Morphological Analyses and Apoptosis Genes Expression Evaluation in Vitrified Human Ovarian Tissue after Warming, Long Term Culturing and Xenotransplantation

Headnote

Keywords: Vitrification, Apoptosis Gene Expression, Transplantation, Long Term Culture, Human Ovarian Tissue

Background: In vitro culturing and retransplantion of vitrified-warmed ovarian tissue are two ways to restore fertility after radiation or chemotherapy.This study aimed to evaluate the incidence of apoptosis in vitrified human ovarian tissue after warming, long term culturing and xenotransplantation by morphological analyses and apoptosis genes expression evaluation.

Materials and Methods: We obtained human ovarian tissue biopsies from 30 women who underwent elective caesarean sections. Tissues were transported to the laboratory, then were cut into small pieces and were divided into two groups, vitrified and non-vitrified. Apoptosis incidence was assessed by light microscope and apoptosis related gene expression in both groups after warming, after 14 days in vitro culture and 30 days after xenotransplantation to γ-irradiated mice.

Results: We observed no morphological differences between nonvitrified and vitrified samples before and after culturing. But vitrified grafted tissues showed significantly less normal follicles than grafted-non-vitrified group (P<0.05).The expression of some pro and anti-apoptotic genes in vitrified-warmed tissues were not changed compared to non-vitrified ones but the expression of Fas and caspase8 was increased and the expression of BRIC5 was decreased in this group (P<0.05). In transplanted vitrified group the Bcl2, FasL and BRIC5 gene expression was high and caspase8 was low (P<0.05). The expression of all genes in both grafted groups was more than non-grafted tissues and lower than cultured groups except for caspase8 and BIRC5 (P<0.05).

Conclusion: This study provides the first evidence for the effects of vitrification on apoptosis in non-cultured, cultured and grafted human ovarian tissue at mRNA level. We concluded that vitrification could induce apoptosis incidence during long term cultivation and we should improve vitrification protocols and culture conditions to achieve favorite result.