جداسازي اسپرمهاي انساني حامل كروموزوم X و Y به وسيله شيب غلظت Puresperm و ارزيابي آن به روش هيبريد كردن در جا با استفاده از مواد فلورسنت (FISH)

Evaluation of Discontinue Puresperm Gradient in Separation of X and Y bearing Human Spermatozoa by Fluorescent In Situ Hybridization (FISH)

Introduction: To evaluate the 8 layer discontinue 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, a control group which was washed with phosphate bufer saline and another one for processed through 8- step discontinue puresperm gradient 35%-84% (7% differences between each subsequent layers). After centrifugation of sperm from top and bottom fraction was aspirated and fixed on slide. Then FISH procedure was performed using X and Y specific probes. DNA probes are bind to alpha satellite complementary sequence from 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 the presence of red or green florescent signals. Results: Statistical analysis by chi-square test revealed no significant difference in the X:Y probration in the 84% layer compared to the controls. But significant difference was observed in the 35% layer in compared to 84% layer and the controls (P=).001, P=0.006 respectively), and X bearing spermatozoa was enriched in bottom layer. Conclusion: The result of this study showed that 8-step discontinue Puresperm gradient is not so reliable for separation of X and Y bearings spermatozoa.