Protective effect of the immunomodulator AS101 against cyclophosphamide-induced testicular damage in mice.

BACKGROUND: Cyclophosphamide (Cy), a widely used anticancer drug, is associated with significant testicular damage and sterility. Co-administration of the immunomodulating compound AS101 during chemotherapy treatments was previously shown to protect organs against cytotoxic damage, without attenuating the drug's anticancer effect. In this animal study, we investigated the effect of AS101 on testicular damage, sperm DNA damage and infertility induced by Cy. Akt and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation were investigated as a possible chemoprotective mechanism. METHODS: Mature male mice, 10 in each group, were injected intraperitoneally with 200 mg/kg Cy once a week for 5 weeks, with or without concurrent treatment with 10 microg per mouse AS101 three times per week. Damage to testicular tubules and sperm production was determined, sperm chromatin damage was analyzed and fertility was gauged. Akt and GSK-3beta phosphorylation were evaluated. RESULTS: Co-treatment with AS101 during the course of Cy administration significantly reduced the percentage of damaged seminiferous tubules (76.0 +/- 10.8% versus 40.3 +/- 2.6%), and reduced sperm DNA fragmentation (%DFI) from 44.7 +/- 1.0% to 25 +/- 6.5%. Co-treatment with AS101 also partially protected against the decrease in numbers of impregnated females and litter size. AS101 increased Akt and GSK-3beta phosphorylation. CONCLUSIONS: Our results indicate that AS101 can significantly protect against Cy-induced testicular damage and sperm DNA damage, probably by acting through Akt/GSK-3beta phosphorylation.

Rat testis mitochondrial adenylate cyclase. Partial purification and characterization.

1. Adenylate cyclase (EC 4.6.1.1) from rat testis mitochondria has been solubilized by treatment with the non-ionic detergent Lubrol PX. The soluble enzyme was further purified by DEAE-cellulose chromatography. 2. The specific activity of the adenylate cyclase eluted from the DEAE-cellulose column was found to be four times higher than that of an intact mitochondrial preparation. At this step the enzyme shows a sedimentation coefficient of 4.2 S and a diffusion coefficient (D) of 3.12 - 10- minus 7 cm-2/sec. 3. Solubilization of the adenylate cyclase resulted in loss of responsiveness to gonadotrophic hormones. Addition of phosphatidylserine to the soluble preparation partially restored the activation of adenylate cyclase by human chorionic gonadotrophin. 4. The results of this study suggest that the activity of the adenylate cyclase may be dependent on the membrane-bound phospholipids and that the enzyme attached to the mitochondrial membranes has some properties which are similar to the adenylate cyclase found to be associated with other membrane systems of the cell.?

Influence of visible light and ultraviolet irradiation on motility and fertility of mammalian and fish sperm.

OBJECTIVE: The effects of visible light irradiation on sperm motility, fertility, and reactive oxygen species (ROS) formation were investigated and compared in ram and fish (tilapia). BACKGROUND DATA: Low-energy visible light has previously been found to modulate various processes in different biological systems. In the literature, it is accepted that the first step following visible light irradiation is the formation of ROS by endogenous cellular photosensitizers. METHODS: Sperm of ram and tilapia were irradiated with various light sources (400-800 nm white light, 660 nm red light, 360 nm blue light, 294 nm UV), and their motility and fertility rates were measured. The amount of ROS generated by irradiation was estimated using electron paramagnetic resonance (EPR) technique. RESULTS: Sperm taken from tilapia showed higher motility and fertility following red and white light irradiation. In contrast, the motility and fertility of ram sperm were slightly increased only by red light. A negative effect on motility and fertility of sperm of both species was obtained following irradiation with UV and blue light. The amount of ROS produced in irradiated tilapia sperm was much higher than that of ram sperm. CONCLUSIONS: The results show that different wavelengths differentially affect tilapia and ram sperm motility and fertilization. The difference in response to the various light sources might be explained by the different amounts of ROS formation by ram and tilapia, which are in agreement with the physiology of fertilization appropriate to each of these species. Based on these results, it is suggested that in vitro fertilization in mammals should be performed in darkness or at least under red light.

Ultramorphological sperm characteristics in the risk assessment of health effects after radiation exposure among salvage workers in Chernobyl.

We present a pilot study of individuals (liquidators) who were engaged in clean-up operations after the disaster at the nuclear power plant at Chernobyl in Ukraine. In the 10 years since the disaster, adverse health effects among exposed individuals have not been clearly defined. There is widespread fear of damage to the reproductive system, with implications for fertility problems and adverse effects on offspring. Bearing this in mind, methods to evaluate the potential for production of fertile semen have been applied using quantitative ultramorphological (QUM) analysis. QUM analysis examines the organization and integrity of sperm organelles by electron microscopy, using both transmission electron microscopy and scanning electron microscopy. Significant differences were observed between clean-up workers and controls of similar age regarding certain ultramorphological parameters of the sperm head. The results of this pilot study suggest that QUM analysis of human sperm is a feasible approach for evaluating the fertility potential of individuals who were exposed to ionizing radiation from the Chernobyl nuclear power plant accident.

Reduced semen quality caused by chronic abacterial prostatitis: an enigma or reality?

OBJECTIVE: To investigate whether there is an inter-relationship between chronic abacterial prostatitis and potential infertility. DESIGN: As part of the eligibility studies for hyperthermia treatment for chronic abacterial prostatitis patients, a large number of chronic prostatitis patients were referred to us from peripheral outpatient clinics. Sperm analysis was a routine portion of the eligibility studies. To exclude bacterial prostatitis, urine cultures, expressed prostatic secretion, and semen cultures were performed. The patient population was not differentiated on the basis of those suffering from either nonbacterial prostatitis or prostatodynia according to the commonly accepted classification. The control group was the laboratory normal standard group. SETTING: Normal human volunteers in an academic and clinical research environment. PATIENTS: The first group includes 86 patients suffering from long-standing (1 to 20 years) chronic abacterial prostatitis, according to the commonly accepted classification, with a mean age of 39.9 +/- 9.5 years. The second group includes 101 normal fertile men with a mean age of 31.4 +/- 5.5 years. INTERVENTIONS: The routine semen analysis performed included biochemical tests of seminal plasma, bacteriology, and light microscopy. MAIN OUTCOME MEASURE: The original hypothesis was based on a reduction in semen quality in these patients caused by chronic abacterial prostatitis. Measurements for sperm motility parameters, morphology characteristics, prostate markers, and white blood cells (WBC) were designed accordingly. RESULTS: Statistical comparisons of the two groups showed that several sperm motility parameters, morphology characteristics, prostate markers, and WBC are outside of the normal value ranges in the chronic abacterial prostatitis group. In addition, there is a correlation between the duration of the disease and two important sperm analysis variables: increased prostatic markers and appearance of sperm morphological defects. CONCLUSION: From the results obtained, the high incidence of secondary infertility in these patients may be explained.

Sperm motility index: a new parameter for human sperm evaluation.

OBJECTIVE: The study was performed to evaluate the correlation between sperm motility index, a novel parameter of semen quality, and routine semen analysis parameters by microscopic evaluation. DESIGN: Sperm motility index was measured by an electro-optical device, the Sperm Quality Analyzer (United Medical Systems Inc., Santa Ana, CA). Human semen samples covering the whole span of qualities were analyzed prospectively and simultaneously by both methods. SETTING: Samples were collected from patients referred to university hospital infertility clinics. PATIENTS, PARTICIPANTS: Nine hundred sixty-eight semen samples of 812 patients and healthy men were analyzed. MAIN OUTCOME MEASURE(S): Sperm motility index is a measurement of optical density fluctuations caused by motile cells; therefore, a positive correlation was anticipated between its values and semen motility parameters. RESULTS: Sperm motility index values demonstrated statistically significant correlation with motile cell concentration, total cell concentration, and percent motile cells. They were also shown to reliably represent semen quality assessment obtained by two experienced andrologists. CONCLUSIONS: The sperm motility index provides a reliable and objective reflection of semen motility parameters and quality.

The relationship between sperm ultrastructural features and fertilizing capacity in vitro.

OBJECTIVE: To evaluate the relationship between ultramorphological features of the human sperm and its fertilizing capacity in vitro. DESIGN: The study was performed retrospectively. Ultrastructural features were assessed using scanning and transmission electron microscopes in sperm samples of individuals who underwent an in vitro fertilization (IVF) treatment cycle no more than 6 months before the study. SETTING: Institutional clinical care. PATIENTS: Fifty-six infertile couples in whom mechanical infertility was diagnosed in the female partner. Patients were categorized as fertilizing when fertilization of at least 30% of the oocytes occurred (n = 27) and nonfertilizing when none of the oocytes fertilized in at least two consecutive IVF treatment cycles (n = 29). RESULTS: The two groups differed significantly only in ultramorphological parameters of the sperm head and acrosome (head, F(8,36) = 2.8, P less than 0.02; acrosome, F(4,40) = 2.8, P less than 0.04), and especially in the following malformation patterns: hyperelongated head, acrosome deficiency, and acrosome damage. The suggested score based on these findings was able to predict 90% and 76% of the cases with and without fertilizing potential, respectively. CONCLUSION: The ultrastructural morphology of the sperm head components is a key parameter for assessing the sperm fertilizing capacity in vitro.

The correlation between sperm cell morphology and fertilization after zona pellucida slitting in subfertile males.

OBJECTIVE: This work was undertaken to evaluate the correlation between sperm cell morphology and fertilization after zona pellucida slitting in subfertile males. DESIGN: Twenty-two couples who failed at least one in vitro fertilization attempt because of lack of oocytes fertilization underwent a zona-slitting micromanipulative procedure. A total of 245 oocytes were retrieved and inseminated by three different modes: 151 oocytes underwent micromanipulation, 2 were damaged, and the remaining 149 inseminated by the husband's sperm (group A). Fifty-five oocytes were not manipulated and inseminated by the husband's sperm (group B), and 39 oocytes were not manipulated and inseminated by a donor sperm (group C). RESULTS: Fertilization rates were 26.8%, 5.5%, and 53.8% in groups A, B, and C, respectively, and differed significantly between group A and group B. The cleavage rates were lower for oocytes fertilized by the husband's sperm (48.6%) than that obtained by donor (90%), suggesting a sperm factor contributing to this phenomenon. The procedure was most efficient in patients with a total motile sperm count after preparation of greater than or equal to 5 million and with either normal sperm morphology or defects localized to the acrosome or tail region only. Sperm with nuclear morphological abnormalities demonstrated a marked reduction in fertilization potential. CONCLUSION: It is concluded that the zona-slitting technique enhances fertilization of severely subfertile sperm, and its efficacy is affected by sperm morphology and a threshold concentration of motile cells.

Sperm quality of subfertile males before and after treatment with human follicle-stimulating hormone.

OBJECTIVE: To assess the potential of short-term systemic administration of FSH for improving sperm quality, including ultrastructure, in teratozoospermic patients having normal endocrine profiles. DESIGN: Semen parameters were assessed prospectively using light microscopy (LM), biochemical analysis, and quantitative ultramorphological analyses within 2 months before FSH administration and within 5 days after the end of treatment. SETTING: Samples were collected from patients who were referred to the male fertility clinic at Bar-Ilan University. PATIENTS: Thirty-one patients with teratozoospermia who exhibited normal hormonal profiles and who failed to fertilize their wives in at least two previous IVF attempts (n = 17) or who had wives with apparent normal fertility unable to conceive for > or = 5 years (n = 14) were classified as subfertile. One hundred one males with no previous history of infertility, whose wives conceived after < or = 12 months of pregnancy expectation, served as the control group. INTERVENTION: Treatment was 75 IU FSH administered daily for 30 days. MAIN OUTCOME MEASURES: Pretreatment and post-treatment sperm evaluation of basic and quantitative ultramorphological analyses parameters. The hypothesis was FSH treatment may improve spermatid morphogenesis by its multiple actions on the Sertoli-gamete cell compartment without interfering with the testicular hormonogenic function. RESULTS: A significant improvement in agenesis of the acrosome and in the amorphous heads was observed, reaching normal values after treatment with FSH. The axonema deteriorated. No significant changes were observed in basic semen analysis parameters. CONCLUSIONS: Because malformations of the fine structure of the sperm head subcellular organelles seem to be prerequisites for the success of FSH treatment, ultramorphological examination of the sperm may serve as an indication for the probability of success of this treatment.

Follicle-stimulating hormone treatment for men with idiopathic oligoteratoasthenozoospermia before in vitro fertilization: the impact on sperm microstructure and fertilization potential.

OBJECTIVE: To assess the effect of FSH on sperm fertilization potential and sperm intracellular structure in men with oligoteratoasthenozoospermia and a proven low fertilization rate in IVF. DESIGN: Prospective, randomized, partial crossover study. SETTING: IVF Unit, Golda Campus, Rabin Medical Center, Petah Tikva, Israel. PATIENT(S): Forty normogonadotropic, normogonadal men with oligoteratoasthenozoospermia and at least one previous IVF attempt in which fertilization failed or the fertilization rate was <30%. INTERVENTION(S): The men were randomly assigned to treatment with daily injections of 75 IU of FSH or 150 IU of FSH for at least 60 days before IVF treatment. A control group of men underwent an IVF cycle without treatment and then were randomly assigned tojoin group 1A or 1B for an additional IVF cycle with treatment. MAIN OUTCOME MEASURE(S): LH, FSH, and testosterone levels during FSH treatment, evaluation of ultramorphologic changes in sperm by electron microscopy, and comparison of fertilization rates in the control and study groups. RESULT(S): After treatment with 75 IU or 150 IU of FSH, the mean fertilization rates were 19.7% and 20.5%, respectively, compared with a 5.8% fertilization rate in the study control cycles. CONCLUSION(S): Prolonged treatment with FSH results in a significant increase in fertilization rates. This effect may be related to improvements in subcellular components of the sperm.

Ultrastructural changes in sperm with improved fertilizability after treatment with egg yolk.

OBJECTIVES: To determine the existence of differences in the ultrastructural parameters between the medium-washed sperm samples of the affected and nonaffected egg yolk (EY) groups and to verify whether ultrastructural changes occur in the EY-affected spermatozoa after EY preincubation. SETTING: The study was performed in the Laboratory of the IVF Unit, Serlin Maternity Hospital, and the Laboratory of Male Fertility, Bar-Ilan University, Ramat Gan, Israel. PATIENTS: The positive group included 12 males who underwent 1.9 IVF cycles with 0% fertilization rate that increased to 68% after EY treatment. The negative group included 11 males with 1.2% fertilization rate in 1.1 IVF cycles with no improvement after preincubation of spermatozoa in EY. RESULTS: Compared with the laboratory standard, patients of both groups exhibited a lower normalcy of the head sperm cell subcellular organelles. With EY treatment, the positive group exhibited a decrease in the frequency of some sperm head organelle specific malformations. CONCLUSIONS: Fertilization capacity of mature spermatozoa might be reduced because of an excess of acrosome malformations, postacrosomal lamina, and chromatin caused by in vitro sperm manipulations. The manipulation effect may be avoided by EY treatment.

The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program.

OBJECTIVE: To study the possible influence of antioxidant treatment on human spermatozoa and the fertilization rate in an IVF program. DESIGN: Prospective study. SETTING: In Vitro Fertilization Unit, Serlin Maternity Hospital, and the Laboratory of Male Fertility, Bar-Ilan University, Ramat-Gan, Israel. PATIENTS: Fifteen fertile normospermic male volunteers who had low fertilization rates in their previous IVF cycles. INTERVENTIONS: Vitamin E (alpha-tocopherol) 200 mg daily by mouth for 3 months. MAIN OUTCOME MEASURES: Lipid peroxidation potential (amount of malondialdehyde [MDA]), quantitative ultramorphologic analysis of spermatozoa, and fertilization rate per cycle. RESULTS: The high MDA levels significantly decreased from 12.6 +/- 9.4 nmol/10(8) spermatozoa to normal levels of 7.8 +/- 4.2 nmol/10(8) spermatozoa after 1 month of treatment. The fertilization rate per cycle increased significantly from 19.3 +/- 23.3 to 29.1 +/- 22.2 after 1 month of treatment. No additional effects on MDA levels and fertilization rate were observed after completion of treatment. With regard to the quantitative ultramorphologic analysis, none of the sperm cell subcellular organelles were affected significantly by vitamin E treatment. CONCLUSION: Vitamin E may improve the fertilization rate of fertile normospermic males with low fertilization rates after 1 month of treatment, possibly by reducing the lipid peroxidation potential, and with no change of the quantitative ultramorphologic analysis of subcellular organelles.

The influence of in vitro caffeine treatment on human sperm morphology and fertilizing capacity.

After publication in the literature that in vitro caffeine treatment causes damage of the normal shape of the sperm head and thereby decreases fertilizing capacity, we carried out a clinical and electron microscopic study to determine the influence of caffeine on the fertilizing capacity and sperm cell morphology. Sixty women (with infertile husbands) underwent artificial insemination by donor with frozen/thawed semen over a period of 12 months, using randomized addition of caffeine in alternate months. Fourteen women became pregnant during the 6 months they received caffeine-treated semen, whereas only 7 pregnancies occurred during the 6 months the women received semen without caffeine. Scanning electron microscopic examinations of fresh proven donor semen showed no morphologic changes caused by the in vitro caffeine treatment. However, quantitative morphologic analysis of the frozen/thawed semen was unsatisfactory because of the freezing technique and the masking effect of the protective medium. It is concluded that in vitro caffeine treatment of fertile donor semen does not damage the spermatozoa; furthermore, it seems to improve the fertilizing capacity.

Dual energy metabolism-dependent effect of Ureaplasma urealyticum infection on sperm activity.

Genital Ureaplasma urealyticum infection is considered a sexually transmitted infection. It has long been debated whether the presence of U. urealyticum in semen may be a possible cause of infertility. Long-term incubation (4 hours or overnight) of sperm cells with U. urealyticum in vitro resulted in a significant inhibition of sperm motility and membrane alteration whereas a short incubation (45 minutes) of sperm cells with ureaplasmas resulted in an acceleration of sperm velocity. The aim of this study was to understand these contradictory reports of U. urealyticum infection on sperm motility. Spermatozoa from fresh ejaculates of normozoospermic semen of men who were referred to the university Male Fertility Laboratory for semen analysis, with no history of genital tract infection, and from normal Assaf breed rams were infected in vitro with U. urealyticum serotype 8, at different pHs and O2 concentrations. Sperm viability and motility and changes in extracellular pH were evaluated. A significant (16%-43%) increase in sperm activity was observed upon infection at alkaline pH (7.8) under aerobic or hypoxic conditions, and a 58% increase was observed under anaerobic conditions and pH 7.2. When the infection was conducted under aerobic conditions and acidic pH (6.3), or under hypoxic conditions at neutral pH (7.2), an 8%-25% inhibition of sperm activity was observed. These results indicate that when sperm activity depends on mitochondrial oxidative phosphorylation, usually at low pHs, U. urealyticum competes with mitochondrial energy production and therefore reduces sperm motility and viability. However, when sperm energy metabolism depends on glycolysis, usually at higher pHs, U. urealyticum stimulates glycolysis and sperm activity.

Sperm Motility Analyzer (SMA), a practical tool of motility and cell concentration determinations in artificial insemination centers.

A description of a new instrument, which analyzed objectively sperm cell concentration and collective motility of a fresh semen sample of bull or ram, is reported. A display read-out of sperm cell concentration for an aliquot of the undiluted semen sample in the range from 200 x 10(6) to 4000 x 10(6) cell/ml with an accuracy of +/- 5% is shown immediately by optical density units. A significant correlation coefficient (0.918, p < 0.01) was obtained between these units and cell concentrations, as determined in four A.I. centers in the U.S. and Canada. The intensity of the sperm collective motility is expressed as an objective index (SMI) which was significantly correlated to the fraction of the live motile cells in the semen sample. The SMI values were corrected for a possible error originating from the difference in sperm cell concentration by an empirical equation. Due to the accuracy, repeatability and objectivity of evaluating the semen quality and also the simplicity of operation, as well as the possibility of utilizing the measured semen sample for further processing, the Sperm Motility Analyzer (SMA) seems suitable as a standard tool for semen quality control in the A.I. centers.

Lincomycin and spectinomycin in the treatment of breeding rams with semen contaminated with ureaplasmas.

Ureaplasma species were isolated from semen samples collected sequentially from one Awassi and three Assaf breeding rams. Each ram was injected subcutaneously with an aqueous solution of lincomycin and spectinomycin for five consecutive days at a dose equivalent to 4.5 mg kg-1 lincomycin and 9.0 mg kg-1 spectinomycin daily. Serum and semen samples were collected at intervals during the treatment and assayed for lincomycin. No Ureaplasma species were isolated from semen samples collected during the course of the treatment and at intervals for 17 days after the last treatment. The concentration of lincomycin in semen ranged from 0.51 microgram ml-1 four hours after treatment to 0.08 microgram ml-1 24 hours after treatment, and these levels were three to nine times higher than the corresponding serum concentrations.

[Ultramorphological changes in sperm cells of infertile men with testicular varicocele].

The ultramorphological characteristics of sperm cells of infertile men with testicular varicocele were evaluated. There was an increased frequency of abnormal changes within their spermatozoa as compared to fertile men. The abnormalities included agenesis, partial agenesis, malformation and degradation of the subcellular organelles of the heads and tails of the sperm cells. These findings suggest the use of these characteristics as a part of a "varicocele score" to determine those who would benefit from varicocelectomy in the treatment of their infertility.

[Severity of testicular varicocele and sperm cell ultramorphologic characteristics].

Ultramorphologic characteristics of sperm cells found in infertile men with varicoceles were compared with the severity of the varicocele. Classification of varicoceles into mild, moderate or marked was determined by spermatic vein diameter and degree of blood reflux. These were evaluated by high resolution duplex sonography. There were no statistical differences between the 3 grades of varicocele severity with regard to abnormalities in the subcellular organelles of the acrosome, karyoplasm, and skeleton of spermatozoal heads, or in the axoneme, outer dense fiber and skeleton of sperm cell tails. The only abnormal ultramorphologic features found to be significant among the 3 groups were differences in the frequencies of karyoplasm agenesis, and of partial agenesis, malformation and degeneration of the acrosome in sperm cell heads. However, these features could not be related to grade of severity and therefore, no ultramorphologic severity pattern could be detected. Thus, no varicocele beneficial score based on grade of severity and sperm cell ultramorphology can be suggested for clinical use.

[Seminiferous tubule cytological pattern in infertile, azoospermic men in diagnosis and therapy].

We determined spermatogenic patterns of seminiferous tubules in azoospermic infertile men and evaluated the prevalence of bilateral testicular homogeneity. 185 azoospermic men underwent bilateral testicular fine-needle aspiration (TFNA) in which each testis was punctured at 3 different positions. Aspirated material was stained and classified according to the most mature spermatogenic cell type present or whether only Sertoli cells were present. 35.7% had spermatozoa in their testes, 36.2% had spermatogenic maturation arrest, and 28.1% had only Sertoli cells in their seminiferous tubules. In 15.6% of all patients, the diagnosis in 1 testis differed from that in the other. In only 73.2% of those with testicular spermatozoa was it bilateral. In the remaining 26.9%, only Sertoli cells, spermatocytes or spermatids were found as the most mature cell type in the other testis. The study definitely indicates that fertilization with retrieved testicular spermatozoa should not be offered to azoospermic patients without prior evaluation of the seminiferous tubuespermatogenic pattern in both testes.