6% of the men had weakly positive ASA (20%– 49% immunobead binding [n 5 11]), and 85.4% of the men did not have ASA (no immunobead binding [n 5 204]). All men with ASA had a significant reduction in linearity, velocity, and percent mo804

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In this prospective study, 13% of the men had IgG ASA in semen, and 9% had IgA ASA. There was a significant difference in sperm motility and morphology between men with and without ASA. Postcoital testing was performed in 190 of these infertile couples. Poor postcoital test results were associated with semen specimens that were MARpositive. This association remained constant after an attempt to control cervical mucus quality by administration of exogenous estrogen. After 12 months, significantly more couples with no ASA were pregnant than couples with ASA. Twenty-five percent of MAR IgG-negative couples were pregnant compared with 7.1% MAR positive couples. Furthermore, 25% of MAR IgA-negative couples were pregnant compared with no pregnancies for MAR IgA-positive couples. The strong association between IgA ASA and impaired fertility also is supported by Kremer and Jager (58) who concluded that the presence of antisperm IgA in either member of an infertile couple impairs sperm interaction with cervical mucus. Menge and Beitner (59) evaluated the relationship between agglutinating and immobilizing ASA by sperm immobilization assay tests and TAT and cervical mucus penetration assays in 849 semen analyses. In all cases, the presence of ASA had a deleterious effect on sperm motility, cervical mucus penetration, and sperm morphology. Clark (60) used IBD to test the relationship between specific immunoglobulin isotype and sperm cervical mucus penetration. He found that the presence of IgA ASA adversely affects sperm motility. Specimens were considered ASA-positive if .20% of motile sperm were bound to immunobeads. Specimens with IgA ASA by direct immunobead assay had less cervical mucus penetration than those without IgA ASA and also exhibited a greater degree of sperm shaking. These findings were not demonstrated when IgG ASA were present. Sperm shaking is observed commonly in specimens with poor forward motility. An important consideration in sperm motility is that the motility itself occurs in three dimensions and is composed of several factors, such as straight-line velocity, curvilinear velocity, and amplitude of sperm head Vol. 70, No. 5, November 1998

movement. Different ASA may bind to different areas on the sperm plasma membrane and, thus, affect individual components of motility. A confounding variable is that the plasma membrane and its component proteins are a fluid structure. Assessment of ASA on motility, therefore, is inconsistent and thus inaccurate (61).

a sperm immobilization assay test in 6.9% of the infertile women. Sperm incubated with ASA-positive sera had significantly less zona binding than sperm incubated with ASAnegative sera. In addition, work by Zouari and De Almeida (69) and Liu et al. (70) using IBD testing confirmed that ASA inhibit sperm binding to the zona pellucida.

Another potential mechanism by which ASA may disrupt fertility, is by disrupting sperm-oocyte recognition and fusion (62). Sperm-immobilizing antibodies may prevent sperm from undergoing capacitation (63). Addition of exogenous sperm-immobilizing antibodies prevented sperm from undergoing both spontaneous or induced acrosome reactions (64). Capacitation is a maturation event during which sperm are altered, allowing for the development of fertilizing capability. Potentially, antibodies may be directed against antigens present on the inner acrosomal membrane that are not exposed until after capacitation when the outer acrosomal membrane is lost. These antibodies may prevent sperm-egg recognition and fusion.

Mathur et al. (48) examined the effect of cytotoxic ASA and native complement in the sera, seminal fluid, and cervical mucus from 93 infertile and 40 fertile couples with the use of a double-fluorochromasia cytotoxicity assay (71). They found a significant reduction in in vitro sperm survival when sperm were incubated with sera from men or women with ASA. Sperm motility and survival also were decreased significantly when sperm were incubated with either seminal plasma or cervical mucus from men or women with ASA. However, although semen does not contain complement under normal physiologic conditions, it may leak into the ejaculatory system after an injury (49).

Antisperm antibodies may act as blocking agents that inhibit penetration of the zona pellucida by sperm (65). Bronson et al. (54) used the zona-free hamster egg penetration assay to demonstrate that ASA decrease in vitro sperm penetration. This effect was not confirmed by Francavilla et al. (66), who found that ASA did not affect either in vitro sperm penetration in the zona-free hamster egg penetration assay or capacitation. Comparing the methodologies between these two studies, Francavilla et al. exposed sperm to ASA and then washed them before capacitation and the zona-free hamster egg penetration assay. In contrast, Bronson et al. did not remove the ASA before hamster egg insemination. The different outcomes between these studies may result from ASA presence during hamster egg insemination in the work of Bronson et al. Wolfe et al. (67) also suggest that IgG and IgA ASA may impair sperm-oocyte membrane fusion. Twenty-nine couples whose previous IVF attempts failed, with $60% of sperm having one class of ASA underwent subzonal sperm insemination (SUZI). Two hundred sixty-three metaphase II oocytes were microinjected successfully, and 57 ultimately fertilized (21.6%). The percent of sperm coated with IgG was significantly higher in cycles with fertilization failure than in cycles in which fertilization occurred (90.2% compared with 75.6%). As the percent of IgG bound to sperm increased, the fertilization rate decreased. Most of the fertilization failures demonstrated ASA binding to the sperm head. The influence of ASA on sperm penetration of the zona pellucida was further evaluated by Tsukui et al. (68) with a sperm immobilization assay test on serum from 160 infertile women. Control sera were obtained from virgin donors. Follicular oocytes and associated cumulous cells were obtained from donor subjects undergoing surgery for another gynecologic process. Antisperm antibodies were detected by FERTILITY & STERILITYt

Complement-dependent, neutrophil-mediated sperm cytotoxicity may provide another mechanism by which ASA affect infertility. The sperm plasma membrane contains specific integrins involved in neutrophil-mediated sperm injury (72, 73). Neutrophils and complement are both present in the female reproductive tract. Under normal conditions, expression of complement inhibitors such as CD35 (C3b-C4b receptor), CD46 (membrane cofactor protein), CD55 (decayaccelerating factor), and CD59 (membrane attack complex inhibitor) on the outer sperm plasma membrane allow sperm to avoid damage. However, these complement inhibitors potentially could be blocked by ASA. D’Cruz and Haas (74) evaluated the expression of these complement-inhibitors in sperm under capacitating conditions. CD55 and CD59 were expressed on acrosome intact and acrosome-reacted sperm. However, CD46 was expressed only after acrosome loss. They suggest that because exogenous CD55 and CD59 can be reincorporated in vitro into cellular membranes, addition of these recombinant proteins to ASA-positive sperm might limit complement-mediated damage. Antisperm antibodies also may have a deleterious effect on postfertilization early embryo development and implantation. Naz (75) suggests that specific sperm cell membrane antigens provide a cleavage signal, ultimately causing the oocyte to divide. In an examination of early embryo cleavage among 64 patients undergoing IVF, he found one couple with ASA directed against a 22-kD protein, which was later found to inhibit cleavage in a murine IVF model. In the human case and in the murine model, gamete recognition and fusion occurred normally, but further cleavage was abnormal. In the original human couple studied, normal cleavage occurred when the woman’s oocytes were fertilized with donor sperm. Finally, ASA may play a significant role in pregnancy 805

maintenance. There is a tenuous association between the prevalence of ASA in women’s sera and recurrent abortion. Witkin and Chaudhry (76) compared ASA prevalence by IBD in 44 women with two or more consecutive unexplained spontaneous abortions to 616 women undergoing an infertility evaluation. They defined a positive test as 40% bead binding to motile sperm. Women with recurrent abortions had a significantly higher incidence of serum ASA (36.4%) than infertile women without ASA (14.6%). In an earlier study, Witkin and David (77) reported an association between spontaneous conception and pregnancy outcome over an 18-month period in 109 couples with unexplained infertility and the presence of ASA. Antisperm antibodies were detected in the woman’s sera by ELISA. Thirty-three women (30%) became pregnant, and 16 experienced a pregnancy loss. Two (11.8%) of the 17 women who became pregnant and later delivered were ASA-positive compared with 7 (43.8%) of the women who experienced a pregnancy loss. Seventy-six women (69.7%) did not become pregnant; 29 (38.2%) were ASA-positive. However, these associations between ASA incidence and recurrent pregnancy loss were not supported by Simpson et al. (78). They compared ASA prevalence by IBD in 111 women (55 diabetic and 56 nondiabetic) with recurrent pregnancy loss in the first trimester with 220 (104 diabetic and 116 nondiabetic) women delivering at term. For this study, IBD binding of either 20% or 50% was considered positive. Women experiencing a pregnancy loss were more likely to have IgA ASA than women not experiencing a loss. However, when all ASA data were pooled, the investigators believed that there were not enough data to support a significant role for ASA in recurrent pregnancy loss.

ANTISPERM ANTIBODY TREATMENT Several strategies are used in an effort to improve the potentially deleterious effects of ASA-mediated infertility. Three basic strategies include the following: [1] methods to decrease ASA production, [2] methods to remove ASA already bound to sperm, and [3] ART. Each of these strategies theoretically reduces gamete exposure to ASA, resulting in improved gamete function. Two methods used to reduce ASA production include condoms and or systemic corticosteroid treatment. Theoretically, repeated or multiple sperm exposure to the female reproductive tract results in ASA formation. Therefore, condom use would decrease sperm exposure, resulting in a concomitant decline in ASA production. In fact, in their original report, Franklin and Dukes suggested that condom therapy was an effective therapy for some patients (2). Mild inflammatory and immune system suppression with corticosteroids also may provide some couples with a limited benefit. However, the risks associated with chronic corticosteroid use may outweigh their benefits. Eleven infertile men 806

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with positive MAR tests were given 20 mg/day of prednisolone for the first 10 days of the partner’s cycle and then 5 mg/day for days 11 and 12 for 3 cycles. Antisperm antibodies were quantitated via flow cytometry. Less than 20% of patients showed a decrease in percentage of sperm bound ASA after treatment (79). In a prospective, double-blind, placebo-controlled study of 43 men with ASA-bound sperm, 24 received methylprednisolone for three cycles and 19 received placebo for three cycles. There was a statistically significant decrease in sperm-associated IgG in the steroid versus the placebo group. However, there was little effect on sperm-bound IgA. Furthermore, in this study there was no statistically significant difference in pregnancy outcome between the two groups (80). A crossover study by Lahteenmaki et al. (81) compared the effectiveness of oral prednisolone to intrauterine insemination (IUI) in 46 couples with male ASA. The men were assigned randomly to receive either 20 mg/day of prednisolone for days 1–10 of the cycle followed by 5 mg/day for days 11 and 12 and timed intercourse or to undergo IUI with no steroid treatment for three cycles. Crossover occurred if the couple was not pregnant. The pregnancy rate before crossover for the IUI group was 16.7%, whereas no pregnancies occurred in the steroid-treated group. After crossover, one more pregnancy occurred in the IUI group, and one also occurred in the steroid treated group. This study concluded that IUI is superior to low-dose steroid therapy for treatment of men with ASA. Another randomized, prospective, crossover study treated 30 men with ASA with either 4 months of oral steroids and timed intercourse or with 4 months of oral steroids and IUI and superovulation (82). Twenty milligrams of oral prednisolone was administered on days 1–10 of the cycle and 10 mg on days 11 and 12. There was a significant decrease in ASA levels by IBD as well as an increase in sperm motility during steroid treatment. The cumulative pregnancy rate was 39.4% for the IUI group compared with 4.8% for the timed intercourse group. Although this study suggests that IUI is superior to timed intercourse, it is difficult to determine whether steroids contributed to the high pregnancy rate in the IUI method or whether the high pregnancy rate in IUI method was due to some other factor, such as superovulation, bypassing the cervical mucus, or improving the uterine environment. Methods that attempt to remove ASA already bound to sperm include immunodepletion, sperm washing, and IgA protease treatment. Vigano et al. (83) attempted to purify sperm populations without ASA by coating magnetic microbeads with human antiimmunoglobulins and mixing them with sperm. The sample then is subjected to a magnetic cell sorter, and the ASA positive sperm are removed from the sample. They reported a significant decrease in the percentage of ASA bound sperm, although only 11 men were Vol. 70, No. 5, November 1998

studied. Verheyen et al. (84) expanded on this and evaluated the use of immunobead adsorption to select sperm without bound ASA for IVF. They found a significant decrease in percentage of ASA-bound sperm after treatment, but there was no improvement in either fertilization rates or embryo quality compared with an untreated group. Agarwal (85) retrospectively reviewed a cohort of 159 couples diagnosed with infertility who were treated with sperm washing and IUI over a 2-year period. Forty-five couples were diagnosed with ASA. Among these couples, there were 15 pregnancies compared with 37 pregnancies for the entire group. Antisperm antibodies were diagnosed by MAR and IBD. In an effort to decrease sperm ASA binding and to determine whether ASA binding occurs before or after ejaculation, Windt et al. (86) examined semen samples from 12 men with positive ASA by MAR and IBD. The first sample served as the control, and the second was diluted rapidly by collection into sterile medium. There were no differences in sperm-bound ASA between either of the two groups. They concluded that pregnancies resulting from washed sperm take place in the presence of ASA, and improved pregnancy rates may be associated with improvements in overall postpreparation sperm quality, elimination of cervical factors, and minimizing the distance between the gametes. Lenzi et al. (87) explored the effect of outer acrosomal membrane loss on ASA binding in 14 semen samples. They noted that ASA bound to the acrosome region were lost after capacitation and loss of the outer acrosomal membrane. The possibility that enzymatic cleavage of ASA may result in improved sperm function was suggested by Bronson et al. (88) who found that the Fc portion of the ASA immunoglobulin was responsible for inhibiting sperm penetration into cervical mucus. They theorized that IgA proteases, which cleave the Fc portion of IgA, could improve cervical mucus penetration. Furthermore, Kutteh et al. (89) added IgA proteases to a mixture of ASA-negative sperm and ASA-positive cervical mucus. The protease-exposed group exhibited an 80% decrease in ASA binding to sperm. There is a growing experience with the interrelationship between ASA and ART. Although ART may be used to treat ASA, ASA may have a detrimental effect on ART. Several studies have examined the use of IUI, intracervical insemination (ICI), IVF, gamete intrafallopian tube transfer (GIFT), subzonal sperm injection (SUZI), and, more recently, intracytoplasmic sperm injection (ICSI). Ford et al. (90) reported an inverse relationship between semiquantified ASA by IBD and IVF fertilization rates. As the concentration of ASA increased, IVF fertilization rates decreased. Kobayahsi et al. (91) correlated ASA titers detected by sperm immobilization assay tests with pregnancy rates. In their retrospective cohort, 96 women were treated by one or more attempts of IUI followed by IVF if unsucFERTILITY & STERILITYt

cessful. They noted a direct correlation between successful treatment and ASA titer. Women with greater ASA titers were less likely to become pregnant by either treatment method than women with lower ASA titers. Lahteenmaki (92) analyzed a retrospective cohort of 33 infertile couples with ASA treated with 47 IVF cycles. He reported that couples with high MAR ASA titers had a lower fertilization rate than those with lower ASA titers. Antisperm antibodies titers determined by TAT did not affect the fertilization rate. He noted that after controlling for the fertilization rate, ASA titers did not affect the pregnancy rate. Similar results were reported by Rajah et al. (93) in an analysis of 16 couples with ASA. Although the fertilization rates of men with ASA detected by IBD or MAR were significantly lower than men without ASA, the pregnancy rates did not differ after controlling for the fertilization rates. They suggest that ASA interfere with sperm-egg fusion but not early embryo development or pregnancy. Antisperm antibodies binding to the acrosome or sperm head may decrease fertilization and disrupt sperm motility more than ASA bound to the midpiece or tail. A retrospective analysis of 21 couples undergoing IVF for ASA revealed that the couples with successful fertilization had significantly more ASA binding to the tail than the head (94). Daitoh et al. (95) assessed IVF outcome in women with ASA in comparison with women with tubal factor infertility. It is surprising that the ASA group had an increased rate of implantation and pregnancy continuation compared with the control group even though the average age of the ASA group was significantly greater. The investigators concluded that ASA do not necessarily result in preimplantation embryo cytotoxicity. However, this study was limited because it did not include a comparison with a group of women with unexplained infertility or with a group of women not undergoing IVF. Acosta et al. (96) retrospectively examined the combined effects of ASA and sperm morphology on fertilization and pregnancy rates in 85 couples with male factor infertility, treated with 38 cycles of IVF and 57 cycles of GIFT. The ASA-negative group had greater fertilization and pregnancy rates than the ASA-positive group. Poor sperm morphology also was associated with a decrease in fertilization and pregnancy rates. The physical location and isotype of immunoglobulin involved are important factors that need to be assessed when attempting to examine the effect of treatment of ASA. Yeh et al. (97) retrospectively evaluated the impact of immunoglobulin isotype and location of binding by IBD in 48 ASApositive couples undergoing 80 IVF cycles. They noted that IgA significantly reduced fertilization rates only when it was associated with IgM and was present on the sperm head. Furthermore, IgM located either on the sperm head or end of the tail adversely affected fertilization rates. 807

Pagidas et al. (98) compared the IVF outcome of 31 couples with ASA with that of 312 couples with tubal infertility. There were no statistically significant differences between either of the two groups. They concluded that ASA in either female sera used to supplement IVF culture medium or ASA bound to sperm do not affect IVF. Intracytoplasmic sperm injection is a method that may allow some couples to avoid fertilization failure secondary to an autoimmune mechanism. Lahteenmaki et al. (99) treated 29 infertile ASA-positive couples with ICSI after 22 of them demonstrated a poor fertilization rate (6%) during IVF. After ICSI, the fertilization and cleavage rates for the ASA positive group (79% and 89%, respectively) were similar to the ASA-negative group (68% and 93%, respectively). It is notable that 46% of the pregnancies occurring in the ASApositive group ended in spontaneous pregnancy loss compared with none in the ASA-negative group. A retrospective analysis of 55 ICSI cycles for 32 different couples with high levels (.80%) ASA-bound sperm demonstrated no difference in pregnancy rate (30%) between the ASA-positive group undergoing ICSI and an ASA-negative group undergoing ICSI (100). There is evidence that IgG and IgA ASA in male sera or seminal fluid affect fertility by reducing sperm concentration, morphology, and motility. There is also evidence that IgG and IgA ASA bound directly to sperm significantly affects cervical mucus penetration. The available data also support the supposition that it is the ASA bound directly to the sperm head antigens that have the greatest impact on impaired sperm function. These ASA may interfere directly with gamete recognition and subsequent fusion. However, routine ASA screening in couples with unexplained infertility is limited because there is no well-accepted consensus about which assay should be used. Much of the data support the use of either IBT or MAR to evaluate whether IgG or IgA ASA are present. Mixed antiglobulin is technically easier to perform but is less sensitive than IBT. There is not enough evidence to support the use of systemic immunosuppressive methods to treat ASA. However, there are promising animal data that suggest that ASA formation may be prevented by prompt antibiotic treatment in the case of suspected genital tract infection in men. The use of ART in many couples with ASA and unexplained infertility is beneficial because it minimizes impaired gamete recognition and fusion. In summary, there is sufficient evidence to support the hypothesis that ASA play a role in selected couples with unexplained infertility. Both the prevalence and magnitude of this role remains controversial. Conflicting outcomes may result from variable specimen preparation, different testing methods, subjective test interpretation, and natural biologic variation. Of paramount importance, therefore, is a need to 808

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standardize methods used in ASA detection, interpretation, and reporting. Furthermore, a clear relationship between a positive test for ASA and clinical significance should be sought. Continued ASA research will help elucidate the events surrounding fertilization and embryo development as well as further contraceptive development.

Acknowledgment: The authors thank Edward E. Wallach, M.D., for his patience, perseverance, and persistent mentoring; the publication of this paper would not be possible without him.

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Vol. 70, No. 5, November 1998