Assessment of Estrogen Receptors and Apoptotic Factors in Cryopreserved Human Ovarian Cortex

The aims of this study were: (i) to examine frozen-thawed ovarian tissues for features of follicular health and atresia by histology; 60 to assess the expression of estrogen receptors alpha (ER alpha) and beta (ER beta) by real-time PCR; (iii) to evaluate the Bax/Bcl-2 ratio, as an apoptotic index, in the ovarian tissues before and after cryopreservation. Ovarian cortical biopsies were obtained from 11 patients. The fragments were subdivided into two groups, fresh (control tissues) and cryopreserved tissues obtained by direct plunging into liquid nitrogen. Both tissue groups were subjected to a histological evaluation of the healthy and atretic follicles, immunohistochemical localization of the ER, and a real-time PCR (qPCR) to evaluate the expression of ER, Bax, Bcl-2 as well as beta-actin, as control gene. Damage was observed in 31% of primordial, 45% of primary, and 75% of secondary follicles in the cryopreserved tissue group. The qPCR analysis showed that the level of ER beta was greater in fresh than cryopreserved tissues, whereas the ER alpha expression and Bax/Bcl-2 ratio were similar in both tissue groups. A significant inverse association was observed between ER alpha mRNA levels in the fresh tissue group and subjects' ages. The results show that cryopreservation and thawing of human ovarian tissue does not affect the morphology of primordial or primary follicles and that cryopreservation does not affect apoptosis. However, cryopreservation seems to have an inhibitory effect on the level of ER beta. Additional studies are needed to evaluate the differential effects of freezing follicles at different stages of follicular development and ovarian steroidogenesis.