Vitrification using dimethyl sulfoxide free cryoprotectant as a cryopreservation method for fertility preservation of immature mouse testicular tissue.

e20672 Background: Loss of fertility is a major problem for childhood cancer survivors treated with gonadotoxic therapy. Cryopreservation of testicular tissue is a promising approach to preserve fertility for prepubertal boys, as sperm banking cannot be considered for these patients. Slow freezing (SF) and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration for clinical application. Methods: Fragments of imamature testicular tissue from 6-day-old mice were assigned to one of the following cryopreservation procedures: SF using dimethyl sulfoxide (DMSO) as cryoprotectants, SF using DMSO free cryoprotectant, vitrification using DMSO, vitrification using DMSO free cryoprotectant. Histological and immunohistochemical analyses were performed to evaluate cell viability, intratubular proliferation (Ki-67), apoptosis (caspase-3) after freezing and thawing. Thawed testicular tissue were cultured for in vitro production of mature sperm using previously described organ culture method. The fertility of induced spermatozoa was tested by intracytoplasmic sperm injection (ICSI). Results: Seminiferous tubules showed good integrity after cryopreservation and thawing in SF with DMSO group and vitrification with DMSO free cryoprotectant group. In these groups, cell viability and their ability to proliferate were observed by immunohistochemistry, and mature spermatozoa were induced after 35 days of organ culture. ICSI showed fertility of induced spermatozoa only in vitrification using DMSO free cryoprotectant group. Conclusions: An optimal cryopreservation protocol enhances the chances for successful fertility restoration.The findings of the present study have potential implications for cryobanking of immature testicular tissue and fertility preservation. Vitrification using DMSO free cryoprotectant would be a preferable cryopreservation method for later induction of functional spermatozoa.