Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: A tool for evaluating cryopreservation utility

Fertility preservation is of major importance for women with cancer in whom ovarian function may be disturbed by the use of potentially sterilizing chemotherapeutic drugs and/or pelvic irradiation. Cryopreservation of ovarian cortical tissue is one of the potential options for preserving fertility among these women. Cryopreserved thawed human ovarian tissue can be autografted either orthotopically or heterotopically, but may also be transplanted first into an animal host with subsequent maturation and collection of oocytes. The objective of this study was to investigate the prevalence of ovarian follicular apoptosis in fresh and frozen/ thawed human ovarian tissue as a measure of follicular viability. The study group included 6 women with cancer who underwent ovarian tissue cryopreservation (OTCP). Ovarian tissue samples (n=2) were obtained from each woman with one sample undergoing evaluation for apoptosis immediately following removal (control, group A) and the other evaluated for apoptosis following freezing/thawing (group B). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and 4'6' diamido-2-phenylindole hydrochloride (DAPI) staining methods were used to investigate follicular apoptosis. Morphological changes in the same samples were evaluated in hematoxylin and eosin (H&E)-stained sections. In each slide, only primordial and primary follicles were evaluated for abnormal morphology and apoptosis. Abnormal morphology was demonstrated in 23.8±8.7% of group A follicles compared to 48.3±11.2% of group B follicles (p<0.05). Apoptosis was demonstrated in 25.4±8.4% of group A follicles compared to 60.9±6.0% of group B follicles (p<0.05). We have shown that the ovarian follicles in group B demonstrated a higher incidence of apoptosis compared to those of group A. Therefore, the data suggest that follicular apoptosis might be a consequence of the freezing and thawing procedure. This may be used as a method for evaluating and comparing the outcome of different freezing/thawing protocols.