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.     

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.     

Seminal sperm antibodies exhibit an unstable spontaneous course and an increased incidence of leucocytospermia

The prognostic significance of seminal sperm antibodies for male fertility is difficult to define. Among other factors, spontaneous remissions and transient induction through genito-urinary infections may change their significance for male fertility considerably. We therefore investigated their spontaneous course over time and their association with leucocytospermia. For the investigation of possible relationships between the mixed antiglobulin reaction (MAR) test results for IgA and IgG sperm antibodies and leucocytospermia, 138 patients with <1 million leucocytes/ml ejaculate were compared with 1051 patients with <1 million leucocytes/ml ejaculate (WHO normal range). In a second part of the study the spontaneous course of MAR IgG and IgA test results was investigated in 58 of the 1189 patients who had three or more MAR tests and a leucocyte concentration of <1 million/ml ejaculate. The mean interval between the first and second MAR test was 4.1 (SD ±4.2) and between the second and third MAR test 6.0 (SD ± 6.7) months. In total, 64% of patients with an MAR IgG test result >40% showed a stable spontaneous course. Patients with lower MAR IgG test results and the majority of all MAR IgA-positive patients were found to have an undulating course of MAR test results. There was a higher incidence of leucocytes <1 million/ml ejaculate in patients with higher MAR results ( p <0.05 for IgG, p <0.001 for IgA). The unstable spontaneous course of MAR IgG results <40% and all MAR IgA results suggests that evaluations of the in-vivo significance of seminal sperm antibodies for male fertility should be based on repeated MAR determinations. Moreover, the association of leucocytospermia (<1 million leucocytes/ml ejaculate) with an increased incidence of positive MAR test results suggests a thorough evaluation, treatment and follow-up of possible genital tract infections in MAR-positive patients with <1 million leucocytes/ml ejaculate.     

Identification of the sperm antigen interacting with antibodies in serum from an infertile woman

Serum (IS) obtained from an infertile woman induced head-to-head agglutination of human sperm. The immunoglobulin G (IgG) fraction of the IS was prepared by ammonium sulfate fractionation and DEAE cellulose chromatography. The IgG localized to the post-acrosomal region of the sperm, determined by indirect immunofluorescence and interacted with a human sperm protein with an estimated Mr of 80 kDa, determined by immunoblotting. The identity of the interacting sperm antigen was verified by isolating the 80 kDa sperm protein by affinity chromatography. The present results suggest that the anti-80 kDa antibodies may be responsible for the infertility.     

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.     

Evaluation of the direct and indirect mixed antiglobulin reaction with latex particles for the diagnosis of immunological infertility

The direct Mixed Antiglobulin Reaction (MAR test) is a simple method for the detection of auto-antibodies attached to spermatozoa in fresh semen. The test was improved by using IgG-coated latex particles (SpermMAR) instead of coated red blood cells. A positive direct MAR test with adherence of latex particles to 40% or more of the motile spermatozoa was found in 16 out of 312 men (5%) consulting for infertility, but never in fertile controls. The percentage of motile spermatozoa reacting with the coated latex particles was correlated significantly with the serum titre of sperm agglutinins assessed by the tray agglutination test. The direct MAR test on semen was highly specific but rather insensitive, particularly if the serum titre of agglutinins was low. The same kit (SpermMAR) can be used to detect circulating sperm antibodies in serum of male or female patients with the indirect mixed antiglobulin reaction. The indirect MAR test discriminated clearly between serum with low (less than 1/32) or high (greater than 1/32) titres of circulating agglutinins assessed by the tray agglutination test. Considering their simplicity and accuracy, both the direct test on semen and the indirect spermMAR test on serum should be included in the routine evaluation of the infertile couple.     

Prostasome antigens as targets for sperm agglutinating antibodies demonstrated by 1-D gel electrophoresis and immunoblottings

Many immunoinfertile men have sperm agglutinating antibodies that are directed against prostasome-derived antigens, but these antigens have not been defined so far. We selected serum samples with high ELISA titres against prostasomes from a group of immunoinfertile patients with sperm agglutinating antibodies and used the sera for immunoblottings on 1-D SDS-PAGE of prostasomes and sperm cells. The immunoblottings with individual antiprostasome antisera on 1-D SDS-PAGE of prostasomes, revealed three to 10 bands for each serum. Eighty-five per cent of the serum samples contained bands in the 70-75 kDa region and 80% of the samples contained bands in the 50-55 kDa region. Immunoblottings of extracted sperm cells, revealed one to six bands in the molecular weight range 25-82 kDa and two of the samples recognized two bands with molecular weights (50 and 43 kDa) similar to immunoblottings of prostasomes. The prostasomal antigens recognized by the high titre-antisera of immunoinfertile men were generally different from the sperm antigens recognized by the same sera. This suggests that prostasomes offer a new set of antigens available for research on male immunoinfertility and immunocontraception.     

精子膜表面抗精子抗体与精液参数的关系

目的 探讨精子膜表面AsAb导致男性不育的机制。方法 对102例不孕夫妇的丈夫精液进行混合球蛋白反应试验(MAR),其中48例同时进行了血清AsAb的检测。结果 48例同时进行MAR和ELISA的不育男性,精液MAR阳性率为14.6%(7/48);血清ELISA阳性率为50%(24/48)。两种方法的阳性率有非常显著性差异(P<0.001)。102例进行MAR检测的不育男性中,随着精液量、密度、存活率、活动力、畸形率的变化,精子膜表面AsAb的阳性率均无显著性差异(P>0.05)。根据精子膜表面AsAb阳性与否分成两组组间的病人年龄、精液量、密度、存活率、活动力、畸形率均无显著性差异(P>0.05)。结论 血清ELISA阳性率过高,不宜用于不育男性免疫因素的检测;精液精子膜表面AsAb并不影响精液分析的各项参数。因此,精液各项参数的分析对于男性免疫因素不育的筛查没有太大的意义。     

Evaluation of human sperm membrane integrity using the water test and the hypoosmotic test

The objective of the study was to compare the water test and the hypoosmotic test (HOS) in the assessment of the human sperm membrane. A total of 686 semen samples from human male donors were subjected to water and HOS tests after routine semen evaluation. The mean percentage of swollen spermatozoa was 71.8 +/- 9.6% in the HOS test and 67.8 +/- 9.4% for the water test; these values were not statistically different. The correlation of coefficients between the water test and the HOS test was highly significant whether the values for the HOS test were higher or lower than 60% (P < 0.001). A poor correlation was obtained when the two tests were compared for sperm counts either higher or lower than 20 x 6 ml-1 and when the results for both tests were compared with the percentage of eosin-Y staining spermatozoa. A poor correlation was also obtained when the results of each test were compared with eosin-Y staining spermatozoa in normal and abnormal semen samples. The coefficient of regression between the two tests showed a high correlation (P < 0.001). In conclusion, even though a high correlation between the HOS test and water test was observed in this study, it is not possible to recommend assessment of sperm membrane integrity using the water test and the consequent replacement of the HOS test in routine practice. Further studies are necessary to establish the best test for sperm vitality.     

Effect of zinc on human sperm motility and the acrosome reaction

This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 microM) were added for periods of 2, 4 or 6 h. Other samples were incubated with zinc (1000 microM), and after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was greater than or equal to 100 microM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed.     

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.     

Characterisation of a subpopulation of sperm with massive nuclear damage,as recognised with the sperm chromatin dispersion test

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7‐dibrom‐4‐hydroxy‐mercury‐fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two‐dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double‐ and single‐strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele.     

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.     

Assessment of released acrosin activity as a measurement of the sperm acrosome reaction

Aim： To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction （AR）. Methods： Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization （WHO） criteria. Calcium ionophore A23187 and progesterone （P4） were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin （FITC-PSA）. The percentage of sperm undergoing AR （AR%） was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. Results： The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 （P 〈 0.001）. There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining （r= 0.916, P 〈 0.001）. Conclusion： Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR. （Asian J Androl 2008 Mar, 10： 236-242）     

Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction

Summary.  Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo , p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.     

IgA抗精子抗体对精子顶体反应的影响

目的 探讨精浆中IgA抗精子抗体对人精子顶体反应的影响。方法 利用免疫珠法（IBT）筛选出IgA抗精子抗体阳性精浆标本同正常人精子孵育，以孕酮诱发精子顶体反应；以特异性荧光标记物．络合异硫氰酸荧光素的花生凝集素（FITC-PNA）标记精子顶体，通过流式细胞仪检测精子顶体完整性。结果 与IgA抗精子抗体阳性精浆孵育的精子，其孕酮诱发的顶体反应发生率明显低于正常精浆及精子培养液组（P〈0．01），正常精浆组及精子培养液组间无显著性差异（P〉0．05）；IgA抗精子抗体阳性精浆组、正常精浆组、精子培养液组自发顶体反应的发生率无显著性差异（P〉0．05）。结论 免疫性不育患者精浆中的IgA抗精子抗体可以明显抑制孕酮诱发的顶体反应的发生，可能是导致不育的原因之一。     

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.     

Induction of the acrosome reaction in sperm by exposure to low temperature increases their rate of fusion with zona-free hamster oocytes

The fusion rate of human sperm with zona-free hamster ova was investigated after induction of the acrosome reaction by exposure to a low temperature (4 degrees C). Sperm were collected from 14 patients, and selected by the 'swim-up' method. The sperm were incubated for 24 h at either room temperature (control group) or at 4 degrees C (low temperature group), followed by additional incubation at 37 degrees C for 3 h. The mean sperm penetration rate, number of swollen sperm heads as well as the number of sperm attached to the oocyte increased significantly after exposing sperm to low temperature. The sperm penetration rate showed a significant correlation (Spearman test, r = 0.572, n = 28, P less than 0.0035) with the acrosome reaction in the low temperature group. These results were associated with an increase in the rate of penetration of hamster ova observed in this study, presumably due to the increase in induction of the acrosome reaction by low temperature. Incubation of sperm at low temperature might be useful in the evaluation of so-called false negative results in the zona-free hamster test.     

Immunoglobulin Class of Antispermatozoal Antibodies from Infertile Men and Inhibition of in vitro Sperm Penetration into Cervical Mucus

The presence of IgG and IgA on motile spermatozoa from normal semen donors and men from infertile couples was studied with mixed antiglobulin reaction (MAR) tests.
The percentage of motile spermatozoa with IgG (IgG MAR%) was found to be related to the circulating antispermatozoal IgG. No direct relation could be detected between the IgG MAR% and the sperm agglutinating activity in seminal plasma (SP) or the percentage of motile spermatozoa showing the shaking phenomenon (S%) in the sperm cervical mucus contact (SCMC) test. The percentage of motile spermatozoa with IgA (IgA MAR%) showed no direct relation to the sperm agglutinating activity in serum and SP, but was roughly proportional with the S%.
It was discerned that the shaking phenomenon in the SCMC test was probably due to presence of IgA on the motile spermatozoa. Previously it had been demonstrated that also the sperm agglutinating activity in SP is caused by IgA that is probably locally produced in the male genital tract. In conclusion it was thought that the reduced ability of penetration into cervical mucus by spermatozoa from infertile men is caused by locally produced IgA.     

酸性磷酸酶检测评价人精子获能和顶体反应

目的 探讨酸性磷酸酶(ACP)检测评价人精子获能和顶体反应(AR)的可行性。方法 32名健康生育男性的精子悬液，于37℃下孵育lh获能，用孕酮在有无苯甲基磺酰氟(PMSF)下诱导AR15、30、45、60min，分别检测获能前后及AR各时间点的精子悬液上清中ACP活性，同时用CASA分析精子运动参数证实精子发生获能和AR。结果 精子获能前后的ACP活性无显著性差异；AR(有PMSF)各时间点的ACP活性显著高于获能前后及不加PMSF时的ACP活性；CASA分析证实，精子运动参数的改变与其发生获能和AR的运动特性一致。结论 ACP分析为一种简便易行、耗时少、能反映精子群体AR状态的较为客观的方法，但蛋白酶的潜在影响不容忽视。