Effect of isotypes of antisperm antibodies on semen quality

A direct immunobead test (IBT) was performed on 233 men who attended an immunological centre. Thirty-four (14.6%) of these men were found to be positive (greater than 20% binding) for antisperm antibodies (ASA). IgA, IgG and IgM were the most common sperm-associated immunoglobulins. In 50% of men with ASA asthenozoospermia, teratozoospermia, leukocytospermia or hypofunction of the seminal vesicles was observed. Semen parameters were altered most frequently when IgM was present in association with IgA and/or IgG. This suggests that there is an active inflammatory process in the reproductive tract, as evidenced by leukocytospermia, and this could be responsible for the abnormal semen parameters. ASA generation could be a consequence of this process rather than being the cause of the abnormal semen quality. If ASA do affect fertility, this could take place in the female reproductive tract.     

Effect of antispermatoeoal antibodies in seminal plasma upon spermatozoal function

The indirect immunobead test for antispermatozoal antibodies of the class IgA, IgG and IgM was applied to the seminal plasma of male partners of infertile couples. The presence of both IgA and IgG was associated with a decreased incidence of good post-coital test results and a reduced rate of fertilization of human oocytes. No significant differences were found for men with IgA or IgG alone when compared to men with no detectable antispermatozoal antibodies.     

Antisperm antibodies are more prevalent in men with low sperm motility

Fifty-six men with low sperm motility (less than or equal to 40% of sperm with forward progression) who attended our infertility clinic were tested for antisperm antibodies (IgG and IgA) using the indirect immunobead test. Nineteen (34%) of these men were found to be positive (greater than 10% binding). This was significantly higher (P less than 0.01) than the incidence (5%) of antibodies in men who attended our infertility clinic with high sperm motility (greater than 40% of sperm with forward progression). It is concluded that significantly more men with low sperm motility have antisperm antibodies. The functional significance of these antibodies warrants further investigation.     

Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.     

The incidence and influence upon fertility of antisperm antibodies in seminal fluid following vasectomy reversal

Seminal plasma samples from men undergoing vasovasostomy were analysed for antisperm antibodies using the indirect immunobead test. A pre-operative assessment showed antisperm antibodies of either IgA or IgG class to be present in 9/27 (33.3%) men. A significant increase (P less than 0.05) in the post-operative incidence of the antibodies was seen in the men who achieved patency (27/45, 60%) but not in those men for whom no sperm were seen in the ejaculate (4/10, 40%). After follow-up for a minimum of 1 year, conception rates for couples in which the male partner had achieved patency were similar in the groups with no antibodies detected post-operatively (12/18, 66.7%) or with IgA alone (2/3, 66.7%), but was reduced significantly in the presence of IgG (1/9, 11.1%; P less than 0.05) or IgA + IgG (3/15, 20.0%; P less than 0.01).     

不育相关AsAb的免疫印迹分析

用精子膜抗原Ｗｅｓｔｅｒｎｂｌｏｔ分析法，对７８例生育、１９８例不孕、２２例流产者及４４例中晚期妊娠孕妇血清抗精子抗体（ａｎｔｉｓｐｅｒｍａｎｔｉｂｏｄｉｅｓ，ＡｓＡｂ）进行研究。结果发现，抗分子量８５０００、５１０００、３００００精子抗原的ＡｓＡｂ与不孕相关；抗分子量８５０００、７９０００、６９０００，５１０００精子抗原的ＡｓＡｂ与女性不孕相关；不孕女性ＡｓＡｂ种类（在Ｗｅｓｔｅｒｎｂｌｏｔ上的反应条带数）明显多于生育女性。抗分子量８５０００、７９０００、５８０００精子抗原的ＡｓＡｂ与女性ＲＳＡ相关。本研究未发现不孕组男性、流产组男性及中晚期妊娠孕妇各种分子量精子抗原的ＡｓＡｂ检出率与生育组同性别相比有显著不同。所发现的这些ＡｓＡｂ可能与不育有密切关系，需要进一步研究和证实。     

The detection of antisperm antibodies in serum: a comparison of the tray agglutination test, indirect immunobead test and indirect Spermcheck assay

Testing for antisperm antibodies (ASAs) is an important part of the work-up of the sub-fertile couple, yet there is little consensus regarding the most appropriate methods. The Spermcheck assay (GSC; Bio-Rad Laboratories Inc., Diagnostics Division, Hercules, CA, U.S.A.) is supplied with wash buffer, controls and bead reagent which detects all three major classes of ASAs (IgA, IgG and IgM) in a single test. This study compared results on a bank of samples using the tray agglutination test (TAT), immunobead test (IBT), GSC and a modified Spermcheck assay to detect a single isotype in each test (SISC). The IBT and SISC showed excellent correlation, with 127/141 (90.1%) tests agreeing. There was an apparent lack of sensitivity to IgM with GSC as 8/15 (53.3%) samples testing positive with IBT and 7/15 (46.7%) testing positive with SISC were negative with GSC. Of the 24 IBT-negatives, seven (29.2%) were positive for TAT, indicating a high incidence of non-immunological agglutination, though this decreased as the TAT titre increased. The proportion of samples testing positive for IBT increased with TAT titre: 3/20 (15.0%) for TAT-negative samples, 6/10 (60.0%) for low titres and 21/24 (87.5%) for high titres. This was also observed when comparing the GSC with TAT. The TAT therefore appears useful as a first-line screen, whilst the inability of the GSC to adequately detect IgM limits its use as an indirect test. Both the IBT and SISC can be used to further investigate the type and class of ASA present.     

Sperm antigens recognized by antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure

Immunoblotting of a repertoire of sperm antigens reacting with antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure was performed. In the subgroups selected for this study, 55% of examined infertile women, 65% of infertile men and 64% of prepubertal boys with gonadal failure gave positive results by Western blotting with extracted sperm antigens. Sperm antigens with molecular weights of 57, 58, 62, 63 and 66 kDa were the most immunodominant entities recognized by antisperm antibodies from prepubertal boys. No positive reactions were detected by Western blotting in a control population of fertile adults, whereas in a group of prepubertal healthy boys only one sample revealed reactivity against sperm antigens of 58 and 70 kDa.     

Assessment of antisperm antibodies in a sample of Egyptian patients with hepatitis C virus infection

Association of hepatitis C virus (HCV) with autoimmune phenomena and impaired semen parameters has been previously reported. The aim of this study was to investigate the influence of HCV infection on the development of antisperm antibodies (ASAs) in HCV‐positive males. The study was conducted on 30 HCV‐infected individuals and 30 healthy control subjects. In both patients and control groups, liver enzymes and reproductive hormones were measured; computer‐assisted semen analysis (CASA) was performed; HCV‐RNA in serum was measured and IgG and IgA ASAs in semen were determined. Free testosterone, sperm concentration, progressive and total motility were significantly lower in HCV patients than in the control group, whereas ASAs of the IgG and IgA classes were significantly higher in HCV patients. However, correlations between viral load and the examined semen parameters and ASAs were nonsignificant. In conclusion, HCV may be responsible for the increased ASAs detected in HCV patients in the present study, possibly providing another plausible explanation for the decreased sperm motility reported in HCV patients. These findings could be of value in fertility management of HCV patients.     

Prevalence of antisperm antibodies by SpermMARtest in subjects undergoing a routine sperm analysis for infertility

To evaluate the prevalence of antisperm antibodies (ASA) attached to the sperm plasma membrane in male partners of infertile couples, the binding of latex particles to spermatozoa was investigated using SpermMARtest, included routinely in semen analysis. A total of 860 men were examined, who were referred consecutively for semen analysis. Of these, 750 men were referred because of infertility (0.6–10 years in duration) whereas 110 were volunteers with a history of previous fertility. Samples were assessed by the SpermMARtest kit using latex particles sensitized with human IgG. Sperm-latex binding was read after 3 min and samples scored as negative, positive or highly positive when < 10, > 10–40, or >40% binding occurred, respectively. Of the samples 132 (17.3%) were excluded because of azoo- or severe oligo-asthenozoospermia. IgG attached to spermatozoa were detected in nearly 13% of semen samples from the infertile population and in one of 110 fertile men (0.90/,). From the infertile group, 6.2% of samples showed > 40% binding, and 6.7% intermediate binding, with an overall ASA prevalence of 12.9% in subjects undergoing semen analysis for infertility.     

Analysis of human sperm membrane antigens reacting with sera from antisperm antibody positive and negative patients by Western blotting

Summary. Immunological infertility is thought to be caused by the binding of antibodies to 'fertility-related' antigen(s) on the sperm membrane. We compared antibody profiles in sera from 20 ASA (+) and ASA (-) men, using a sperm membrane extract as an antigen. Antigens were separated by SDS-PAGE under reducing conditions. The patients were classed as ASA (+) by the MAR (> 50%), d-IBT (> 20%) and TAT (> 1:64). The results showed that immunoreactive bands in both the ASA (+) and ASA (-) groups were heterogenous and included bands covering the whole molecular weight range. Statistical analysis showed significantly more patients in the ASA (+) group having immunoreactive bands at molecular weights of 32 Kd ( P > = 0.006) and 79 Kd ( P > = 0.02) when compared to the ASA (-) group. In the ASA (-) group significantly more patients had reactive bands at 81 Kd ( P > = 0.01) when compared to the ASA (+) group. The 32 Kd antigen reacted only with sera from ASA (+) patients. We conclude that differences exist between the ASA (+) and ASA (-) groups when this extraction method is used and that the isolation and purification of the 32 Kd protein may justify further investigation.     

A new test for immunological infertility: an ELISA based on prostasomes

Antisperm antibodies (ASA) are present in patients with immunological infertility, but the antigens are poorly characterized. Prostasomes adhere to sperm cells and are recognized as antigens for ASA. This investigation aimed to study the prevalence of antiprostasome antibodies in ASA-classified sera. We studied the reactivity of ASA-positive sera from 116 immunoinfertile patients. Ninety-seven per cent (113 of 116) of the patients' sera contained IgG antibodies against seminal prostasomes. Accordingly, prostasomes are one of the major targets for ASA. An enzyme-linked immunosorbent assay based on prostasomes is simpler to perform than ASA tests presently in use. It is also easier to achieve reproducible and standardized results.     

Antisperm antibodies detected by mixed agglutination reaction and immunobead test are not associated with chronic inflammation and infection of the seminal tract

The association between chronic inflammatory/infectious diseases of the male reproductive tract and the presence of antisperm antibodies (ASA) in semen is still controversial. We compared the results of the mixed agglutinin reaction (MAR) test and immunobead test for detecting ASA type IgG and IgA in 133 patients attending our special outpatient department for andrological infections and evaluated the differences in the detection rate of ASA. Patients were divided into three groups: a study group that included 79 patients with symptomatic nonacute inflammatory/infectious diseases of the seminal tract, a control group ( n  = 44) and a third group of men with a history of successful vasectomy reversal ( n  = 10). The two tests correlated in a statistically significant manner for the detection of IgG and IgA in all groups. The overall positive detection rate of clinical significant levels of IgG and IgA was 2.5% and 1.3% (respectively) in the patients with inflammation/infection of the seminal tract. No statistical significant difference in the detection rate of ASA levels between the inflammatory/infectious group and the controls was detected. The results of the MAR test and immunobead test have a statistical significant correlation and their results provide evidence that there is no association between inflammatory/infectious diseases of the male reproductive tract and the presence of ASA in semen.     

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.     

Male antisperm antibodies: association with a modified sperm stress test and lipid peroxidation

We previously reported a modified sperm stress test (MOST), low scores (< 0.39) in which were associated with sperm-related abnormal in vitro fertilization. Preliminary observations suggested that the presence of male sperm antibodies (ASA) could give low MOST scores. It was therefore decided to undertake a study to verify this possible association and also to ascertain if such a relationship was causal in nature. Six hundred and fifty semen samples from patients consulting for infertility were assessed for basic seminal characteristics, motion parameters (CASA), ASA and MOST. Thirty-nine samples (6%) were ASA-positive. Samples with and without ASA showed similar characteristics, except for percentage of normal forms and MOST scores (0.35 +/- 0.03 vs. 0.67 +/- 0.01, P < 0.001, for ASA-positive and -negative, respectively). There was a strong statistical association between presence of ASA and low MOST scores (P < 0.0001). One-hundred per cent of ASA-positive samples displayed low MOST scores. To verify the nature of this relationship, we incubated ASA-free spermatozoa with ASA-positive and -negative (control) sera. Despite an increase in the percentage of ASA-bearing spermatozoa in those aliquots incubated with ASA-positive serum, their original (pre-incubation) MOST scores remained unchanged. Furthermore, the rate of lipid peroxidation, indirectly reflected in MOST scores, was not different in the aliquots incubated with ASA. In conclusion, there seems to be a strong association between presence of ASA and low MOST values in semen samples of infertile patients; however, the relationship does not appear to be causal.     

Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies

The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies‐containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.     

Sperm antigens and reactivity of antisperm monoclonal antibodies in elisa

Summary. Several types of sperm antigenic suspensions as well as the whole sperm, either methanol-fixed or air-dried, were checked for intensity of binding to monoclonal antisperm antibodies with known characteristics of reactivity to sperm. The activity of sperm antigen—antibody binding was measured by elisa (enzyme-linked immunosorbent assay) and compared in several variations (parallely run) of the assay where different types of sperm antigen preparations were applied. The obtained results were then evaluated for statistical significance in Wilcox test. It was shown that antibody reactivity was markedly higher in experiments where the whole sperm was coated in a solid-phase in comparison to results obtained with adhered different sperm antigenic suspensions. However, one exception was noted, where the results from elisa, run with sperm organic extract, were (statistically) insignificantly lower than those obtained with the whole sperm. Therefore, organic sperm extracts (containing mostly glycolipids) can be a valuable alternative to screening for antisperm antibody activity and/or infertility background.     

Production and characterization of new monoclonal antibodies to human osteoclasts

Summary Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts.     

Serial follow-up study of serum testosterone and antisperm antibodies in patients with non-obstructive azoospermia after conventional or microdissection testicular sperm extraction

Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection is becoming a first-line treatment even for non-obstructive azoospermia. The current focus of TESE is the identification of seminiferous tubules that contain spermatozoa and minimization of testicular damage. Although microdissection TESE has been introduced as a preferred procedure for sperm retrieval, no serial follow-up studies of testicular damage have been reported. In the present study, we assayed serum testosterone concentrations and for the presence of antisperm antibodies (ASA) for 1 year after conventional multiple TESE or microdissection TESE and compared postoperative testicular damage between procedures. Thirteen patients who underwent conventional multiple TESE and 12 patients who underwent microdissection TESE were included in this study. Serum total and free testosterone concentrations were evaluated before operation and 1, 6 and 12 months after TESE. Serum ASA was also evaluated before and 12 months after TESE. Serum total and free testosterone concentrations in all patients in both groups showed no significant postoperative decrease. A comparison between the two groups of serum total and free testosterone concentrations showed no significant difference (total testosterone, p = 0.2477; free testosterone, p = 0.3098). No incidence of new ASA formation was identified in the present study. In conclusion, TESE procedures cause neither a decrease of serum testosterone nor formation of ASA. Serum testosterone concentration are similar between patients in the conventional multiple TESE and microdissection groups. Therefore, microdissection TESE is safe with respect to testicular damage, particularly for patients with hypogonadism.