Can frozen-thawed human ovary withstand refreezing-rethawing in the form of cortical strips?

Abstract

The aim of this study was to elucidate whether ovarian tissue is able to withstand a double freezing-thawing procedure. Human ovarian cortical biopsies from 4 thawed whole ovaries were divided into 4 experimental subgroups: (a) frozen-thawed non-grafted group, (b) frozen-thawed xenografted group, (c) refrozen-rethawed non-grafted group, and (d) refrozen-rethawed xenografted group. Xenografting was performed using 8 severe combined immunodeficient mice for a total duration of 21days. The following analyses were conducted: classic hematoxylin and eosin staining, Ki67 immunolabeling, transmission electron microscopy, Masson's green trichrome, and double CD34 immunostaining. Morphologically normal preantral follicles were detected in all groups. We observed a dramatic decline of more than 65% in early preantral follicle survival rates after grafting of both frozen-thawed (p< 0.0001) and refrozen-rethawed (p< 0.0001) ovarian tissue. However, mean follicle densities remained comparable between the frozen-thawed and refrozen-rethawed non-grafted groups, as well as both grafted groups. Equivalent proportions of proliferating early preantral follicles were identified in frozen-thawed and refrozen-rethawed samples, whether the tissue was grafted or not. Furthermore, we did not observe any significant difference in atretic follicle rates between any of the four groups, and the ultrastructural quality of follicles appeared unaffected by the refreezing procedure. Similar proportions of fibrosis were noted in the frozen-thawed and refrozen-rethawed groups, irrespective of grafting. Finally, no significant differences were witnessed in terms of vascularization. We were able to demonstrate, for the first time, that refrozen-rethawed ovarian tissue has the same functional characteristics as frozen-thawed ovarian tissue.