Fetal calf serum versus human serum: ultrastructural evaluation of protein support influence on human ovarian tissue cryopreservation.

The aim of this study was to examine the effect of the different protein supports in the cryopreservation solution on improving human ovarian tissue preservation after frozen-thawed procedures. Biopsies of ovarian cortical tissue were obtained from 14 subjects. All specimens were cryopreserved using a slow freezing/rapid thawing method in a solution consisting of propanediol and sucrose in different proportions of 3 protein supports: 30% human serum (HS) (solution A), 20% HS (solution B), or 20% fetal calf serum (solution C). After thawing, 191 follicles and a total of 70 samples were analyzed using transmission electron microscopy (TEM). The post-thaw preservation rate of the follicles in solution A was significantly higher with respect to solution C (p < 0.05). Unlike the follicles, the stromal cell morphology was not affected by any of the solutions investigated. By comparing stromal morphology and the patient age, it was found that HS better preserved the tissue in patients over 20 years of age with respect to younger ones, which showed a wider variability in ovarian preservation. TEM evaluation showed that 30% HS is more suitable for human ovarian tissue cryopreservation, and research should be focused on defining cryopreservation protocols specific to young patients.

Comparison of quartz vials with polypropylene vials for rapid cryopreservation of human ovarian tissue.

BACKGROUND: Because higher survival of follicles during the freezing/thawing procedure improves the quality of cryopreserved tissue reimplanted after oncological therapies, defining an optimal method for human ovarian tissue cryopreservation remains a major issue in this field. One option to improve the cryopreservation procedure is to use better materials, i.e., vials with better conductivity. The aim of this study was to compare polypropylene (PP) with quartz vials. Between September 2012 and January 2013, eight patients were recruited. The ovarian cortex was cut into 3 slices, assigned randomly to a fresh and a cryopreserved group in PP (method B) or quartz vials (method C). Histological and immunohistochemical (IHC) analysis were used. For IHC three antibodies were analyzed: Ki67 (proliferation index), Bcl2 (anti apoptotic index) and Hsp70 (stress index). RESULTS: The majority of GCs showed positive staining for Bcl2 in both cryopreservation device, with higher expression in group C than in group B. Oocytes and their nuclei showed intense positive staining for ki67 in both methods B and C, and also a patch positive stromal cells staining for Ki67. Expression of hsp70 was not increased after cryopreservation. CONCLUSIONS: Cryopreservation using quartz vials led to larger numbers of good follicles while maintaining consistent preservation for stromal cells and vessels.