Apoptosis in cryopreserved ovarian tissue—a tool of evaluation cryopreservation utility

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Objective: Ovarian tissue cryopreservation (OTCP) is an emerging technology, offered for young cancer patients prior to chemotherapy. The optimal techniqe of OCTP is not determined yet. It has been demonstrated that 40–50% of preserved tissue is damaged following freezing and thawing. The aim of this study was to examine the utility of OCTP by investigating apoptosis of follicles in frozen thawed ovarian tissue. Design: Prospective study. Materials and Methods: Ovarian tissue samples were obtained from 6 women with cancer who underwent cryopreservation of ovarian tissue prior to chemotherapy. Ovarian cortex samples measuring 2mm X 5 mm were removed by laparoscopy. The study was approved by the institutional ethical committee and the women signed an inform consent. One tissue sample was evaluated for apoptosis immediately following removal and served as a control. A second tissue sample was frozen, thawed and then evaluated for apoptosis. Light microscopy using hematoxylin and eosin (H&E)-stained paraffin slides and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) methods were used to confirm the incidence of apoptosis. The number of apoptotic follicles is scored on micrograph of at least 4 random sections stained by TUNEL. Follicles were defined apoptotic if they contain at least 6 cell nuclei per section, which are positive to the TUNEL reaction. Results: Apoptosis was demonstrated in 70±6 % of follicles in frozen-thawed ovarian samples compared to 25±12% in the control samples. The same results were obtained with H&E. Conclusion: Higher incidence of apoptosis has been demonstrated in ovarian follicles in frozen-thawed ovarian tissue compared to unfrozen ovarian tissue. This findings reflect a severe tissue damage, which occurs during the freezing thawing process. The investigation of tissue apoptosis may be used as a tool in evaluating the utility of various freezing-thawing prtocols.