Evaluation of Discontinues Puresperm Gradient in Separation of X And Y Bearing Human Spermatozoa by Fluorescent in Situ Hybridization (FISH)

Introduction: To evaluate the 8 layer discontinues puresperm gradient in separation of human spermatozoa according to sex chromosomes by fluorescent in situ hybridization (FISH). Material and Methods: This study was carried out on three patient referring to Royan fertility and infertility center. Semen analysis was assessed according to WHO criteria. Each sample was divided into two aliquots, one as a control was washed with phosphate bufer saline and another one for processing through 8- step discontinues puresperm gradient 35%-84% (7% differences between each subsequent layers). After centrifugation, sperm from top and bottom fraction was aspirated and fixed on slide, FISH procedure was performad by using X and Y specific probes. DNA probes bind to alpha satellite complementary sequence on centromeric region of X and Y chromosomes. More than 1000 sperm were scored for each sample. The proportion of X and Y bearing sperm chromosomes was determined by presence of red or green florescent signals. Results: Statistical analysis by chi-square test revealed no significant differences in the X:Y probration in the 84% layer compared to the controls. But there were significantly differences in the 35% layer compared to 84% layers and the controls (P=0.001, P=0.006 respectively), and X bearing spermatozoa was enriched in bottom layer. Conclusion: The result of this study showed that 8-step discontinues Puresperm gradient is not so reliable for separating X and Y bearings spermatozoa.