Effect of intravesical immunotherapy on sperm parameters in young patients with non--muscle-invasive bladder carcinoma: prospective analysis.

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.

The role of cytokine expression in different subgroups of subfertile men.

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.

Seminal plasma--immunomodulatory potential in men with normal and abnormal sperm count.

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.

High androgen receptor immunoexpression in human "Sertoli cell only" testis.

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.

Morphometrical quantification of spermatogonial germ cells with the 57B anti-MAGE-A4 antibody in the evaluation of testicular biopsies for azoospermia.

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.

Susceptibility gene for non-obstructive azoospermia located near HLA-DR and -DQ loci in the HLA class II region.

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.

Serial ultrasonography, hormonal profile and antisperm antibody response after testicular sperm aspiration.

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.

Ultrasound, antisperm antibody, and hormone profiles after testicular Trucut biopsy.

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.

Involvement of serum and lipopolysaccharide in the production of interleukin-1- and interleukin-6-like molecules by human sperm cells.

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.

Secondary treatment failure without anti-human chorionic gonadotropin antibody in a patient with Kallmann syndrome.

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.

Evaluation of beta-endorphin and interleukin-6 in seminal plasma of patients with certain andrological diseases.

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.

The use of a seminal vesicle specific protein (MHS-5 antigen) for diagnosis of agenesis of vas deferens and seminal vesicles in azoospermic men.

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.

Incidence of antisperm antibodies in patients with carcinoma of the testis and in subfertile men with normogonadotropic oligoasthenoteratozoospermia.

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.

Prospective study of leukocytes and leukocyte subpopulations in semen suggests they are not a cause of male infertility.

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.

Maintenance of sexual function with testosterone in the gonadotropin-releasing hormone-immunized hypogonadotropic infertile male rat.

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)

A solid-phase assay for the detection of anti-sperm antibodies.

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.

Dyspermia in men with localized Hodgkin's disease. A potentially reversible, immune-mediated disorder.

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.

Immunoneutralization of gonadotropin-releasing hormone and subsequent treatment with testosterone Silastic implants in rats: an approach toward developing a male contraceptive.

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.

Quantitative restoration of advanced spermatogenic cells in adult male rats made azoospermic by active immunization against luteinizing hormone or gonadotropin-releasing hormone.

The ability of testosterone to quantitatively restore spermatogenesis in rats made azoospermic by active immunization against LH or GnRH was examined. Sexually mature adult male rats (n = 15/group) were actively immunized against ovine LH or GnRH-human serum globulin conjugate, while control rats (n = 10) were injected with saline. After 10 weeks of immunization, five rats per group were euthanized. For each rat, trunk blood was collected for determination of LH, FSH, and testosterone by RIA; seminiferous tubule fluid (STF) was collected from one testis per rat, and testosterone concentration was measured by RIA; the number of advanced spermatids per testis was determined from the contralateral testis. The results obtained after 10 weeks of treatment were as follows. 1) Serum LH and FSH were undetectable by RIA in GnRH-immunized rats. 2) Serum testosterone was undetectable in both the LH- and GnRH-immunized groups. 3) The testosterone concentration in STF (STF-T) was reduced from the control value of about 64 ng/ml to about 2 ng/ml in the LH- and GnRH-immunized rats. 4) LH- and GnRH-immunized rats were azoospermic. After the initial 10-week treatment period, five rats in each of the LH- and GnRH-immunized groups received 24-cm testosterone-filled polydimethylsiloxane (PDS-T) capsules (3 x 8 cm long) sc. The remaining immunized rats (n = 5/group) received empty capsules. Two months later, all rats were euthanized. Testis weights, serum testosterone, and STF-T concentrations remained significantly reduced in LH- and GnRH-immunized rats that did not receive testosterone supplementation, and the rats remained azoospermic. STF-T concentrations rose significantly (P less than 0.05) in the LH- and GnRH-immunized rats that received PDS-T, but were still significantly less (by approximately 80%) than the concentration in intact controls. Nonetheless, implantation of PDS-T caused restoration of advanced spermatogenic cells in the testes of both LH- and GnRH-immunized rats to numbers that were not significantly different from the number in controls. These data indicate that 1) testosterone is capable of quantitatively restoring spermatogenesis in rats actively immunized against LH or GnRH, suggesting that FSH may not be required for the restoration of spermatogenesis in adult rats; and 2) quantitatively complete restoration of spermatogenesis can occur at STF-T concentrations that are significantly reduced compared to those in intact controls.

Analysis of HLA antigens on germ cells in human semen.

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.