Enrichment of Human Spermatogonial Cells by Cul-ture of Testicular Cell Suspension in Obstructive Azoosper-mic Patients

Background: 50 million of men are infertile, and notably, azo-ospermia comprises 25% of male infertility cases. Therefore, it is of great interest to generate functional gametes for patients with male infertility especially for azoospermia. Spermatogen-esis is a complex process regulated by multiple interactions between developing germ cells and the surrounding microenvi-ronment. Production of sperm in vitro would not only provide male gametes for azoospermic patients but also offers various methods for the in vitro derivation of male germ cells have been developed. So, in the present study, we investigated different methods to proliferation of SSCs.Materials and Methods: Human testicular samples were ob-tained from men with obstructive azoospermia. after enzymatic digestion process, cells assigned to following groups: culture of SSCs in the dish without cover (control group), co-culture of SSCs with infertile sertoli cells (I), co-culture of SSCs with fertile sertoli cells (II), culture of SSCs on nanofiber (covered with laminin) (III), culture of testicular cell suspension (IV). Then cells were cultured for two weeks in 34-StemPro medium and evaluated colony formation of human spermatogonial cells and gene specific methylation and quantitative genes expres-sion of pluripotency (Nanog, C-Myc, Oct-4) and specific germ cell (Integrin α6, Integrin β1, PLZF) genes in five different cul-ture systems.Results: We found that the highest number and diameter of colonies and cellular proliferation associated with IV group which were significantly different with control group and other groups while it was fewest in control group, III, II, I groups respectively. Expression of germ specific genes in IV group were significantly increased (P≤0.05) and levels of expression of pluripotency genes were significantly decreased in this group (P≤0.05) compared with other groups. Gene specific methyla-tion pattern of examined genes did not show any changes dur-ing culture period in culture systems.Conclusion: Our findings indicate that testicular cell suspen-sion can reconstruct a microenvironment capable of regulating proliferation of cell colonies.

کلیدواژه ها:

Spermatogonial Stem Cells (SSCs) ، Colonization ، Obstructive Azoospermia ، Culture Systems