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Production of anti-sperm antibody (ASA) often suffers from autoimmune reaction against sperms in human infertility. The antibodies are measured in both blood and seminal plasma of males. Here, we reported a simple protein biochip methodology that takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS) and enables identification of anti-sperm antibody in Chinese male infertility. To validate this biochip platform, we immobilized purified sperm protein on the biochip surface and tested a variety of parameters in quality controls for the protein assay, respectively. Then, we analyzed serum samples from 368 patients with infertility and 116 healthy donors by means of this biochip simultaneously. We found that positive rate of serum ASA was 20.92% (77/368) in the cases and 1.72% (2/116) in the controls, respectively. Furthermore, we further corroborated the biochip assay in comparison with ELISA method. We found that both methods were compatible for the detection of serum ASA in the patients. In addition, a follow-up study for natural conception in ASA-positive and ASA-negative patients was conducted. The result showed a significant correlation between serum ASA expression and natural pregnancy rate 6.5% in ASA-positive patients while 18.9% in ASA-negative patients, indicating the potential roles of ASA in naturally reproductive processes., (© 2020 The Author(s).)

Introduction: To investigate the effects of intravesical immunotherapy on semen parameters in young patients with non-muscle invasive bladder tumour., Methods: A total of 17 sexually active male patients < 45 years of age underwent transurethral resection of bladder tumour (TURBT) from Jan 2010 to Dec 2012. On HPE analysis, T1 high grade was found in 16 patients and Ta grade high grade in 1 patient. Associated CIS was found in 4 patients. Induction course of 6 weeks of adjuvant BCG therapy was given. Semen analysis was done 1 week prior to BCG therapy and 3 months after BCG therapy. Serum levels of hormones like total testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were also measured., Results: Mean age of patients at diagnosis was 34.6 (29-43) years. Total semen volume was found to be decreased in 2 patients. Main parameter which was deteriorated was total sperm concentration which was significantly decreased in 12 patients and 5 patients even had their counts below oligospermia levels. Seven patients had associated decrease in sperm motility. However, no patient showed significant difference in other semen parameters. Also no patient had any change in androgen hormonal status except 2 patients in which serum testosterone was found to be non-significantly decreased., Conclusion: Intravesical therapy with BCG was found to adversely affect spermatogenesis and cause oligospermia. It is important that relatively young patients must be informed of these effects and advised to have sperm preservation before instillation of BCG therapy to avoid fertility issues in future., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Problem: The aim of this study was to evaluate the levels of seminal plasma cytokines interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) in male subfertility., Method of Study: A total of 73 male partners of an infertile couple attending a regional andrology unit were recruited into this prospective study and subdivided into the various groups based on semen analysis. Concentrations of cytokines such as IL-6, IL-8, IL-10, IL-11, IL-12, TNF-alpha and IFN-gamma in the seminal plasma were determined using enzyme linked immunosorbent assay (ELISA)., Results: Significant higher concentrations (P < 0.05) of IL-6 in the mild and severe oligospermic group, IL-8 and IL-10 in the asthenospermic group and IL-6, IL-10, TNF-alpha and IFN-gamma in the obstructed azoospermic group were determined. IL-10 concentrations correlated significantly with other cytokines in the obstructed azoospermic group and the asthenospermic group., Conclusion: Our study confirms that cytokines rarely act in isolation, but rather in a network of other cytokines and may affect sperm function directly or indirectly. The presence of increased levels of cytokines in the obstructed azoospermic group suggests that the cytokines may not originate from the testis.

CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. Humans express only one, whereas mice express two CD59 genes. We previously reported the targeted deletion of the mCd59b gene in which absence of mCd59b together with an unintended down regulation of mCd59a caused hemolytic anemia with spontaneous platelet activation. To confirm the complement role in the hemolytic anemia caused by abrogation of mCd59 function, we have developed a mCd59a and mCd59b double knock out mice and analyzed its phenotype in complement sufficient and deficient (C3(-/-)). We report here that total abrogation of mCd59 function in mCd59ab(-/-) mice results in complement-mediated hemolytic anemia that is rescued by the deficiency of C3 in compound mCd59ab(-/-)/C3(-/-) mice.

Objectives: Seminal plasma elicits recruitment of immune cells into the cervix. It increases in mice in vivo and in humans in vitro the endometrial epithelial expression of those cytokines and growth factors, which play an essential role in implantation. To analyse if the stimulatory effect of seminal plasma correlates to the quality of the sperm count, the immunomodulatory potential of seminal plasma of fertile and infertile men was studied., Study Design: Seminal plasma from 34 volunteers with normal sperm count und from 28 men with oligozoospermia or asthenozoospermia was studied. Firstly, the concentrations of IL-6, IL-8, VEGF, TNFalpha, IL-1beta, TGFbeta1 und G-CSF were analysed by ELISA. Secondly, the immunomodulatory potential was studied by bioassays. Bioassays were set-up by isolation of peripheral mononuclear blood cells (PMBC), sensitized by stimulation with LPS. The assays were incubated with seminal plasma of both patient groups and secretion of IL-6, IL-8 and TNFalpha was analysed by ELISA., Results: IL-6, IL-8, VEGF, TNFalpha, IL-1beta, TGFbeta1 and G-CSF were detected in seminal plasma. The bioassays revealed a significant increase of IL-6 and IL-8 and a decrease of TNFalpha by incubation with seminal plasma. The concentrations of all factors and the stimulatory and inhibitory potential of seminal plasma from men with oligozoospermia, asthenozoospermia and normozoospermia were not significantly different in ELISA- and bioassays., Conclusion: The experiments revealed a similar immunomodulatory potential of seminal plasma from men with normal and abnormal sperm counts, suggesting that male infertility is probably not caused by differences in the activity of seminal plasma.

Objective: To assess the types, distributions, and numbers of immune cell infiltrates in the testes of men with azoospermia., Design: Prospective study., Setting: University research and clinical institutes., Patient(s): Thirty-one men with azoospermia showing normal spermatogenesis (n = 10), germ cell maturation arrest (GA, n = 12), and Sertoli cell-only syndrome (SCO, n = 9)., Intervention(s): Testicular tissue biopsies. Sections were stained with routine (hematoxyline and eosin), special (Masson Trichrome, Gordon, Periodic Acid Schiff, Aldehyde Fuchsin, and Orcein stains), and immunoperoxidase stains (using monoclonal antibodies for B and T cells and CD68 macrophages)., Main Outcome Measure(s): Serum levels of T, FSH, and LH in addition to histopathological analysis., Result(s): Hormonal profiles were unremarkable in all patients. Marked deposition of the reticular and collagen fibers was seen more in abnormal than normal spermatogenesis. The immune (B and T lymphocytes and CD68 macrophages) and mast cells were found in the interstitium, tubular walls, and lumens of all the testes analyzed. The differential counts of these cells (B and T lymphocytes, CD68 macrophages, and mast cells, respectively) were higher in SCO (1.66 +/- 0.46, 9.14 +/- 1.30, 2.26 +/- 1.68, 3.35 +/- 0.23) and GA (2.03 +/- 0.48, 4.70 +/- 1.00, 2.61 +/- 0.70, and 4.18 +/- 0.13) when compared with those in normal spermatogenesis (1.22 +/- 0.19, 5.41 +/- 0.58, 1.55 +/- 0.33, and 2.26 +/- 0.13). Increased cellular counts were not statistically significant for T and B cells and macrophages. However, these differences were statistically significant for mast cells., Conclusion(s): Abnormal spermatogenesis is associated with increased numbers of the immune and mast cells. Our findings may reflect an exaggerated immune response in these cases.

Problem: To determine the ability of monoclonal antibodies (MoAbs) against acrosomal antigens to detect physiology and pathology of human spermatozoa and to detect spermatids in ejaculates of infertile male with azoospermia., Method of Study: Sperm antigens detected with prepared MoAbs were partially characterized by biochemical and immunocytochemical methods. The acrosomal status of spermatozoa was compared in men with normal and pathological spermiograms and in sperm before and after induced acrosome reaction (AR). Ejaculates from patients were tested for the presence of spermatids., Results: MoAbs specifically bind to intra-acrosomal sperm antigens with quantitative difference between ejaculates with normal and pathological spermiograms. These antigens are released from the acrosome after induced AR. MoAbs labeled acrosomal proteins in round and elongated spermatids in the ejaculates of patients with azoospermia., Conclusion: MoAbs against intra-acrosomal sperm antigens are useful for human sperm diagnosis and prediction of spermatogenesis. The spermatids can be utilized in assisted reproduction.

Our purpose was to evaluate cellular androgen receptor (AR) distribution and intensity of immunostaining in the human azoospermic testis. Thirty six biopsy specimens from azoospermic men were immunostained, using a monoclonal antibody of human AR. The localization and the intensity of AR immunostaining was evaluated in Sertoli Cell Only (SCO) testis (G1, n = 21), in spermatogenesis arrest testis (G2, n = 11) and in histologically normal testis (G3, n = 4). We found an AR immunostaining in Sertoli, peritubular myoid and Leydig cells, but not in germ cells. The intensity of the immunostaining varied substantially between biopsy specimens of different patients. Sertoli and Leydig cells AR immunostaining (score and intensity) in SCO group was higher than in the other groups. For Sertoli cells, the score means of AR immunoreactivity were 20 +/- 2.36, 10.18 +/- 1.0 and 1 +/- 1, for G1, G2 and G3 groups, respectively. For Leydig cells, the score means were 10.24 +/- 1.37, 6 +/- 0.71 and 0, for G1, G2 and G3 groups, respectively. We found significant differences between G1 and G2 (p = 0.0008), between G1 and G3 (p = 1.54 10-7) and G2 and G3 (p = 0.00032). These results suggest that in the testis AR is located exclusively in somatic cells and its expression is higher in SCO syndrome than in normal and in arrest spermatogenesis testes.

Effects of plasmapheresis and hemosorption on efficacy of the treatment of autoimmune male infertility (AMI) were studied in 289 AMI males with oligoasthenozoospermia aged 19 to 37 years. The males were divided into three groups by the levels of antisperm antibodies (ASAB) in the blood and ejaculate. The study was made of cellular and humoral immunity, intensity of protein and lipid free radical oxidation and antioxidant defense. Patients of group 1 with high ASAB in the blood but low in ejaculate received a course of plasmapheresis. Patients of group 2 with high ASAB both in the blood and ejaculate were subjected to hemosorption and plasmapheresis in one contour. Group 3 patients with high ASAB in ejaculate but low in the blood received efferent therapy only after medication and photomodification of the blood. The treatment reduced elevated levels of ASAB in the blood and ejaculate, normalized free radical oxidation, cellular and humoral immunity, antioxidant defense. This resulted in improvement of spermogram parameters, efficacy of assisted reproductive technologies, higher probability of natural pregnancy in the patients' wives.

The melanoma-associated antigen (MAGE) gene family of cancer-testis antigens is expressed in certain malignant neoplasms and the testis, but not in other healthy tissues. The aim of this study was to determine the usefulness of immunohistochemical staining with the 57B anti-MAGE-A4 mouse monoclonal antibody (MAb) in testicular biopsy specimens from patients with nonobstructive azoospermia and obstructive azoospermia (OA). Fifty-four cases of Sertoli cell only (SCO), 30 cases of spermatocytic arrest, 15 cases of hypospermatogenesis, and 10 testicular biopsy specimens with OA (normal spermatogenesis) were evaluated. Immunohistochemistry was performed using the 57B MAb, which primarily recognizes the MAGE-A4 antigen in paraffinized tissues. The cells were quantitated by a computerized image analysis system. Testicular biopsy specimens with normal spermatogenesis exhibited strong nuclear and cytoplasmic MAGE-A4 staining of spermatogonia and weak staining of spermatocytes, but not spermatids or Sertoli or Leydig cells. No staining was detected in SCO cases. In five cases of SCO with focal spermatogenesis, spermatogonial cells that were initially missed by hematoxylin and eosin staining were detected by MAGE-A4 immunohistochemistry. Immunostaining with the 57B MAb greatly enhanced identification of spermatogonia in cases of spermatocytic arrest and hypospermatogenesis. The number of MAGE-A4-positive spermatogonia was significantly decreased in hypospermatogenesis, as opposed to the OA group (12.1 +/- 4.3 and 30.3 +/- 10.0, respectively). The number of MAGE-A4-positive primary spermatocytes was significantly increased in early maturation arrest, as compared with the OA group (48.2 +/- 10.8 and 16.9 +/- 9.8, respectively). The 57B anti-MAGE-A4 MAb is a useful marker for the detection and quantitation of spermatogonial germ cells. It also facilitates automated image analysis and provides greater accuracy in the histopathologic evaluation of testicular biopsy specimens.

Problem: The objective of this study was to evaluate the possible relevance of cytokines in seminal plasma (SP) of patients with accessory gland infection and oligoterato-asthenozoospermia., Method of Study: Semen samples were obtained by masturbation from 90 men and were examined for the presence of interleukin (IL)-2, IL-6, IL-8, IL-11 and soluble CD23 (sCD23) by enzyme-linked immunosorbent assay. Five groups were included: (1) fertile men (n = 20), (2) infertile men with varicocele and oligo-teratoasthenozoospermia (V-OTA, n = 20), (3) infertile men with genital infection and OTA (INF-OTA, n = 20), (4) infertile men with idiopathic testicular lesion and OTA (ITL-OTA, n = 20) and (5) infertile men with azoospermia (AZOO, n = 10)., Results: We found that the mean level of IL-2 was higher in SP from infertile men compared with SP from fertile men (P < 0.05). Mean levels of IL-6, IL-8, IL-11 in SP of INF-OTA were higher than that of all other groups (P < 0.05, P < 0.05, P < 0.001, respectively). However, no significant differences could be detected between other groups. A significant increase was noted in sCD23 levels in SP from men with ITL-OTA compared with all other groups (P < 0.01). We have not observed any correlations between IL-2, IL-6, IL-8, IL-11 and sCD23 levels in SP and semen parameters. Spearman's correlation coefficient revealed that there was a significant association between IL-6, IL-8, IL-11 levels in men with INF-OTA., Conclusion: The measurement of each cytokine separately in the SP of men with INF-OTA, in spite of the existing significant differences, does not have a diagnostic value in male infertility. However, a combined determination of IL-6, IL-8, IL-11 in the SP of men with genital infection and oligo-terato-asthenozoospermia may provide clinically useful information for the diagnosis of male accessory gland infection.

The technical developments and expanded indications for testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) provide great advantages for patients with non-obstructive azoospermia. Such success, however, also means that genetic abnormalities in non-obstructive azoospermia can be transmitted to the next generation, demonstrating the importance of being able to understand the genetic background of non-obstructive azoospermia. We have previously reported that human leukocyte antigens (HLA)-A33 and -B44 in the HLA class I region and the HLA-DRB1*1302 allele in the HLA class II region are linked to susceptibility to non-obstructive azoospermia in Japanese men. However, strong linkage of HLA-DRB1*1302 with HLA-A33 and -B44 is also evident in the Japanese population. Thus, uncertainty prevails as to whether the HLA class I or class II molecule is more directly associated with non-obstructive azoospermia. In the present study, we performed association analysis with 21 polymorphic microsatellite markers identified near the HLA genes to map the gene involved in the development of non-obstructive azoospermia more precisely. Microsatellite markers located in the HLA class I region or the class III region showed no statistically significant association with this disorder, although once again the HLA-A33 and -B44 alleles showed a significant association. In contrast, some of the microsatellite markers in the HLA class II region and at the HLA-DRB1 and -DQB1 loci displayed strong associations with non-obstructive azoospermia. Taken together, our previous and present data suggest that the critical region for development of non-obstructive azoospermia is near the HLA-DRB1 and -DQB1 segments in the HLA class II region.

Background: In many fertility centres, intracytoplasmic sperm injection (ICSI) with epididymal or testicular spermatozoa is a routine treatment for men with azoospermia. In this prospective study, the physiological consequences after testicular sperm aspiration (TESA), using suction and a 19 gauge needle, were evaluated., Methods and Results: Thirty-five consecutive men with azoospermia underwent TESA. Testicular ultrasonography with Doppler flow imaging was performed and testicular volumes were evaluated pre-operatively and 3 months after aspiration. If focal testicular lesions were found, further examinations were performed 6 and 9 months after TESA. Serum FSH, testosterone and antisperm antibodies (ASA) were analysed. Focal testicular lesions were seen in four out of 61 testes (6.6%) at the 3 month investigation point. Three lesions were resolved after 6 months and all after 9 months. Testicular echogenicity remained unchanged in 50 cases (82%) 3 months after TESA. Four men (11.4%) reported severe subjective discomfort post-operatively, but only one had a medical consultation where an intratesticular haematoma was diagnosed. There were no significant changes in FSH and testosterone after surgery and testicular volumes were similar after 3 months. There were three borderline cases of ASA in serum, but none was classified as ASA-positive., Conclusions: The puncture method of testicular sperm aspiration seems to be a safe method for sperm retrieval, with minimal physiological consequences.

Objective: To assess the effect of Trucut needle biopsy on the ultrasound appearances of the testis in obstructive and nonobstructive azoospermia to test serum samples for antisperm antibodies and gonadotropin and testosterone levels., Design: Prospective case analysis., Setting: IVF unit., Patient(s): Sixteen subjects with obstructive azoospermia had postbiopsy ultrasound scans, 18 had assessment of hormone profiles, and 20 had evaluation of antisperm antibodies., Intervention(s): Trucut needle testicular biopsies under local anesthetic., Main Outcome Measure(s): Postbiopsy testicular ultrasound, the presence of serum antisperm antibodies, and follicle stimulating and luteinizing hormone and testosterone levels., Result(s): There were no postbiopsy hematomas or scars, antisperm antibodies did not develop, and pituitary gonadotropins did not rise nor testosterone levels fall., Conclusion(s): Trucut needle testicular biopsy in men with obstructive azoospermia is not associated with defects of parenchymal structure or function.

Problem: To examine the capacity of sperm cells from fertile and infertile men to secrete interleukin (IL)-6, and the involvement of serum factors and lipopolysaccharide (LPS) in the regulation of IL-6 and IL-1 production by sperm cells., Methods: Swim-up sperm cells from fertile (donors) and oligoteratoasthenospermic (OTA)-infertile men were incubated with or without 5% fetal calf serum (FCS) and LPS (10 microg/mL) for 2-24 hr. After incubation, IL-6 and IL-1 bioactivities were measured in supernatants and lysates by specific bioassays (B9 cell proliferation assay and 1A-5 system, respectively)., Results: IL-6- and IL-1-like activities were observed to be produced by swim-up sperm cells from both study groups. Stimulation of swim-up sperm cells with either LPS or FCS or both together did not affect their capacity to produce IL-1. However, LPS, but not serum increased the secretion levels of IL-6 by swim-up sperm cells., Conclusions: Swim-up sperm cells from both study groups constitutively produce IL-6 and IL-1, and serum components did not affect this capacity. However, LPS was shown to increase the capacity of swim-up sperm cells of both study groups to secrete IL-6, but not IL-1. Cytokines may be involved in the physiology and pathophysiology of sperm functions and, thus, may affect male fertility.

We previously reported that the HLA-A33, -B13, and -B44 antigens, which are major histocompatibility complex class I molecules, are involved in the susceptibility of nonobstructive azoospermia in Japanese men. In this report, HLA-DR antigens, which are class II molecules, are investigated by advanced DNA typing in addition to classical serological typing to study a more complex genotype of HLA-DRB2. Genotyping was performed by the polymerase chain reaction-sequence-specific primer (PCR-SSP) method of analysis and/or by a commercial rapid assay based on the polymerase chain reaction (PCR), followed by reverse dot-blot hybridization of PCR products (the Inno-LiPA assay). The allele frequencies of the HLA-DR13 antigen and the -DRB1*1302 allele were significantly higher in Japanese subjects with nonobstructive azoospermia compared with a control group of healthy Japanese men, and these alleles were associated with relative risks for nonobstructive azoospermia of 4.2 and 4.9, respectively. If we suppose this strong linkage to both HLA class I and II antigens is due to linkage disequilibrium, it may suggest the existence of a novel gene involved in spermatogenesis in the class III region, which is located between the class I and class II regions and contains several genes other than HLA.

A 29-year-old man with Kallmann syndrome suddenly developed decreased semen volume, azoospermia, and facial hair loss after 11 years of successful human chorionic gonadotropin (hCG) and human menopausal gonadotropin (hMG) treatment. Anti-hCG antibody was not detected in the patient's serum. A high serum level of luteinizing hormone (LH) with nasal LH-releasing hormone analogue administration failed to increase serum testosterone to a sufficient level. Testosterone injection after cessation of hCG and hMG therapy was able to improve semen volume, but not azoospermia. Resumption of hCG and hMG therapy after 6 months cessation partially restored spermatogenesis. The secondary failure of hCG and hMG therapy suggests a decrease of testicular sensitivity to LH as well as hCG.

Purpose: Approximately 15 to 20% of infertile men have azoospermia. In the Y chromosome a deletion, termed the azoospermic factor, has been found in some cases of idiopathic azoospermia. We investigate the relationship of factors in autosomal chromosomes (HLA class I antigens) to spermatogenesis failure in idiopathic azoospermia., Materials and Methods: We evaluated 65 infertile Japanese men with idiopathic azoospermia. The frequency of the HLA allele reported in 1,216 healthy Japanese men was used as a control. HLA class I typing was performed by the National Institutes of Health standard serological method or polymerase chain reaction-sequence specific primer analysis. Allele frequencies were calculated. We determined statistical significance in the frequency of each allele in patients and controls using the chi-square test. The relationship of HLA antigens to idiopathic azoospermia was expressed as relative risk., Results: In Japanese men with idiopathic azoospermia the frequency of HLA-A33, B13 and B44 was significantly increased compared with controls. The relative risk of HLA-B44 was 8.4, an extremely high value compared with that of other diseases and HLA antigens., Conclusions: We suggest that HLA class I antigens are important genetic markers that represent a risk factor for idiopathic azoospermia.

This review describes the role of the epididymis in human infertility, by analysing the results of epididymovasostomies which confirm that the more distal the site of obstruction the greater the chance of fertility. The use of epididymal spermatozoa for in vitro fertilization (IVF) yielded poor results in contrast to intracytoplasmic sperm injection using either epididymal or testicular spermatozoa. The nature of the pathology causing obstructive azoospermia is examined reviewing in particular the possible role of mercury toxicity in Young's syndrome. This review describes the results of studies that show that the level of obstruction within the epididymis is correlated with the presence of sperm antibodies and distal obstructions are associated with the presence of sperm antibodies. The demonstration that necrozoospermia decreases with increased sperm transport through the epididymis, when combined with the observation of normal testicular sperm morphology, represents the basis for the disorder termed epididymal necrozoospermia. However, to date the nature of the epididymal pathology causing this disorder remains obscure.

Complete semen analyses including computer-assisted sperm motility and morphology assessments were performed to determine if semen and sperm differed between HIV-seropositive men and fertile controls, or differed with symptoms, or CD4+ peripheral cell count categories. Previous studies included small numbers of men and presented conflicting conclusions. Two hundred and fifty non-vasectomized HIV-seropositive men and 38 fertile controls each provided one semen sample. Non-parameteric statistics were used to analyse both continuous and nominal data. Fertile men had significantly greater semen volume, sperm concentration, percent motility, percent rapid and linear motility and total strictly normal spermatozoa than HIV seropositive men. Neither total number nor subtypes of leukocytes in semen differed between the two groups. Among the HIV seropositive men, significant differences in semen analyses were found between CD4+ cell count, clinical, and AIDS categories. Lower CD4+ cell counts (< 200 mm-3) were associated with significantly lower percent motility, percent normal sperm morphology by strict criteria, significantly more spermatids in semen, and higher percentages of teratozoospermia, oligoasthenoteratozoospermia and leukocytospermia. Healthier men, based on clinical categories, had significantly more normal shaped spermatozoa and fewer had azoospermia, oligoasthenoteratozoospermia or leukocytospermia. Many HIV-seropositive men have normal semen analyses, but as the disease progresses more defects are found, particularly in strict criteria sperm morphology.

Problem: Sera from infertile patients with elevated reactivity against normozoospermic seminal plasma (NSP) have been selected to investigate human antibody binding to seminal fluid antigens present in abnormal ejaculates., Method: Sera from 32 idiopathically infertile patients and 44 control sera from fertile individuals were examined by ELISA against: 1) pooled seminal plasma from asthenozoospermic ejaculates (AsthSP), 2) pooled seminal plasma from patients with aspermatogenic azoospermia (AzooSP), and 3) chromatographic fractions from NSP, AsthSP and AzooSP., Results: Of 32 patients positive for anti-NSP antibodies, only four exhibited increased reactivity to whole AsthSP and/or AzooSP, while 14 recognized antigens of different Mr and various distributions in the corresponding chromatographic fractions., Conclusion: Targets of human anti-NSP antibodies might be lacking, less concentrated, and/or modified in AsthSP and AzooSP. These findings suggest their physiological importance and the possible relevance of the observed auto- and iso-immune responses to infertility.

Human semen contains large amounts of opioid peptides and cytokines. We have measured the concentrations of interleukin (IL)-6 in 140 semen samples and of beta-endorphin in 77 semen samples. The median concentration of beta-endorphin in seminal plasma from normozoospermic men (n = 23) was 154.7 pg/ml (10th-90th percentiles, 42.0-774.6), and there was no significant difference in the beta-endorphin concentration among normozoospermic, oligozoospermic (n = 28), asthenozoospermic (n = 15), azoospermic (n = 4) and post-vasectomy (n = 7) samples. There was no correlation between beta-endorphin concentration and sperm characteristics, nor with blood hormones. beta-Endorphin concentration was lower in cases with immunological infertility, as revealed by a positive direct mixed antiglobulin reaction test (n = 12) (P < 0.01), than in matched controls. The median concentration of IL-6 in samples with normal sperm concentration, motility and morphology with or without white blood cells (n = 39) was 26.1 pg/ml (10th-90th percentiles, 7.3-172.3), and there was no significant difference in the IL-6 concentration among normozoospermic, oligozoospermic (n = 46), asthenozoospermic (n = 32), azoospermic (n = 13) and post-vasectomy (n = 10) samples. The IL-6 concentration was significantly higher in cases of varicocele (n = 22) without white blood cells in semen (P < 0.001) than in matched controls without varicocele (n = 23). In addition, the IL-6 concentration was elevated (P < 0.0001) in cases with accessory sex gland inflammation (n = 40). IL-6 concentration was positively correlated with white blood cells in semen (n = 60, r = 0.59, P < 0.0001), but there was no correlation with beta-endorphin concentration. The IL-6 concentration chosen to differentiate between cases with and without accessory gland inflammation was 45.3 pg/ml, with a specificity of 80.6% and a sensitivity of 92.5%. It is concluded that beta-endorphin in seminal plasma plays an immune suppressive role, and that increased IL-6 concentration may be related to testicular dysfunction in cases with varicocele. Furthermore, IL-6 is an accurate marker of accessory sex gland inflammation.

Azoospermia is the cause of infertility in 8% of infertile male patients. Ten percent of those patients suffer from agenesis of the seminal vesicle (SV) and vas deferens (VD) agenesis. Currently, the diagnosis of SV and VD agenesis is based on low semen volume, low pH, and low fructose content of the seminal fluid of azoospermic men who have normal serum gonadotropins. In this study, an SV-specific sperm-coating antigen, the MHS-5 antigen, was used as a marker for the presence of SVs. The SV-specific protein (SVSP), MHS-5, was present in the control group but was not found in any of the seven samples from azoospermic men with proven agenesis of SV and VD. Another semen component, the prostate-specific antigen (PSA), whose presence in the semen is not influenced by the SV and VD agenesis, was found in both the study and the control groups. Its presence ruled out the possibility of azoospermia due to ejaculatory duct obstruction. The absence of MHS-5 antigen in seminal fluid can be used as a tool for a reliable diagnosis of agenesis of SV and VD in azoospermic men.

An enzyme immunoassay for detecting antisperm antibodies directly on sperm was compared to immunobeads in a prospective cohort design. In a series of 112 divided ejaculates evaluated for assisted reproductive technologies, there was moderate overall agreement (kappa = .64) between the methods. All ejaculates positive by immunobeads were also positive by enzyme immunoassay. The enzyme immunoassay could be performed even on oligospermic specimens. When both assays could be employed, the enzyme immunoassay was positive more often (P = .001). Positive specimens by enzyme immunoassay often bound IgA-class antibodies alone, without IgG. Enzyme immunoassay results may represent false-positive results or a new, lower threshold for antibody detection.

To investigate whether cell-mediated immunity (CMI) against sperm and/or antisperm circulating antibodies are associated with poor semen quality, a leukocyte migration inhibition factor (LMIF) assay and an enzyme-linked immunosorbent assay (ELISA) were performed in groups of men from infertile couples, men from fertile couples and sperm donors. Twenty-five of 102 men (25%) revealed positive CMI against sperm and 10 (10%) had positive antisperm antibody titers in their sera. Fifteen of 28 asthenozoospermic men (53%) from infertile couples revealed positive antisperm CMI. The incidence of antisperm CMI was significantly increased (P < 0.05) in the infertile men with asthenozoospermia compared with the men from the other two groups (men from fertile couples and sperm donors). No significant differences between migration indices were seen when such a comparison was done for oligoasthenoterato- and teratozoospermics. The results indicate that increased antisperm CMI is associated with asthenozoospermia in a significant number of men from infertile couples. The importance of these findings is discussed.

The incidence and the clinical relevance of sperm-reactive antibodies in subfertile men and in testicular cancer patients were assessed in a pilot study. The sera of 42 men with normogonadotropic oligoasthenoteratozoospermia (OAT syndrome, n = 20) or carcinoma of the testis after inguinal semicastration (n = 22) were analyzed for agglutinating antisperm antibodies using fluorescein-labeled antiglobulin. In the group with the OAT syndrome, the incidence of sperm-reactive antibodies was only 5%, which is comparable to that in normal fertile men. Although the incidence of 18% in the testicular cancer patients was markedly higher, only 2 of the patients in question had abnormal spermiograms, which in one case could, moreover, be explained by previous radiation therapy. In summary in this small group of patients, serum monitoring for sperm-reactive antibodies appeared to be of limited clinical relevance in patients with the OAT syndrome and in testicular cancer patients.

We have previously shown that active immunization against GnRH in the mature male rat can predictably produce hypogonadotropic hypogonadism and azoospermia and, further, that normospermia and normal fertility can be restored by testosterone (T) administration alone. The objective of this study was to explore the hypothesis that GnRH-immunized azoospermic rats could be supplemented with T doses sufficient to restore sexual behavior, but insufficient to support adequate spermatogenesis or to allow restoration of fertility. Adult male rats of proven fertility were immunized against GnRH and supplemented with 2-, 4-, or 8-cm T implants or with empty implants. Eight weeks later, fertility was evaluated; concentrations of serum T, LH, FSH, growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) were determined; sperm number was obtained from the testis; and weights of androgen-dependent organs were measured. GnRH immunization and T supplementation resulted in restoration of organ weights and of fertility in a dose-dependent manner. GnRH immunization with or without T supplementation resulted in the absence of circulating gonadotropins, but had no effect on serum GH, PRL, or TSH levels. Whereas all control animals were fertile, rats that received either empty or 2-cm T implants were completely infertile. Rats that received 4-cm or 8-cm T implants were fertile in 60% and 100% of cases, respectively. Sexual behavior of rats with empty and with 2-cm T implants was compared at 10-18 wk after immunization with GnRH. GnRH-immunized rats given empty implants displayed negligible sexual activity, but those with 2-cm T implants displayed sexual activity equivalent to that of untreated controls despite complete infertility.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: To determine the effects of leukocytes in semen on sperm quality and the ability to achieve conception., Design: A prospective analysis of 512 couples attending a regional infertility clinic. Leukocyte subsets were quantified using a monoclonal antibody-based staining procedure. In addition to basic seminal parameters (density, motility, morphology, and antisperm antibodies), reactive oxygen species and immature germ cells were also quantified in the semen of each patient. The presence or absence of a treatment-independent conception was determined 22 months after the start of the study. Semen parameters were then related to the ability to conceive., Setting: University-based center for reproductive medicine., Participants: Success or failure to conceive was recorded from 512 couples. Couples were then selected to minimize the influence of any pathology of the female on outcome. A final study group of 229 couples, in which the women had regular menstrual cycles, ovulatory midluteal serum P levels of > 18 nmol/L, and patent fallopian tubes was finally selected for analysis., Main Outcome Measure: Pregnancy., Results: Leukocyte concentration (total or individual subsets) was not associated with either reduced semen quality or conception rates. Similarly, neither reactive oxygen species or antisperm antibody (immunobead) concentration had any bearing on the outcome. Of all semen parameters measured, only the level of immature germ cells was found to be negatively associated with the rate of conception., Conclusion: Measurement of seminal leukocytes in routine semen analysis appears to be of little prognostic value with regard to male fertilizing potential. As reactive oxygen species and antisperm measurement were of similar predictive value, the term "immunologic male infertility" should be redefined.

An artificial spermatocoele was constructed at the level of the caput epididymis in a patient with obstructive azoospermia. Morphologically normal spermatozoa were harvested but motility and progression were inadequate for intra-uterine insemination or in vitro fertilisation. In addition, there was an antispermatozoal antibody response which transuded back into the reproductive tract and coated spermatozoa with antibody. It is recommended that if harvesting of epididymal spermatozoa is contemplated, the serum should initially be assessed for spermatozoal antibody.

Problem: ELISA is an ideal assay method for a large-scale screening of anti-sperm antibodies among a large number of infertile males. However, conventional ELISA with whole spermatozoa needs time-consuming steps of centrifugation., Method: A solid-phase assay used for detecting anti-sperm antibodies was established. This assay is suitable not only for detecting circulating anti-sperm antibodies of IgG, IgM, and IgA subclass simultaneously but also for screening hybridomas secreting anti-sperm monoclonal antibodies (mAbs). The microtiter plates, on which solubilized sperm antigens are fixed, can be stored at -80 degrees C for up to six months without losing reactivity with anti-sperm antibodies., Results: Using this assay, 53 sera (13 were proven positive and 40 were proven negative for sperm agglutination antibody) were tested. Although the false-negative rate was 0%, the false-positive rate was 32%. One thousand one hundred sixty-five supernatants from hybridomas constructed with splenocytes of mice who were hyperimmunized with human sperm and nonsecreting myeloma cells were tested by this solid-phase assay and two anti-sperm mAb secreting clones were selected and established., Conclusions: It is recommended that for research work this assay could be used for the first screening of the hybridoma secreting anti-sperm mAb, and for clinical use this assay might be suitable for the first screening of sera of infertile patients. However, conventional bioassays should follow to confirm the biological meaning of the positivity.

Involvement of the cytokines in male infertility were considered from in vitro studies, however the nature and the role of the mediator susceptible of arising in vivo is not completely elucidated. The purpose of this study is to measure the concentrations of interleukin-2 (IL-2) in normal and pathologic seminal fluid and to analyse their relationship with semen parameters. Our results demonstrate the presence of IL-2 in seminal fluid. Comparatively to normal seminal fluid, significant lower IL-2 concentrations were observed in infected semen samples or with sperm antibodies. No correlation was found between the cytokines values and the sperm parameters.

Localized Hodgkin's disease (HD) is curable in the great majority of patients. However, common programs of chemotherapy for this disease render most men permanently azoospermic. In studies of seminal cryopreservation prior to treatment, it has been recognized that many men with HD are dyspermic at diagnosis. It is hypothesized that this abnormality reflects a change in the cellular regulation of spermatogenesis; specifically an alteration in the balance between distinct sub-populations of lymphocytes which normally either inhibit or stimulate the production of sperm. This change in regulation within the testes is believed to be part of a systemic perturbation which is unrelated to the extent of HD but is potentially reversible with effective treatment of the primary disease. Recognizing the distinction between sperm analyses and fertility status, it is proposed that radiotherapy of localized HD, delivered in a manner that is not toxic to the male gonad, will restore useful spermatogenesis in patients who are dyspermic (and probably subfertile) before treatment is initiated.

Study Objective: To determine the extent to which increasing doses of exogenous testosterone (T) administered via Silastic implants can restore spermatogenesis and fertility to rats made azoospermic by active immunization against gonadotropin-releasing hormone (GnRH)., Design: Male rats were made azoospermic by active immunization against GnRH. Increasing doses of exogenously administered T (via Silastic implants) were administered for 8 weeks, and testicular sperm concentration and ability to impregnate female rats were evaluated., Setting: Reproductive Endocrinology Laboratory, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado., Animals: Sexually mature male Sprague Dawley rats (SASCO, Omaha, NE)., Results: Suppression of gonadotropins and azoospermia was achieved by actively immunizing rats against GnRH. Testosterone was capable of restoring quantitatively complete spermatogenesis and fertility in GnRH-immunized azoospermic rats. This relationship was dose-dependent, as evidenced by the partial restoration of spermatogenesis and fertility observed in animals replaced with smaller T Silastic implants., Conclusion: Gonadotropin-releasing hormone immunization and T-filled Silastic implants may provide a model to study isolated gonadotropin deficiency and for the development of a reversible male contraceptive.

Associations between sterility in varicocele and HLA antigens have been studied. The results show increased incidence of antigens B13 and B22 in sterile males with varicocele. Specific distribution of HLA antigens, their relations with varicocele were characterized. The findings can be of use in identification of risk groups among varicocele patients.

Antisperm antibodies and their relationship with infertility has been investigated in semen and serum samples of 100 infertile and 25 fertile males. The investigation of antisperm antibodies has been done by latex agglutination test in semen samples and by ELISA procedure in serum samples. In addition, the semen samples have been investigation for several characteristics. Antisperm antibodies have been found 7% positive in blood and 30% positive in semen. In 28.5% of the patients with positive antisperm antibodies in blood, antisperm antibodies in semen have also been found to be positive. For cases with infertility of unknown cause the antisperm antibodies ratio has been found to be significantly high (p less than 0.002). These results have shown the importance of further investigation of antisperm antibodies in cases with infertility of unknown reasons.

Germ cells isolated from semen of oligospermic human donors were found to react with GDA-J/F7 monoclonal antibody (MoAb). Their reactions with this antibody were demonstrated by using fluorescein activated cell sorter (FACS) analysis and indirect immunofluorescence (IIF) test. In the IIF test, the MoAb recognised an antigen on the surface of the sperm cell precursors (SpP) as well as on mature spermatozoa. The specificity of the antibody reaction with the SpP was further confirmed by immunoelectron microscopy. The MoAb did not react with peripheral blood lymphocytes or polymorphonuclear cells but did show cross-reactivity with monocytes. This antibody therefore provides the first marker for the SpP and could be used as a probe for their distinction from leucocytes. This could have clinical application in seminal analysis.

On the basis of elevated levels of seminal plasma immunoglobulins and a decreased production of LMI in the presence of PPD, 6 patients (out of the idiopathic normogonadotrophic oligospermic group) were selected for a therapeutic trial with levamisole. The following regime of treatment was applied: 3 days on levamisole (150 mg per day) followed by a 10 day interval without treatment, for a period of three months. The sperm count increased in 5 out of 6 patients and motility increased in 2. At the same time a reduction in seminal plasma IgG levels was observed in all treated patients. A significant increase of EA rosettes was observed after treatment.

Nucleated cells other than sperm (NCOS) were isolated from human semen by centrifugation on a Ficoll density gradient. Using tissue-specific monoclonal antibodies (mAb) greater than 99% of the NCOS were found to be sperm cell precursors (SpP). These cells were tested for the expression of class I and II (DR, DP and DQ) HLA antigens by using specific mAb. The anti-HLA class I and II and anti-beta 2-microglobulin mAb reacted with less than 1% of the NCOS. This was demonstrated by indirect immunofluorescence microscopy and fluorescence-activated cell sorter analysis. These results were similar to those obtained from testing germ cells in frozen sections of normal adult testis using the same panel of mAb. In mixed lymphocyte-NCOS cultures, the SpP failed to stimulate allogeneic lymphocytes even when different concentrations of cells were used. These results indicate little or no expression of HLA class I and II including the HLA-D (T cell-defined) determinant on the SpP, a phenomenon which could be of biological importance.

An immunocytochemical method was used to label the kinetochores on human synaptonemal complexes. Synaptonemal complex spreads were labelled with autoimmune CREST serum, followed by a second antibody labelled with colloidal gold, and examined by electron microscopy. Clusters of gold particles were found at discrete sites which were identified as kinetochores on the autosomal synaptonemal complexes, as well as on the XY pair. This method was used to investigate the extent of pairing of the human X and Y chromosomes at pachytene. Our observations confirm earlier work, based purely on measurements, that the pairing of the sex chromosomes sometimes extends beyond the centromere of the Y chromosome into the long arm. At the same time we showed that the centromeric indices of the X and Y at pachytene are highly variable, so that measurements alone are not sufficient to estimate the degree of pairing of the sex chromosomes.

Biopsy specimens of human testes from 190 men of infertile couples were treated with fluorescein-labelled antibodies to demonstrate tissue-bound IgG, IgA, IgM, IgD, C3, and C4. In 24 cases (12.6%) autoantibodies and/or complement could be detected. Most often IgG bound to walls of seminiferous tubules was found. Three men had antibodies or complement in germinal cells. In one case each IgA immunofluorescence was found in spermatids or Leydig cells. The results suggest that autoantibodies to testicular antigens may be implicated in the pathogenesis of some cases of infertility in man.

To study the diagnostic value of various serological tests in the detection of serum sperm antibodies a group of 50 patients with azoospermia was studied. Cases were divided by testicular biopsy into: 32 obstructive and 16 non obstructive cases. Tests applied included a microagglutination and a macroagglutination test as well as 2 complement dependent tests; a sperm cytotoxic and a sperm immobilization tests. Applied separately the macroagglutination was the most sensitive test but used together, more cases were detected. Because of this and the different value and significance of each test, it is advisable to use more than one test in the investigations of cases of male infertility.

The levels of complement components (C1q, C4, C3 and C3d) were measured in the sera of nine azoospermic Nigerian men and compared with those of 12 oligospermic and 19 normospermic Nigerian men. The mean values of C1q, C4, C3 and C3d for azoospermic men showed no significant difference from those obtained for the other men. Generally, the individual sera from the azoospermic men did not show abnormally lowered levels of any of the complement components when compared with values obtained for the normospermic group.

A notable feature of the male gametes is the presence in them of proteins that are "foreign" to the immune system of both male and the female. It is there that are considered responsible for the elicitation of auto- and iso-antibodies in certain natural infertility cases. By virtue of their dual application in both sexes the sperm antigens have interesting potential for exploration as possible agents for control of fertility.

We measured immunoreactive epidermal growth factor (EGF) by a homologous RIA in seminal plasma (SP) from 31 fertile and 52 infertile men to determine the relationship between SP EGF levels and total sperm count in the ejaculates. The mean SP EGF levels in fertile and infertile men were 41.7 +/- 21.5 (+/- SD) and 53.1 +/- 30.8 micrograms/L, respectively. Infertile men with sperm-associated immunoglobulin G (n = 9), immunoglobulin A (n = 6), or both (n = 8) had mean SP EGF levels of 48.9 +/- 26.1, 47.9 +/- 17.5, and 56.5 +/- 32.1 micrograms/L, respectively. Seven men with severe oligospermia had a mean SP EGF level of 58.5 +/- 35.9 micrograms/L. There was no correlation (r = 0.14; P greater than 0.05) between SP EGF levels and total sperm counts in these men. Fractionation of SP by high performance liquid chromatography on a size exclusion (TSK G2000 SW) column revealed a single immunoreactive peak with an approximate mol wt of 8000, slightly higher than the mol wt of circulating human EGF (6000). We conclude that SP EGF may be distinct from peripheral plasma EGF.

The outcome of 584 AID pregnancies was analysed in order to examine the possible role of immune mechanisms in the development of pre-eclampsia (PE). The overall incidence of PE was high (9.3%) compared with the expected incidence of 0.5-5.0%. The incidence was similar in both multigravid and primigravid women. The expected protective effect of a previous pregnancy was not seen, with a 47-fold increase in PE (observed versus expected) in AID pregnancies after a previous full-term pregnancy and a 15-fold increase after a previous pregnancy of short duration. In the context of AID pregnancies these findings tend to support the proposed 'fresh mating' concept of PE although the incidence is lower in multigravidas after a full-term (4.7%) than after a partial pregnancy (15.7%). This suggests that it may only be full-term pregnancy which protects against the development of PE. THe category and duration of infertility and the duration of marriage did not affect the incidence of PE except where the male partner was oligozoospermic. In this case the incidence of PE was higher where infertility was 3 years or longer. Further analysis of the findings failed to provide clear evidence to discriminate the role of HLA and tissue-specific non-HLA antigens in the feto-maternal relationship as determinants of the development of PE. Neither did the results support the operation of immunological enhancement induced by prior coital antigen exposure in the genesis of PE, but raised instead the possibility of the operation of a mechanism of sensitisation of the female partner to seminal components over a long period of time when the husband was oligozoospermic compared with one whose husband was azoospermic.

Modern knowledge of the HLA system structure and its role in the organism supports the statement that the presence of HLA antigens in a person increases his/her predisposition to some diseases. Association of HLA antigens with some diseases has already been confirmed by many authors. This study was an attempt to obtain insight into the possibilities of the association of HLA antigens with azoospermia and oligospermia of an unknown aetiology. HLA groups were allocated to 71 men with azoospermia and oligospermia. The condition of disordered spermiogenesis was confirmed on the basis of anamnestic data, objective genital examination and spermiogram, by which secondary causes were excluded and unknown aetiology was confirmed. To standardize HLA antigens the authors used 14 sera for locus A antigens and 26 sera for locus B antigens. The results obtained were compared with findings obtained from 670 healthy persons (controls). These results showed higher frequency of the following antigens in these persons: A26 in 15.4% of cases with r.r. 9.2, P less than 0.001 and chi 2 = 37.74 (control group = 1.9%), A28 in 23.9% of cases with r.r. 2.8, P less than 0.005 and chi 2 = 8.23 (control group = 10%) and B18 in 18.3% of cases with r.r. 2.5, P less than 0.005 and chi 2 = 8.23 (control group = 8%).

In 168 azoospermic males with normal or only slightly raised serum FSH levels, serum antisperm antibodies were measured, and the site of obstruction or the nature of the failure of spermatogenesis was defined by exploratory scrototomy with inspection of epididymes, vasography and testicular biopsy. When possible, surgical reconstruction was done by side-to-side epididymovasostomy, with vasovasostomy when necessary using 6/0 Prolene and no splints. Acquired blocks of cauda epididymis (34 cases) and vas (23 cases) were significantly more commonly associated with serum antisperm antibodies than congenital bilateral absence of vasa (29 cases) or blocks at the caput epididymis (48 cases), most of which were associated with sinusitis, bronchitis or bronchiectasis (Young's syndrome). Many of the former patients came from abroad, whereas most of the latter came from the British Isles. Sperm counts of 10 million per ml or more were produced by 23 (45%) of 51 adequately followed up patients with acquired blocks following surgical reconstruction, and 11 pregnancies (21.5%) were produced. Amongst those with spermatozoa in the ejaculate after surgery, serum antisperm antibodies were found significantly less often in those whose wives became pregnant compared with those who failed to produce pregnancies. It is concluded that failure of surgical treatment in some of these cases may have an immunological basis. No success was achieved with other groups.