Sperm Antibodies Research Articles

Status of spermatogenesis and sperm parameters in langur monkeys following long-term vas occlusion with styrene maleic anhydride.

Vas occlusion by styrene maleic anhydride (SMA), trade name RISUG (one of the promising male contraceptive procedures currently in phase III clinical trials), at 60 mg/vas deferens dissolved in 120 micro L dimethyl sulphoxide (DMSO) at up to a 540-day study period caused severe oligospermia in the first 2 to 3 ejaculations and uniform azoospermia in the subsequent ejaculations without toxicity in langur monkeys. The ejaculated spermatozoa were necroasthenoteratozoospermic, suggesting instant sterility. Routine hematology and clinical chemistry parameters and the serum testosterone and sperm antibody titers remained unchanged from their pretreatment values until 540 days vas occlusion. Histology of testes revealed continued spermatogenesis throughout the study period. The stages of spermatogenesis appeared normal until 300 days of vas occlusion. At 360 days of vas occlusion, germ cells appeared in the lumen. Degeneration of seminiferous epithelium was evident in some of the tubules. Following 420 days of vas occlusion, the central portion of the testis showed regressed seminiferous tubules depicting various shapes and devoid of germ cells, which continued until 540 days of vas occlusion. Ultrastructure of the testes after 540 days of vas occlusion revealed vacuolization in the cytoplasm of Sertoli cells and degenerative features in the membranes of the spermatocytes and spermatids in the affected seminiferous tubules. The sub-cellular features of the normal tubules were similar to those of controls. The results suggest focal degeneration of seminiferous epithelium in the central portion of the testis following long-term vas occlusion with SMA.

Effects of sera from infertile women with sperm immobilizing antibodies on fertilization and embryo development in vitro in mice.

This study was performed to investigate if patients' sera with anti-human sperm antibodies show inhibitory effects on in vitro fertilization (IVF) and embryo development in mice. Patients' sera were collected from eight infertile women having sperm immobilizing antibodies and 17 infertile women without the antibodies. Male ICR mice and female F1 mice (BALB/c X C57BL/6J) were used. In mouse IVF, pre-incubated sperm were cultured in the medium containing patient's serum with or without sperm immobilizing antibodies, or bovine serum albumin (BSA) as a control. The fertilization rates and the incidences of blastocyst formation were compared. A mouse sperm immobilization test was established. Five (62.5%) of eight serum samples with sperm immobilizing antibodies and nine (52.9%) of 17 serum samples without the antibodies showed sperm immobilizing activities in mice. There was no significant difference between the two groups. Five sera with sperm immobilizing activities in human and mice, and five sera without sperm immobilizing activities in human or mice were used for the further experiments. The fertilization rates in BSA, patient's serum with sperm immobilizing antibodies, and that without the antibodies were 82.5% (746/904), 43.6% (508/1165), and 64.5% (669/1037), respectively. There were significant differences between the groups. The incidences of blastocyst formation were 59.9% (447/746), 31.7% (161/508), and 47.7% (319/669), respectively. There were also significant differences between the groups. Some of the patient's serum with and without sperm immobilizing antibodies could immobilize sperm with complement. However, as compared with control, sera with sperm immobilizing activities against human and mouse sperm significantly blocked IVF and inhibited embryo development in mice. Further studies are required to investigate the mechanisms of the blocking effects of antisperm antibodies on fertilization and embryo development using the mouse model.

Head (H) And Tail (T) Monoclonal Antibodies of Boar Spermatozoa

Semen collected from three boars were pooled and the sperm cells separated from the semen, washed, sonicated and mixed with equal volume of Freund's adjuvant. The mixture of sperm with complete Freund's adjuvant was used to immunize five mice of the BALB/C strain intraperitoneally on Day O. The sonicated sperm cell immunize the mice the same tourte on Day 14. The mice were immunized with suspension in Incomplete Freund's adjuvant was used to sonicated sperm without adjuvant on Day 21 before they were sacrificed on Day 24. Spleen cells prepared from the mice were fused with myeloma cells (Ag.8.653) by addition of polyethylene glycol (PEG). Two distinct monoclonal antibodies (antihead (H) and anti-tail (T) Mabs) were produced after cloning of the hybridoma cells. There were intermediate forms that are believed to have arisen as a result of incomplete cloning. The Mabs were identified by the use of indirect immunofluorescence method and their specificity confirmed by their non-reaction with cells from some other organs. These Mabs can be used for further work on the mechanisms of sperm antibody effect on swine infertility. It is speculated that the H. Mab and the T Mab may block acrosome reaction and sperm motility respectively. Keyword: Boar spermatozoa monoclonal antibodies Trop. Vet. Vol. 21 (2) 27-34 (2003)

Comparison of detection methods for antibodies to boar spermatozoa

Sera collected from each animal at weekly intervals and sera from two 3-month old virgin gilts were tested for sperm antibody utilizing indirect immunofluorescence, immunoperoxidase, indirect immunobead, Staphylococcus Aureus protein a agglutination techniques and enzyme linked immunosorbent assay (ELISA). Sera from the virgin gilts and preimmune sera of immunized animals were negative for sperm antibody with all methods. The different methods varied in their sensitivity to determination of sperm antibody titers. The ELISA was the most sensitive of the methods. The ELISA and Protein A agglutination were the most suitable in terms of specificity and repeatability for sperm antibody detection in swine. (Tropical Veterinarian: 2003 21(1): 1-13)

Immune response of the tammar wallaby (Macropus eugenii) to sperm antigens.

In the present study, male and female tammar wallabies were immunised with whole tammar wallaby sperm in adjuvant. An assay for sperm antibodies using a live sperm ELISA has been developed to detect sperm surface antigens and used to validate an assay using a 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate (CHAPS) membrane extract of whole tammar wallaby sperm. The tests were used to monitor the immune response to whole sperm in both male and female tammar wallabies. Antisera with a limited array of specificities were generated, with those locating to the midpiece region of the sperm appearing the most likely candidates for targets for fertility perturbation based on immunofluorescence of fixed and non-fixed sperm. These systemically generated antibodies were demonstrated to have access to both the female and male tammar reproductive tracts and were found on ejaculated sperm and antibodies from female sera and follicular fluid-labelled fresh ejaculated sperm from non-immunised males. Preliminary sequencing of these proteins has identified some possibilities for further investigation.

Which test of sperm quality is clinically useful in the subsequent evaluation of normozoospermic infertile men?

Objective: Diagnosing defective sperm function is difficult by routine semen analysis because spermatozoa are highly specialized cells that express a diverse array of biological properties to achieve fertilization. A subset of men with normal standard sperm parameters, i.e. normozoospermic, may still be infertile. They are diagnosed as idiopathic or unexplained infertility when examination of the female partner is normal. In this study, we examined the diagnostic significance of hypo-osmotic swelling (HOS), acrosome reaction (AR), acrosin activity, and antisperm antibody (ASA) tests in the evaluation of normozoospermic infertile men. Design: Prospective study in a tertiary care facility in an academic institution. Materials/Methods: Study included 40 men attending our infertility clinic with a history of infertility of more than 12 months. All men had normal standard semen parameters, on repeated analysis, according to the World Health Organization guidelines (WHO, 1999) (sperm concentration 20 × 106/mL, motility ≥50%, normal sperm forms ≥30%, and seminal leukocytes ≤1 × 106/mL). A group of normal fertile donors (n = 20) served as a control. Semen samples, collected by masturbation, were subjected to HOS test. Assessment of AR was performed by a triple-staining technique and acrosin activity using a spectrophotometric enzyme assay. Mixed antiglobulin reaction (MAR) test was performed to detect ASA (IgG). Results: Standard semen parameters were not significantly different in normozoospermic patients compared to the fertile donors (p >0.05). Similarly, no significant differences were observed in results (mean ± standard deviation) of HOS and AR in the infertile patients (74 ± 7. 9 and 19.4 ± 2.7; respectively) versus fertile donors (77.7 ± 5.5 and 20.5 ± 1.4; respectively) (p >0.05). However, acrosin activity in the patient group (17.7 ± 4) was significantly lower than in the donors (32.6 ± 5; P = 0.001). Sperm antibodies (IgG) were positive in 3 patients, whereas all donors were negative. Sperm motility was relatively reduced in the 3 ASA-positive cases compared to the remaining ASA-negative patients. Conclusions: The results of the 4 additional tests of sperm quality, performed in this study, indicate that levels of acrosin activity were significantly reduced in normozoospermic infertile men compared to the fertile donors. Therefore, the integration of acrosin activity test in the infertility work-up may help detect an abnormality in a subset of infertile men with a diagnosis of idiopathic or unexplained infertility based on apparently normal standard sperm characteristics, i.e. normozoospermia. Supported by: None.

Serum Sperm Antibodies Are Not Elevated After Mumps Orchitis

Serum Sperm Antibodies Are Not Elevated After Mumps Orchitis

Serum Sperm Antibodies Are Not Elevated After Mumps Orchitis

Serum Sperm Antibodies Are Not Elevated After Mumps Orchitis

Endometriosis in reproductive immunology.

Endometriosis is suggested to represent an autoimmune disorder, but what is the prevalence of autoantibodies to antigens relevant to reproduction? The humoral immune response to the women with endometriosis (stage I-II: 261 women; stage III-IV: 62 women) in serum and in peritoneal fluid was investigated compared with 101 healthy women. Enzyme-linked immunosorbent assay (ELISA) was used in all the women for the detection of seven antiphospholipid antibodies [antiphospholipid antibodies (aPLs) against cardiolipin, L-phosphatidyl (ph)-serine, ph-glycerol, ph-inositol, ph-ethanolamine, phosphatidic (ph)-acid and against beta2-glycoprotein I] of class IgG, IgA, and IgM. A passive haemmagglutination method and ELISA (BioGen) was used for assessment of antizona pellucida antibodies (aZP), tray agglutination test (TAT) and indirect mixed anti-imunoglobulin reaction test (MAR-test) for the determination of sperm antibody levels. Endometriosis I-II were associated with higher serum and peritoneal fluid levels of aPLs against inositol, cardiolipin, ethanolamine, and beta2-glycoprotein I. Forty percent of patients were positive for aZPA. Patients with lesions of endometriosis stage I-II had more autoantibodies than those with stage III-IV, and may be immunologically more active. This result may be significant for future treatments such as in vitro fertilization and embryo transfer.

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.

The age-related changes in the incidence of 'natural' anti-sperm antibodies suggest they are not auto-/isoantibodies.

Establishing the age-dependent patterns of sperm antibody levels among normal humans. Sera samples from 498 healthy subjects aged 0-97 years - 246 males and 252 females - were tested by the gelatin agglutination test of Kibrick, tray agglutination test of Friberg, sperm immobilization test of Isojima and enzyme-linked immunosorbent assay (ELISA). We found a significant increase in the level of sperm agglutinins after 40 years, which decreased after 88 years. The antibodies detected by ELISA were the highest among prepubertal subjects and also declined with aging. No age-dependent changes were established for the sperm immobilizins. With few exceptions, there were no significant differences between male and female sera, as well as between sera of newborn and their mothers. These data are similar to those established for the age-dependent changes of antibodies towards exogenous antigens, suggesting that the 'naturally occurring' antibodies against human spermatozoa are not auto-/isoantibodies.

Serum sperm antibodies are not elevated after mumps orchitis

Objective: To assess the level of serum sperm antibodies after mumps orchitis. Design: Controlled descriptive study. Setting: Academic research environment. Patient(s): Seventy-four mumps orchitis patients. Intervention(s):Sampling of serum at different intervals after the onset of orchitis symptoms: 1 to 7 days, 31 to 60 days, and 61 to 431 days. Main Outcome Measure(s): Level of serum sperm antibodies, using Kibrick’s gelatin agglutination test, Friberg’s tray agglutination test, Isojima’s sperm immobilization test, and ELISA. Result(s):Clinically relevant sperm antibody values were detected by the Friberg method among patients tested from 1 to 7 days (10.5%) and 61 to 431 days (10.5%) after the onset of disease. The Isojima test revealed a statistically insignificant higher incidence among patients at 61 to 431 days (31.6%) as compared with those sampled at 1 to 7 days (10.5%). None of the orchitis sera tested positive by the Kibrick and ELISA techniques. The established incidences did not differ significantly from the results for negative controls (blood donors) and were lower than the values acquired from positive controls (males with unexplained infertility). Conclusion(s):Mumps orchitis does not cause enhanced humoral immunity to spermatozoa.

Serum sperm antibodies unrelated to mumps orchitis

In order to determine whether there is an association between mumps orchitis and serum sperm antibodies, we tested patients at the time orchitis was diagnosed (n = 7) and individuals who had had orchitis at least 1 month previously (n = 14). Data were compared with the results for a control group of blood donors (n = 20). Sperm antibodies were detected by the gelatin agglutination test of Kibrick, the tray agglutination test of Friberg and the ELISA. Clinically significant sperm antibody levels were not found in patients in the early stages of the disease. Four subjects tested post-disease were positive in the Friberg test and one was positive in the ELISA. One control serum was also positive in the latter test. Significant differences were not found between levels in patients in the early stages of the disease and levels in individuals post-disease, although the results of the Friberg test differed significantly between controls and former mumps orchitis cases. These data do not support the assumption of an involvement of humoral immunity against spermatozoa in mumps orchitis patients.

Effect of corticosteroids on sperm antibody concentration in different biological fluids and on pregnancy outcome in immunologic infertility.

In the last fourteen years, detection of free spermagglutinating antibodies has been performed by tray agglutination test (TAT) and by direct/indirect mixed antiglobulin reaction test (MAR) in 696 infertile couples aged 23-42 years (female) and 26-52 years (male) with previously undiagnosed infertility. Oral decreasing doses of prednisone or dexamethasone for three months in the case of serum or seminal plasma spermantibodies, plasmapheresis in one patient with serum spermantibodies were used. Hydrocortisone to the ectocervix was applicated in patients with spermagglutinating antibodies in cervical ovulatory mucus. Serum IgG spermagglutinating antibodies totally disappeared in 3 out of 11 men, and four out of 15 women. But serum IgM sperm antibodies persisted. One female patient was treated with corticosteroids and also with plasmapheresis. Seminal plasma IgG spermagglutinating antibodies were greatly influenced in 57.8% of the patients, IgA in 38.9% and each case by oral corticosteroids. A decrease of IgG and IgA spermagglutinating antibodies in ovulatory cervical mucus during hydrocortisone local application was registered in 61.3%, IgG in 50.7% and IgA in 65.9%. Levels of IgM spermagglutinating antibodies in cervical ovulatory mucus were not influenced, spermantibodies in IgA and IgE were affected very little. The corticosteroid influence of immunocompetent cells in each case must be chosen individually with regard to the localisation of spermantibodies. We often combine the long lasting corticosteroids treatment with in vitro fertilization. The therapy seems to be very promising for the improvement of immunological causes of infertility.

Polycystic ovary syndrome as an autoimmune disease: a new concept

Background: Polycystic ovary syndrome (PCOS) is characterized by a chronic hyperandrogenic anovulatory state. Several mechanisms have been proposed, but the pathogenesis is still under discussion.Objectives: To determine whether an autoimmune reaction could contribute to this syndrome, and to determine the association between this syndrome and one of immunological upset (ie, the antisperm antibodies).Methods: Thirty-five cases of proven PCOS were enrolled in the study (mean age 25.3 ± 5.2). The criteria of diagnosis are based on hyperandrogenemia, high LH, ultrasonographic appearance of polymicrocystic ovaries with stromal hyperthropathy detection of circulating antiovarian antibodies. Antisperm antibodies in the serum and cervical mucus were detected by ELISA using human ovaries as antigens were expressed as the ratio of the optical density (OD) of each serum to the reference OD equivalent to the mean ± 2 SD of that obtained by a reduced variable method with control samples from 25 normal healthy control women with a mean age of 26.5 ± 3.7. The controls were without any autoimmune disease. Values >8.1 were considered as positive.Results: There was a statistically significant higher mean ratio for the patients with PCOS than for the control: IgG, P <0.0002; IgA, P <0.001; and IgM, P <0.001.The specificity of these antiovarian antibodies was significantly higher in 15 women with positive antinuclear antibodies; and a positive ratio for IgG was present in 52%, IgA in 22%, and IgM in 38%. Positive association of cervical antisperm antibodies was found in 20 of 35 patients with PCOS, which is highly significant compared with normal controls (P <0.001). Serum antisperm antibodies were found in 15 cases of PCOS, mixed 2 types cervical, and serum sperm antibodies were found in 17 cases (highly statistically significant, P <0.005).Conclusions: A high concentration of antiovarian antibodies suggests that immune reaction is associated with PCOS; a high concentration of antisperum antibodies suggests an association of the two conditions with PCOS. A new theory has been proposed that this immunological reaction leads to inflammatory reaction, and they will lead to abnormal production of cytokines that will in turn lead to overt biochemical changes in PCOS. Steroid therapy is well known in the treatment of antisperm antibodies and it can be used as adjuvant in ovulation induction in PCOS.

The intra- and inter-assay variation of the indirect mixed antiglobulin reaction test: is a quality control suitable?

The test most commonly used to detect sperm antibodies is the mixed antiglobulin reaction (MAR), standardized by the World Health Organization. The indirect MAR test detects soluble sperm antibodies in seminal plasma by using healthy donor spermatozoa as antigen. In this study we systematically investigated the influence of donor spermatozoa and the source of sperm antibodies upon the results of the indirect MAR test, and calculated the intra- and inter-assay variations. Using one individual seminal plasma and the same donor semen, results of the indirect MAR test are highly reproducible (low intra-assay variation). Two dimensions of inter-assay variation must be considered: (i) serial ejaculates of an individual donor may be used at different times; (ii) different donors may be applied to identical antibody sources. Donor spermatozoa strongly influenced the results of the indirect MAR test. Using multivariate statistical tests, highly significant main effects between the different donors (P < 0.001) and specific reciprocal effects between donor spermatozoa and seminal plasma samples (P < 0.001) were observed. The high inter-assay variation of the indirect MAR test will lead to incorrect results. There is urgent need of a reliable and reproducible test for sperm antibody detection to improve quality control of the methods.

Sperm antibodies in rat models of male hormonal contraception and vasectomy.

The presence of sperm antibodies correlates with nearly every pathological condition of the male reproductive tract. In the seasonal breeder, mink, a decrease in gonadotrophin secretion and testicular regression also induces sperm antibodies. Because the Sertoli cells and the principal cells of the epididymis (i.e. the cells mainly responsible for protection of germ cells from autoimmune destruction) are dependent on androgens, and because the androgen concentration decreases in both the testis and epididymis during male hormonal contraception, the presence of IgG class sperm antibodies in serum was studied in rats during the suppression and recovery phases of testosterone contraception and after vasectomy. Five-centimetre long testosterone implants were placed under the dorsal skin of rats under pentobarbitone anaesthesia. The control rats received empty implants. All implants were left in the rats for 27 or 53 days. The total number of testicular antigens detected by sera from the vasectomized rats increased significantly until 66 days post-operation, and then decreased to the levels of intact rats. The number of testicular antigens detected by sera from rats receiving contraceptive doses of testosterone did not increase before the testosterone capsules were removed, but at 40 days post removal of the silastic capsules, the number of antigens detected by the sera was significantly higher than in intact rats and at 77 days post removal of the silastic capsules, the number of antigens detected by the sera was significantly higher than at 27 days after starting testosterone administration. No significant changes in the number of antigens detected by the sera could be observed after the implanting of empty capsules or after their removal. Vasectomy mostly induced antibodies against testicular antigens in the molecular ratio ranges of 70-82, 25-33 and 21-24.5 kD. Antibodies against antigens in these molecular ratio ranges were not significantly induced during or after treatment with contraceptive doses of testosterone. Cell nuclei with apoptotic morphology could be observed in the seminiferous tubules of the vasectomized rats, but DNA in situ 3'-end labelling of testes could not confirm any differences between the testes of vasectomized and sham-operated rats or between testosterone-treated and empty implant-treated rats. CD3+ T cells could not be observed in the testes of any of the treatment groups. These results suggest that the immunological conditions remain stable in the testes after vasectomy and during testosterone treatment, but that the animals are more prone to develop autoantibodies after vasectomy and during recovery from treatment with exogenous testosterone.

Altered sperm function or sperm antibodies are not associated with chlamydial antibodies in infertile men with leucocytospermia

Leucocytospermia, defined as a concentration of more than 10(6) leucocytes/ml of seminal fluid in patients without clinical symptoms due to an adnexitis, is seen in about 10% of patients in an infertility department. Infection with Chlamydia trachomatis is possibly relevant as other pathogenic bacteria were not cultured from the semen in significant numbers. University Clinic, Department of Andrology. Two hundred and seven patients attending the department for male infertility investigation. Analysis on each semen sample included determination of leucocyte count and the MAR test for the detection of sperm antibodies. Chlamydial antibodies in semen were determined using an on-slide enzyme immunoassay. No differences between leucocyte counts in patients with and without chlamydial antibodies were detected. In addition, no differences in the sperm parameters or results of MAR-tests in these two groups was seen. There were no correlations between the leucocyte count and sperm parameters, including the MAR-test results. We conclude that antibodies to chlamydiae in semen are not associated with leucocytospermia. Leucocytospermia per se does not appear to be significant for the sperm functions and immune responses to sperm.

Altered sperm function or sperm antibodies are not associated with chlamydial antibodies in infertile men with leucocytospermia

Objective Leucocytospermia, defined as a concentration of more than 106 leucocytes/ml of seminal fluid in patients without clinical symptoms due to an adnexitis, is seen in about 10% of patients in an infertility department. Infection with Chlamydia trachomatis is possibly relevant as other pathogenic bacteria were not cultured from the semen in significant numbers.Setting University Clinic, Department of Andrology.Patients Two hundred and seven patients attending the department for male infertility investigation.Methods Analysis on each semen sample included determination of leucocyte count and the MAR test for the detection of sperm antibodies. Chlamydial antibodies in semen were determined using an on-slide enzyme immunoassay.Results No differences between leucocyte counts in patients with and without chlamydial antibodies were detected. In addition, no differences in the sperm parameters or results of MAR-tests in these two groups was seen. There were no correlations between the leucocyte count and sperm parameters, including the MAR-test results.Conclusions We conclude that antibodies to chlamydiae in semen are not associated with leucocytospermia. Leucocytospermia per se does not appear to be significant for the sperm functions and immune responses to sperm.

Semen culture, leukocytospermia, and the presence of sperm antibodies in seminal hyperviscosity.

Seminal hyperviscosity is generally thought to reveal genitourinary infection. The aim of the present work was to study this hypothesis. A total of 65 semen samples were obtained from males presenting for infertility screening. The samples were evaluated according to WHO criteria and microbiologically investigated, including culturing for Mycoplasma hominis and Ureaplasma urealyticum, and microscopic observation of Chlamydia trachomatis by a direct fluorescence assay. Determination of local antisperm antibodies was performed. Semen was categorized according to consistency: normal (n = 31) and high (n = 34). No difference was recorded either in the number of positive cultures, or in the number of species found in each sample. The number of white blood cells and the percentage of antibody-bound sperm showed no difference in the groups under study. There was no association between seminal hyperviscosity, positivity in semen cultures, number of species isolated in semen cultures, leukospermia, or presence of sperm antibodies. Further studies should be performed considering the evolution of the genital-infected patients to clarify the etiology of the hyperviscosity.