Evaluation of the effect of human testicular cell conditioned media on the in vitro development of follicles from cryopreserved human ovarian cortical pieces. A potential approach for fertility preservation for cancer patients

Abstract

If in vitro culture conditions could be improved, in vitro growth of cryopreserved ovarian tissue and isolated follicles could become an alternative to ovarian tissue transplantation. This study evaluated two cryopreservation methods, slow cooling and vitrification, and two in vitro culture methods, to determine their effect on the in vitro growth of human ovarian follicles. After warming, the OT pieces (1 × 10 × 3 mm) were cultured in either routine culture medium (DMEM), or DMEM supplemented 50:50 with media previously used for human testicular cell culture (hTCCM) to serve as a source of growth factors. Several parameters were evaluated during culture including follicle recruitment, growth, morphology, diameter, hormone production, and gene expressions (PTEN, BMP-15, PDGF, GDF-9, and GAPDH). The follicular morphology and hormonal secretion were comparable for both cryopreservation methods and both culture methods (P > 0.05). Follicle recruitment was accelerated in the vitrified group, in the presence of hTCCM (P > 0.05). After 7 days of culture, the follicle diameter was greater in the slow/hTCCM compared to the vitrification DMEM group. Similarly only one gene expression profile, BMP-15, in the slow/hTCCM differed significantly from another treatment group (Vit DMEM). At the end of culture (over 21 days), three immature, low-quality oocytes were the achievement. In conclusion, a very quick and easy vitrification protocol gave comparable results to the slow cooling method. We also established that the laboriously prepared hTCCM supplement did not benefit the outcomes. Further work is needed before in vitro maturation of cryopreserved ovarian cortical pieces can be used clinically.