Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality.

BACKGROUND: We showed that in men with a constitutional chromosomal abnormality, DNA fragmentation was significantly higher in chromosomally unbalanced spermatozoa than in spermatozoa with a normal or balanced chromosomal content. These results could be explained by a phenomenon already described in infertile men: abortive apoptosis. OBJECTIVES: To determine whether magnetic-activated cell separation could select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men carrying a structural chromosomal abnormality. MATERIALS AND METHODS: The spermatozoa of ten males with a chromosomal rearrangement were separated into two populations using magnetic-activated cell separation (annexin V (-) and annexin V (+) fractions), in order to study meiotic segregation by fluorescence in situ hybridization, the percentage of spermatozoa with an externalization of phosphatidylserine by annexin V staining and DNA fragmentation by TdT-mediated dUTP nick-end labeling on the whole ejaculate and on selected spermatozoa in the same patient. RESULTS: For all patients, the percentage of spermatozoa with externalization of phosphatidylserine decreased in the annexin V (-) fraction and increased in the annexin V (+) fraction as compared to the frozen-thawed semen sample. The rates of DNA fragmentation were statistically much lower in the annexin V (-) fraction when compared to the rate before magnetic-activated cell separation for all but one patient. Conversely, we observed a statistically significantly higher rate of DNA fragmentation in the annexin V (+) fraction for six patients. After magnetic-activated cell separation, there was a significant increase of normal/balanced spermatozoa in the fraction of annexin V (-) for all patients. Conversely, we observed a significant decrease in the fraction of annexin V (+) for seven patients. DISCUSSION AND CONCLUSIONS: Magnetic-activated cell separation is a promising tool for increasing the selection of healthy spermatozoa, with a decrease in the number of spermatozoa with externalization of phosphatidylserine, DNA fragmentation, and chromosome unbalance, for use in assisted reproductive technologies such as intracytoplasmic sperm injection for males with a chromosomal structural abnormality.

In vitro assessment of the adverse effects of antiretroviral drugs on the human male gamete.

This study was designed to investigate the in vitro effects of didanosine, zidovudine, saquinavir and indinavir, commonly used in highly active antiretroviral therapy, on human sperm fertility parameters. Thirty semen samples from healthy men were collected and prepared by gradient density method. Aliquots of 90% fractions with >80% motile spermatozoa were incubated for 1, 3, and 6h with different concentrations of the antiretroviral drugs (20, 40, and 80 µg/ml). Sperm motility was evaluated by computer assisted sperm analysis system. Sperm mitochondrial potential was evaluated using 3,3'-dihexyloxacarbocyanine iodide (DIOC(6)) and the acrosome reaction was examined using pisum sativum agglutinin method. A dose-dependent decrease in sperm motility was observed with saquinavir. Saquinavir also induced a significant time and dose-dependent decrease in mitochondrial potential and an increase in spontaneous acrosome reaction. These findings indicate that, in vitro, higher doses of saquinavir have adverse effects on sperm motility, mitochondrial potential and acrosome reaction.

Incoming human cytomegalovirus pp65 (UL83) contained in apoptotic infected fibroblasts is cross-presented to CD8(+) T cells by dendritic cells.

Human cytomegalovirus (HCMV) infection is well controlled mainly by cytotoxic CD8(+) T lymphocytes (CTL) directed against the matrix protein pp65 despite the numerous immune escape mechanisms developed by the virus. Dendritic cells (DCs) are key antigen-presenting cells for the generation of an immune response which have the capacity to acquire antigens via endocytosis of apoptotic cells and thus present peptides to major histocompatibility complex class I-restricted T cells. We examined whether this mechanism could contribute to the activation of anti-pp65 CTL. In this study, we show that infection by HCMV AD169 induced sensitization of MRC5 fibroblasts to tumor necrosis factor alpha-mediated apoptosis very early after virus inoculation and that pp65 contained in apoptotic cells came from the delivery of the matrix protein into the cell. We observed that immature DCs derived from peripheral monocytes were not permissive to HCMV AD169 infection but were able to internalize pp65-positive apoptotic infected MRC5 cells. We then demonstrated that following exposure to these apoptotic bodies, DCs could activate HLA-A2- or HLA-B35-restricted anti-pp65 CTL, suggesting that they acquired and processed properly fibroblast-derived pp65. Together, our data suggest that cross-presentation of incoming pp65 contained in apoptotic cells may provide a quick and efficient way to prime anti-HCMV CD8(+) T cells.