Analysis of Apoptosis in Cultured Human Vitrified Ovarian Tissue in the Presence of Leukemia Inhibitory Factor

For improving the human ovarian tissue culture, this study was designed to assess the incidence of apoptosis in this tissue following vitrification and in vitro culture in the presence of leukemia inhibitory factor (LIF) as an anti-apoptotic factor. After collecting the ovarian tissue samples they were divided into non-vitrified and vitrified groups and cultured for 14 days in the presence and absence of LIF then morphological, ultrastructural and steroidogenesis studies, TUNEL and caspase-3/7 assays, and apoptosis analysis by real time RT-PCR were done in all groups. The data were analyzed by independent t-tests and the real time RT-PCR results were compared by one-way ANOVA (p-values of <0.05 were considered significant). No significant difference was observed between non-vitrified and vitrified groups in normality rate of follicles, the levels of hormones, TUNEL positive cells and caspase-3/7 activity. But in all LIF-treated groups, the levels of 17-β estradiol and progesterone were higher and TUNEL signals and caspase-3/7 activity were lower than non-LIF treated groups. The expression of Fas and FasL genes was higher in vitrified group in comparison with non-vitrified group but the expression of other genes was not significantly different. In LIF- treated groups, the expression of pro-apoptotic genes was significantly lower and the expression of anti-apoptotic genes was higher than non-LIF treated group. The vitrification of human ovarian tissue did not increase the incidence of apoptosis at the morphological and molecular levels during long term culture and LIF improves the survival and development of cultured follicles.