Importance of oxygen tension in human ovarian tissue in vitro culture

STUDY QUESTION Is there any difference between 20% and 5% oxygen (O-2) tension in vitro culture (IVC) on the viability and quality of human follicles contained in cultured ovarian cortex? SUMMARY ANSWER An O-2 tension of 5% yields higher follicle viability and quality than does 20% O-2 tension after 6 days of IVC. WHAT IS KNOWN ALREADY The primordial follicle (PMF) pool resides within the ovarian cortex, where the in vivo O-2 tension ranges between 2% and 8%. Some studies suggest that lowering O-2 tension to physiological levels may improve in vitro follicle quality rates. STUDY DESIGN, SIZE, DURATION This prospective experimental study included frozen-thawed ovarian cortex from six adult patients (mean age: 28.5 years; age range: 26-31 years) who were undergoing laparoscopic surgery for non-ovarian diseases. Ovarian cortical fragments were cultured for 6 days at (i) 20% O-2 with 5% CO2 and (ii) 5% O-2 with 5% CO2. Non-cultured fragments served as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS Cortical fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; Ki67 staining to evaluate PMF proliferation; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine and gamma-H2AX (gamma H2AX) immunolabeling to detect oxidative stress damage and DNA double-strand breaks (DSBs) in oocytes and granulosa cells (GCs); and beta-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore the gene expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase 4 (GPX4) from the antioxidant defense system and cyclin-dependent kinase inhibitors (p21 and p16) as tissue senescence-related genes. MAIN RESULTS AND THE ROLE OF CHANCE Apoptosis (P = 0.002) and follicle senescence (P < 0.001) rates were significantly lower in the 5% O-2 group than in the 20% O-2 group. Moreover, GCs in follicles in the 20% O-2 group exhibited significantly (P < 0.001) higher oxidative stress damage rates than those in the 5% O-2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (P = 0.001) in the 20% O-2 group than in the 5% O-2 group. SOD2 expression was significantly greater in the 5% O-2 group compared to the 20% O-2 group (P = 0.04) and the non-cultured group (P = 0.002). Expression of p21 was significantly increased in both the 20% O-2 (P = 0.03) and 5% O-2 (P = 0.008) groups compared to the non-cultured group. Moreover, the 20% O-2 group showed significantly greater p16 expression (P = 0.04) than the non-cultured group, while no significant variation was observed between the 5% O-2 and no culture groups. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This study focuses on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue. The impact of O-2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that 5% O-2 tension culture is a promising step toward potentially solving the problem of poor follicle viability after IVC. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0064.22, CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.). The authors have nothing to disclose.