RESUMEN
This study aimed to evaluate the incidence of apoptosis in vitrified and non-vitrified human ovarian tissue by the use of morphological analysis and apoptosis assay techniques. We obtained human ovarian tissue biopsies from 30 women who underwent elective caesarean sections. Tissues were transported to the laboratory in pre-warmed, equilibrated Leibovitz L-15 medium within 2 hours. The tissues were cut into small pieces and divided into two groups, vitrified and non-vitrified [control]. Apoptosis incidence was assessed by light microscope and the TUNEL assay and DNA laddering. Evaluation of caspase 3/7 protein levels was performed by the luminescent assay. We observed no morphological signs of apoptosis in the vitrified samples. There were no apoptosis signals as evidenced by TUNEL staining and no DNA laddering pattern observed in the vitrified group. Caspase 3/7 activity was 2294 +/- 169.19 RLU/microg protein in the non-vitrified control group and 2231 +/- 89.271 RLU/microg protein in the vitrified group, which was not significantly different. The structure of human vitrified ovarian tissue was well preserved. Vitrification could not increase apoptosis and caspase 3/7 activity in human ovarian tissue
RESUMEN
In this study, we evaluated the incidence of apoptosis at the ultrastructural levels and expression of some apoptosis-related genes in vitrified human ovarian tissue just after warming. Human ovarian tissue biopsies from 23 women after caesarean section were transported to the laboratory within 2 hours, and then they were cut into small pieces. Some pieces were vitrified and warmed and the other samples were considered as control. Apoptosis was assessed by a transmission electron microscope and also by molecular analysis of pro-apoptotic [Fas, FasL, Bax, p53, caspase8, and caspase3] and antiapoptotic [Bcl-2 and BIRC5] genem RNA levels using real-time RT-PCR before and after vitrification. No sign of apoptosis was shown ultrastructurally in vitrified samples. The level of FasL, Bcl-2, Bax, p53, and caspase3 mRNA and Bax:Bcl-2 ratio were similar in non-vitrified and vitrified groups; however, the expression of Fas and caspase8 genes was higher and BIRC5 was lower in vitrified samples compared to non-vitrified group [P<0.05]. The fine structure of human vitrified ovarian tissue was well preserved; moreover, vitrification was shown to affect the expression of some apoptosis-related genes. However, additional study is needed to confirm this observation