Bioengineering of Human Artificial Ovary From Cryopreserved Tissue: Optimization of the Follicles Enzymatic Isolation.

Abstract Background: This study aims to evaluate the effectiveness of Tumor Dissociation Enzyme (TDE) for the isolation of cryopreserved human ovarian cortex tissues, proposing an optimized applicable for artificial ovary.Methods: This is a prospective experimental study. We present a comparative analysis among the outputs of follicles isolated from cryopreserved ovarian biopsies of ten young women undergoing laparoscopy. Follicles were isolated by Tumor Dissociation Enzyme (TDE) or Liberase Dispase High (DH) enzymatic digestion. Follicles were assessed by evaluating the number, viability, morphology, oxidative stress. Moreover, the follicle growth and viability were analyzed after eight days of in vitro culture (IVC) between each protocol.Results: The recovery rate of follicles in Group 1 was significantly higher by 78 follicles compare to Group 2 (p < 0.05), while the difference in weight and volume of biopsies in both groups was no significant (p > 0.05). Group 1 allowed to isolate more primordial follicles (p < 0.05) and smaller diameter of follicles (p < 0.01) than Group 2. Group 1 allowed to isolate 7% more of bright red follicles than Group 2 (p < 0.01). We also found that Group 1 had isolated a significantly higher percent of viable follicles (p < 0.05), more morphologically normal follicles (p < 0.05), and lower oxidative stress levels compared with Group 2 (p < 0.01). The velocity of follicles growth from Day 0 to Day 8 in Group 1 was significantly higher (p < 0.05) than in Group 2. The viability of follicles on Day 8 of in vitro culture in Group 1 was significantly higher than Group 2 (p < 0.05).Conclusions: TDE treatment can be an alternative for Liberase DH, allows the isolation of highly viable follicles from the cryopreserved human ovarian cortex, with an intact morphology and low oxidative stress, and with high proliferation potential after culture in vitro.