Serum Antisperm Antibodies Research Articles

Antisperm antibodies in sera of Chinese infertile subjects and identification of cDNA encoding for a specific antigen

Antisperm antibodies in sera of Chinese infertile subjects and identification of cDNA encoding for a specific antigen

Detection of Testicular Endocrine Abnormalities and their Correlation with Serum Antisperm Antibodies in Men Following Vasectomy

We measured the serum gonadotropin response to gonadotropin-releasing hormone in 25 men who underwent vasectomy 2 to 64 months before the study. Ten age-matched fertile men were used as controls. Baseline serum follicle - stimulating hormone, luteinizing hormone and testosterone levels were not significantly different between vasectomized men and controls. However, mean serum follicle-stimulating and luteinizing hormone responses to an intravenous bolus injection of 100 meg. gonadotropin-releasing hormone were significantly greater in the vasectomy group (p equals 0.008 and 0.003, respectively). There was no correlation between these responses and the interval after vasectomy.Serum antisperm antibodies were present in 13 vasectomized men (52 per cent) using enzyme-linked immunosorbent assay and microagglutination techniques. A significant correlation (p equals 003) was found between the presence of serum antisperm antibodies and a normal follicle-stimulating hormone response to gonadotropin-releasing hormone stimulation. Of 13 patients with demonstrable antisperm antibody titers 9 (69 per cent) had normal follicle-stimulating hormone responses, compared to only 1 of 12 (8 per cent) without identifiable antisperm antibody titers.Our data suggest that certain men following vasectomy have abnormalities in seminiferous tubule and Leydig cell functions of the testes. These abnormalities are unrelated to the interval after vasectomy and are not identifiable with routine static hormonal measurements. In addition, serum antisperm antibodies are most likely to be present in men who demonstrate normal seminiferous tubular activity after vasectomy.

Antisperm antibodies in young oral contraceptive users.

Thirty-five young oral contraceptive users (group A) and twenty-four non-users (group B) of comparable age and background were screened for the presence of serum antisperm antibodies, antibodies to Chlamydia trachomatis and vaginal infections. Twenty-three per cent (8/35) of the oral contraceptive users were found to have significant titers of antisperm antibodies as compared to none in the non-user group (p less than 0.05). In 17% of group A patients (6/35) the presence of antisperm antibodies coincided with the presence of serum antibodies to chlamydia and vaginal candidiasis. One of these patients had E. coli in her vaginal culture in addition to the serum antichlamydia antibodies. The other two patients harboring serum antisperm antibodies were free of any sign of vaginal infection. A significant correlation (r = 0.7038, p less than 0.0000005) was found between the presence of antisperm antibodies and antichlamydia antibodies. There appears to be an increased frequency of serum antisperm antibodies in oral contraceptive users. This finding coincides with infections or colonizations with potential pathogens of the lower genital tract.

Pregnancy after vasovasostomy for vasectomy reversal: a study of factors affecting long-term return of fertility in 282 patients followed for 10 years.

The aim of this study was to determine the eventual fertility of those patients following vasectomy reversal who have no pressure-induced secondary epididymal blockage. These patients underwent simple vasovasostomy because at the time of the reversal surgery there were sperm present in large numbers in the vas fluid. It was possible to obtain long-term follow-up on 326 early patients who underwent vasectomy reversal 8-10 years ago. Two hundred and eighty-two of those patients had sperm in the vas fluid. These patients were studied for pregnancy rate and post-operative semen parameters in relation to presence or absence of sperm in the vas fluid at the time of vasectomy reversal, duration of time since vasectomy, pre-operative serum antisperm antibody titers, the influence of varicocoele and quantitative evaluation of testicular biopsy. All of the 44 patients with no sperm in the vas fluid remained azoospermic following vasovasostomy. Of the 282 patients with sperm in the vas fluid, 228 (81%) eventually impregnated their wives. Twenty-four patients with sperm in the vas fluid (9%) were azoospermic and did not impregnate their wives. Of the 258 patients who had sperm patency, the pregnancy rate was 88%. The number of mature spermatids per tubule in the testis correlated closely with the post-operative sperm count in patent cases. Quantitative evaluation of the testicular biopsy revealed normal spermatogenesis, even in patients with azoospermia or severe oligospermia post-operatively. Technical failures were due to blockage either at the vasovasostomy site, or epididymal blockage unrecognized at the time of vasovasostomy.2+perm count had a minimal impact on the

A prospective study on the lack of development of antisperm antibodies in women undergoing intrauterine insemination

To test the hypothesis that intrauterine insemination with washed spermatozoa induces antisperm antibody formation, we measured serum antisperm antibody levels by the Immunobead technique in a population of women receiving exogenous gonadotropins. Antibody levels were measured before therapy (baseline) and then serially during subsequent stimulation cycles, for a maximum of six cycles. Twenty-eight patients underwent intrauterine insemination; each patient served as her own control. An additional 25 patients were treated with exogenous gonadotropins but did not undergo intrauterine insemination; they served as external controls. Antisperm antibody levels in women who underwent concomitant intrauterine insemination were compared with levels in those who did not. Of the 53 enrolled patients, 18 completed six treatment cycles, and 35 achieved pregnancy before six cycles. Forty-five patients (85%) had less than 10% Immunobead binding, six (11%) had binding between 10% and 25% (mean 16%, range 14% to 20%), and two had binding greater than 25% (28% and 42%, respectively). Mean binding was similar (less than 10%) in the intrauterine insemination and external control groups. Eighteen patients conceived in the intrauterine insemination group and seventeen in the control group. Of patients who conceived, all but one had less than 10% Immunobead binding at the time of conception (mean 1.6 months). In patients who did not conceive, there was no difference in Immunobead binding between control and intrauterine insemination groups after 6 months of therapy. Our data do not support the hypothesis that serum antisperm antibody levels, as detected by Immunobead binding, will increase in menotropin-stimulated women undergoing intrauterine insemination over a prolonged treatment period.

Detection and titration of class-specific antisperm antibodies in serum using an enzyme-linked immunosorbent assay

An antisperm antibody enzyme-linked immunosorbent assay (ELISA) that uses whole unfixed sperm and detects immunoglobulin G (IgG) and IgA antibodies in serum was developed. Donor sperm were washed and plated on poly-L-lysine-treated microtiter plates. The patient's sera were diluted to concentrations of 1:4 to 1:256 and incubated with sperm. Positive and negative sera had been previously tested for IgG antisperm antibody activity with a radiolabeled antiglobulin assay. Samples were considered positive when the mean absorbance of triplicate wells was greater than 2 SD above the pooled negative mean. Intra-assay variation was 7.9 and 9.6% for pooled negative and positive controls, respectively. Identical titers of control positive serum were consistently detected. A correlation of 0.83 was observed between ELISA IgG serum titers and radiolabeled antiglobulin results (N = 12). All negative samples tested negative in both assays (N = 21). Some serum samples showed IgA antisperm antibodies. Determination and titration of class-specific antibodies in serum should facilitate initial screening and follow-up of patients at risk for antisperm antibodies.

The Influence of Vasovasostomy on Antisperm Antibodies in Rats1

Serum antisperm antibodies were studied in Sprague-Dawley rats after vasectomy and vasovasostomy. Animals received a bilateral vasectomy, a vasectomy followed 3 mo later by vasovasostomy, or sham operations. Blood samples were obtained at 1, 3, 4, and 7 mo, and antisperm antibodies were assayed by an enzyme-linked immunosorbent assay. After vasectomy reversal was performed at 3 mo, antisperm antibodies were significantly higher in rats in the vasovasostomy group at 4 mo than in animals that had a persisting vasectomy or sham operations. At 7 mo, the antisperm antibody level for the vasovasostomy group was approximately double that for the vasectomized rats. Spermatic granulomas occurred in 76% of rats after vasovasostomy. Antisperm antibody levels were higher in vasovasostomized animals with granulomas than in those lacking granulomas. The results suggest that vasovasostomy may stimulate an antibody response to sperm rather than lead to a reduced response, as was anticipated upon removal of the obstruction. Spermatic granulomas may serve as sires for continued antigenic challenge. The observed increase in antisperm antibodies after vasovasostomy in Sprague-Dawley rats may be related to their relatively low immunologic responsiveness to vasectomy, with vasovasostomy serving as a second major immunologic challenge, aided by the formation of an additional granuloma. In the more responsive Lewis strain, we previously observed a rise in antisperm antibodies after the initial vasectomy, with no further increase after vasovasostomy.

Clinical evaluation of the enzyme-linked immunosorbent assay (ELISA) kit for antisperm antibodies

A commercially available enzyme-linked immunosorbent assay (ELISA) kit for the detection of antisperm antibodies in serum was compared with standard sperm immobilization test (SIT) with the use of sera from 83 infertile women and 29 control individuals. For the ELISA, 24% of the infertile patients and 10% of the controls showed positive results, whereas 15% of the patients and none of the control were positive in the SIT. Parallel tests carried out on the same sera indicated that these methods detect a different, though often overlapping, spectrum of antibody activity. The presence and number of motile sperm in cervical mucus during postcoital tests were found to be related to the results of the SIT. On the other hand, the ELISA did not appear to be related to the quality of postcoital tests. These data indicate that care must be employed to interpret the results of this ELISA kit for the detection of antisperm antibodies.

Testicular Alterations are Linked to the Presence of Elevated Antisperm Antibodies in Sprague-Dawley Rats after Vasectomy and Vasovasostomy

The relationship between alterations in testicular histology and antisperm antibodies was studied after vasectomy and vasovasostomy in Sprague-Dawley rats, which are immunologically relatively non-responsive to vasectomy. Testes were prepared for histologic study at intervals up to seven months after vasectomy, vasectomy followed three months later by vasovasostomy, or sham operations. Antisperm antibodies were assessed with an ELISA. Testicular alterations, which were observed in a minority of animals after vasovasostomy, consisted mainly of depletion of germ cells. Mean serum antisperm antibody levels were greater for animals with altered testes than for rats with normal testicular histology. In addition, the proportion of rats that showed a positive antisperm antibody response was greater among animals with testicular changes than among those with unaltered testes. When the present results on Sprague-Dawley rats were compared with previous findings on the highly responsive Lewis strain, it was evident that the incidence of testicular changes and the proportion of positive antibody responders were greater in the Lewis strain. However, elevated antisperm antibodies and testicular alterations appeared to be more tightly linked in the less responsive Sprague-Dawley rats. (J. Urol., 140:627–631, 1988)

Results of in vitro fertilization in women with antisperm antibodies in serum, cervical mucus, and follicular fluid.

This study was undertaken to investigate the presence of antisperm antibodies (ASA) in serum, cervical mucus, and follicular fluid (FF) of women undergoing in vitro fertilization and embryo transfer (IVF-ET). IgG and IgA ASA directed mostly against sperm head were found at similar concentrations in serum, cervical mucus, and FF of 2 of 34 patients. Ninety-one percent fertilization and 100% cleavage rates, respectively, were observed in one of the two patients. No fertilization occurred in the second patient. In both women, in vitro sperm penetration tests revealed hostile mucus and repeated postcoital tests were poor. It is concluded that the sperm-cervical mucus penetration test and mucus ASA measurements are useful in establishing the diagnosis of immunological infertility.

The indirect mixed antiglobulin reaction test using a commercially available kit for the detection of antisperm antibodies in serum

A simple procedure is described for the detection of antisperm antibodies of the IgG class in human serum using the indirect mixed antiglobulin reaction (MAR) test. The test uses only one dilution of serum (1/16) and no washing procedures. The test was performed in 361 sera. Comparative studies were conducted with the tray agglutination test (TAT) in 164 sera and with the direct MAR test on semen in 110 cases. In 24 sera with borderline test results, and in 13 sera with discordant findings, evidence of antisperm antibody activity was investigated by means of additional testing, namely, the adenosine triphosphate release cytotoxicity test (ARCT) and the indirect immunobead tests (IB) for IgA and IgM. Using the value of 40% reaction between motile spermatozoa and coated latex particles as the lower limit of significant activity, the indirect SpermMAR test has a sensitivity of 96% and specificity of 87%. The two false-negative indirect MAR results occurred in cases with IgM, the clinical significance of which is doubtful. Hence, the indirect MAR test appears to be an ideal screening procedure for sperm antibodies in serum since it is easy to perform and sensitive. Considering the false-positive rate of 13%, additional tests should, however, be performed on sera with a reaction of 40% or more in order to assess the relevance of the antibodies as a cause of infertility.

Mechanisms of Active Suppression of the Immune Response to Spermatozoa

The production of autoantibodies to spermatozoa in males and isoantibodies in females is inhibited both by the physical isolation of spermatozoa from the systemic immune system and by active immunosuppression mechanisms. Lymphoid cells present in the epithelial lining of the rete testis, epididymis, and vas deferens, as well as the human ejaculate, are predominantly T suppressor/cytotoxic cells. Mononuclear cells derived from semen inhibit the in vitro activation of peripheral blood lymphocytes. Soluble specific T suppressor/cytotoxic cell activators in semen or on the sperm surface may be responsible for the predominance of this T cell subset in the male reproductive tract. The activation of T suppressor/cytotoxic cells following coitus may also limit the immune response to spermatozoa in females. Spermatozoa can also initiate immunosuppression, either by selectively inducing T suppressor cells or through the generation of activated complement components that block antibody production. Antisperm antibodies in sera from females may be associated with either a deficiency in the ability of their T suppressor/cytotoxic cells to be induced by factors in semen or by the occurrence in their husbands' ejaculates of microorganisms, antibodies, or other factors that induce T helper lymphocytes. Activated T cells produce interferon gamma, which induces Ia antigen expression on macrophages and allows the female's T helper cells to recognize processed sperm antigens. Recognition of the role of cell-mediated immune functions in the male and female genital tract identifies possible new target sites for the development of contraceptive agents.

Effect of sperm antibodies on pregnancy outcome in a subfertile population

The relationship between sperm antibodies, conception, and miscarriage was examined in 109 infertile couples. Antibodies present on the surface of husbands' ejaculated sperm and antibodies in husbands' or wives' sera that reacted with a purified population of the husbands' motile spermatozoa were detected by an enzyme-linked immunosorbent assay. During an 18-month period, conception occurred in 33 (30.3%) of the couples; 16 (14.7%) women subsequently suffered a spontaneous miscarriage during the first trimester, whereas 17 (15.6%) women maintained their pregnancies past this time period. Antisperm antibodies were present in sera from only two of 17 (11.8%) women with successful pregnancies, whereas seven of 16 (43.8%) women who miscarried and 29 of 76 (38.2%) who did not conceive had these antibodies in their sera. IgG (22.4%) and IgM (21.1%) antisperm antibodies predominated in sera of women who did not conceive, whereas IgA (37.5%) and IgG (37.5%) antibodies were most prevalent in sera of women with miscarriages. In men, the presence of antisperm antibodies in sera was unrelated to fertility. However, there was a correlation between sperm surface antibodies and an inability to conceive. IgG was identified on ejaculated spermatozoa from eight of 76 (10.5%) men whose wives failed to conceive and in none of 33 men whose wives conceived. Similarly, IgA was present on spermatozoa from 16 (21.1%) infertile and two (6.1%) fertile men. Thus antisperm antibodies in female sera and on ejaculated spermatozoa were associated with a failure to conceive and first-trimester miscarriage.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

Strain variations in anti-sperm antibody responses and anti-fertility effects in inbred mice.

This study provides evidence for polygenic controls of antisperm antibody levels in inbred male mice immunized with syngenic testis and epididymis. H2-linked and non-H2-linked genes were involved. Mice of H-2d haplotype were high responders, whereas those with H-2k haplotype were nonresponders; however, B10.D2/nSnJ mice (H-2d) were also nonresponders. In vitro fertilization inhibition by antisera correlated positively with the serum antisperm antibody levels, particularly with antibody of the immunoglobulin (Ig) G class. Inheritance of antibody response that inhibited in vitro fertilization (IVF) was an autosomal dominant trait, but this was not apparent for the control of antibody levels per se. Since IVF was inhibited by both IgG and fragment antigen-binding (Fab) isolated from immune sera, but not by immune IgG previously absorbed by sperm or testis, the biologic effect is antigen-specific and probably involved blockade of functional antigenic epitopes. Antisera to testis, caput sperm or cauda sperm were found to inhibit IVF to a similar degree. Inbred strains of mice that produced the highest levels of serum antisperm antibodies that inhibited IVF were A/J, SJL/J, DBA/1J and BALB/cByJ mice, and their antisera immunoprecipitated a common sperm antigen molecule of 35,000 to 40,000 Mr. In contrast, C57BL/6 and C57BL/10 mice produced significant antibody levels that had no effect on IVF, and their sera did not react with the 35,000- to 40,000-Mr peak. Moreover, among BALB/c H-2 congenic mice, only antiserum of responder BALB/cByJ (H-2d) mice immunoprecipitated the 35,000- to 40,000 Mr peak. Thus the 35,000- to 40,000-Mr protein may be of functional significance in the fertilization process.

Incidence of sperm-associated immunoglobulins in infertile men with suspected autoimmunity to sperm.

Semen samples from 120 infertile men with suspected autoimmunity to sperm were investigated by a direct immunobead test (IBT). Fifty-three (44%) of them had 10% or more motile sperm coated with anti-IgG and/or anti-IgA immunobeads. Both classes of immunoglobulins were found to be present in 88.7% of the antibody positive ejaculates. These sperm-bound Igs were associated with sperm autoagglutination in 80% of the ejaculates and with decreased sperm penetration into cervical mucus in 97.6% of the cases. The close correlation found between the IBT results and the occurrence of antisperm antibodies in serum and in seminal plasma suggests that sperm-bound Ig's are sperm-specific antibodies. It is concluded that the direct IBT is not only a reliable screening test for sperm antibodies but is also a relevant test to determine whether these antibodies exert an influence on male fertility.

Detection of autoimmunity to sperm: mixed antiglobulin reaction (MAR) test or sperm agglutination? A study on 537 men from infertile couples

Two different ways of testing for antisperm antibodies were compared: the mixed antiglobulin reaction (MAR) test for demonstration of antibodies of the IgG and IgA classes bound in vivo to the sperm membrane antigens and the gelatin agglutination test for detection of nonbound antisperm antibodies in serum and seminal plasma. Samples from 537 men from infertile couples were investigated. Antibodies bound to the sperm membrane were detected in 49 men (9.1%), IgG in 44 (8.2%), and IgA in 38 cases (7.1%). Sperm agglutinins were recorded in seminal plasma from 30 men (5.6%) and in serum (titer greater than or equal to 16) from 43 men (8.0%). The investigation revealed a very close correlation between the results of MAR testing and the occurrence of sperm agglutinins in serum and seminal plasma. However, if one focuses on antisperm antibodies of the IgA class, which seem to play the major role in male immune infertility, the MAR test offered the advantage that a minor group of patients with pure IgG responses could be distinguished, and rare cases with mainly or exclusively locally produced IgA antibodies could be detected.

Effect of cyclosporin A in male autoimmune infertility

Nine infertile men (mean duration of infertility, 8.3 years) with antisperm antibodies were treated with the immunosuppressive drug cyclosporin A for 6 months with a dose of 5 to 10 mg/kg/day. Seminal plasma and serum antisperm antibodies fell in three subjects on treatment, and sperm count and motility increased substantially in one. Three successful pregnancies occurred in the study group: one on treatment, one in the first cycle of artificial insemination with the husband's sperm after treatment (twin infants), and one 3 months after cessation of treatment (twin infants). Successful conceptions with cyclosporin were unrelated to falls in antisperm antibody titer.

Antisperm antibodies in infertile and homosexual men: relationship to serologic and clinical findings

The incidence and significance of antisperm antibodies in different groups of men were evaluated by a modified enzyme-linked immunosorbent assay. In serum, 4.0% of dermatologic patients (n = 223), 9.6% of andrologic patients (n = 178), and 28.6% of homosexual men (n = 42) were positive for IgG and/or IgM antibodies. In seminal fluids, 7.3% of the andrologic patients had IgA (and IgG) antibodies to spermatozoa. Only 1 of 29 positive men had antibodies both in serum and in seminal fluid. No correlation between antisperm antibodies and IgG/IgM concentrations was found in serum, whereas in seminal plasma men with antisperm antibodies showed higher IgG/IgA concentrations than men without (IgA, 3.2 versus 1.7 mg/dl; IgG, 9.8 versus 6.3 mg/dl). It is concluded that there is a high incidence of antisperm antibodies among homosexual men, probably because of contact of spermatozoa with the immune system by passive anal intercourse. There is little correlation between antisperm antibodies in serum and seminal plasma of infertile men because of a lack of relevant antibody transfer from the serum and the formation of local antibodies in seminal plasma. Antisperm antibodies in seminal fluid are associated with elevated local IgG and IgA concentrations.

Human antisperm monoclonal antibodies constructed postvasectomy.

Sperm and spermatogenic cell antigens, escaping the blood-testis/blood-epididymal barrier, elicit an autoimmune response in patients following vasectomy. In this study, antisperm antibody-positive sera and peripheral blood lymphocytes were obtained 6-9 mo following vasectomy. Serum antisperm antibody levels were assessed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence. Lymphocyte-myeloma hybridomas were constructed by fusing peripheral blood lymphocytes, harvested from antisperm antibody-positive sera, with a hypoxanthine guanine-phosphoribosyltransferase (HGPRT)-negative mouse myeloma line. Immunoglobulin-secreting colonies surviving drug selection were detected by ELISA and screened for antisperm activity. Antisperm antibody-producing cultures were cloned and expanded for bulk antibody production both in culture and as ascites in athymic nude mice. Eight mouse-human fusions yielded 205 hybridomas secreting human monoclonal antibody, of which 11 demonstrated antisperm reactivity by ELISA. Two of these hybridomas are described in detail: HAS-1, which secretes human immunoglobulin M (IgM, kappa)-recognizing epitopes located on the sperm midpiece, and HAS-2 (IgM, lambda), which secretes monoclonal antibody-recognizing epitopes located on the entire sperm tail. The results indicate successful capture of human antisperm autoantibody from the postvasectomy autoimmune state using somatic cell hybridization techniques.