TEM and FISH studies in sperm from men of couples with recurrent pregnancy loss.

The role of the male partner in recurrent pregnancy loss (RPL) is not clear. In this study, semen characteristics of 22 men whose partners had experienced RPL were examined by light microscopy. Sperm morphology was analysed by transmission electron microscopy (TEM) and the data were mathematically elaborated to obtain numerical indices expressing the status of an ejaculate: the fertility index and the percentage of apoptosis, necrosis and immaturity. Sperm apoptosis and necrosis were also evaluated by annexin V/propidium iodide assay. To explore the status of meiotic segregation, fluorescence in situ hybridisation (FISH) with probes for chromosomes 18, X and Y, was applied directly on sperm nuclei. Sperm characteristics from a group of men of proven fertility were used as controls. Among the considered sperm characteristics, apoptosis (P < 0.01), 1818YY diploidy (P < 0.05) and 18YY disomy (P < 0.01) scores were significantly higher in men with RPL compared with controls. Our study showed that some patients with normal semen parameters can present a slight increase in aneuploidy compared with controls, indicating a possible involvement of sperm in some cases of RPL. Chromosomal FISH analysis and chromatin tests of sperm could be included in RPL work-ups when no other cause has been detected.

Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C.

Little is known about the effect of chronic hepatitis B and hepatitis C on sperm quality. In this study, we analysed sperm quality from selected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. Semen samples were examined by light and transmission electron microscopy (TEM). TEM data were elaborated with a mathematical formula able to indicate a fertility index and the presence of the three main sperm pathologies: apoptosis, immaturity and necrosis. Meiotic chromosome segregation was investigated by fluorescence in situ hybridisation carried out on sperm nuclei, using probes for chromosomes 18, X and Y. Despite normal sperm concentration, we observed reduced motility. TEM analysis highlighted that 35.7% of patients showed generally good semen quality. However, significantly higher values of apoptosis and necrosis, compared with controls, were observed, demonstrating spermatogenetic alterations. Regarding meiotic segregation, we found an incidence of disomies similar to that observed in control samples, whereas diploidy resulted higher in HCV patients, without reaching statistical significance. In conclusion, sperm quality in the studied group was not impaired; however, apoptosis and necrosis resulted out of normal range and the fertility index was significantly lower in HCV- and HBV-infected patients versus controls.

Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C.

Little is known about the effect of chronic hepatitis B and hepatitis C on sperm quality. In this study we analysed sperm quality from selected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. Semen samples were examined by light and transmission electron microscopy (TEM). TEM data were elaborated with a mathematical formula able to indicate a fertility index and the presence of the three main sperm pathologies: apoptosis, immaturity and necrosis. Meiotic chromosome segregation was investigated by fluorescence in situ hybridisation carried out on sperm nuclei, using probes for chromosomes 18, X and Y. Despite normal sperm concentration, we observed reduced motility. TEM analysis highlighted that 35.7% of patients showed generally good semen quality. However, significantly higher values of apoptosis and necrosis, compared with controls, were observed, demonstrating spermatogenetic alterations. Regarding meiotic segregation, we found an incidence of disomies similar to that observed in control samples, whereas diploidy resulted higher in HCV patients, without reaching statistical significance. In conclusion, sperm quality in the studied group was not impaired, however, apoptosis and necrosis resulted out of normal range and the fertility index was significantly lower in HCV and HBV infected patients versus controls.

Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells.

Resveratrol is a phytoalexin with antioxidant properties. We evaluated resveratrol toxicity in swim-up selected human sperm and in rat spermatocytes and spermatids separated by the STAPUT method. Resveratrol antioxidant activity was tested against lipid peroxidation (LPO) induced by tert-butyl hydroperoxide. LPO was evaluated using the C11-BODIPY(581/591) probe and transmission electron microscopy in samples incubated with and without resveratrol. LD50 for human sperm and rat spermatids was 50 µM; spermatocytes were more sensitive to resveratrol cytotoxicity. Sperm motility increased progressively at 30 µM, 15 µM and 6 µM. 15 µM resveratrol acts against LPO, preserving sperm chromatin and plasma membranes. LPO were more marked in spermatocytes than in spermatids and the effect of resveratrol was more evident in spermatocytes. In this study, the scavenger properties of resveratrol were demonstrated in vitro in human sperm and rat germ cells, thus resveratrol could be added to the media used in assisted reproduction techniques and cryopreservation when oxidative stress is exacerbated.

Retrospective assessment of potential negative synergistic effects of varicocele and tobacco use on ultrastructural sperm morphology.

OBJECTIVES: To investigate, in a retrospective study, whether smoking cigarettes increases the effect of varicocele on sperm morphology. METHODS: The semen quality of 2 groups of patients with varicocele were compared, those who smoked (n = 121) and those who did not (n = 158). The semen parameters were evaluated, and sperm morphology was assessed using transmission electron microscopy and quantitatively elaborated (fertility index, immaturity, necrosis, and apoptosis percentages). RESULTS: In the smoker and nonsmoker varicocele-associated cases, sperm motility and the results from transmission electron microscopy analysis were significantly impaired compared with controls. However, a nonsignificant difference was detected when the semen parameters were compared. Subsequently, we divided the patients into 4 groups: mild (> or = 1 but < or = 10 cigarettes/d), moderate (>10 but <20 cigarettes/d), and heavy (> or = 20 cigarettes/d) smokers and a group of randomly chosen nonsmoker patients with varicocele. The sperm motility, sperm concentration, and fertility index decreased and the percentage of sperm pathologic features increased as the number of cigarettes smoked daily increased. CONCLUSIONS: A detrimental effect of cigarette smoking (>10 cigarettes/d) associated with varicocele on sperm motility and morphology was observed. Because much of reduced fecundity associated with smoking may be reversed within 1 year of cessation, as reported in published studies, effective interventions targeted at helping patients quit smoking should be addressed for the benefit of general health and fertility.