ABSTRACT

Intracytoplasmic sperm injection (ICSI) allows overcoming the natural barrier offered by the oocyte to sperm fertilization in couples with severe male factor infertility. Sperm fluorescence in situ hybridization (FISH) uses fluorescence DNA probes directed to specific DNA sequences in the interphase sperm nuclei. Visualizing the hybridization signals using fluorescence microscopy, it is possible to identify numerical chromosome abnormalities in the nucleus of ejaculated, epididymal, and testicular sperm. Sperm FISH analyses corroborate previous reports with altered meiosis in infertile men, as they have shown higher aneuploidy rates for chromosome 21 and sex chromosomes due to meiotic nondisjunction. Sperm heads have a tightly compacted nucleus due to the presence of disulfide bridges between protamines; this condensation of nuclear chromatin makes it inaccessible to DNA probes. Double-strand DNA denaturation of the sperm and FISH probes is carried out after incubation at high temperature. The hybridization signals are visualized using a fluorescent microscope equipped with specific filters for each fluorochrome.