Importance of oxygen tension in human ovarian tissue in vitro culture.

Abstract

Is there any difference between 20% and 5% oxygen (O2) tension in vitro culture (IVC) on the viability and quality of human follicles contained in cultured ovarian cortex? An O2 tension of 5% yields higher follicle viability and quality than does 20% O2 tension after 6 days of IVC. The primordial follicle (PMF) pool resides within the ovarian cortex, where the in vivo O2 tension ranges between 2% and 8%. Some studies suggest that lowering O2 tension to physiological levels may improve in vitro follicle quality rates. 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% O2 with 5% CO2 and (ii) 5% O2 with 5% CO2. Non-cultured fragments served as controls. 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 (γH2AX) immunolabeling to detect oxidative stress damage and DNA double-strand breaks (DSBs) in oocytes and granulosa cells (GCs); and β-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. Apoptosis (P = 0.002) and follicle senescence (P < 0.001) rates were significantly lower in the 5% O2 group than in the 20% O2 group. Moreover, GCs in follicles in the 20% O2 group exhibited significantly (P < 0.001) higher oxidative stress damage rates than those in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (P = 0.001) in the 20% O2 group than in the 5% O2 group. SOD2 expression was significantly greater in the 5% O2 group compared to the 20% O2 group (P = 0.04) and the non-cultured group (P = 0.002). Expression of p21 was significantly increased in both the 20% O2 (P = 0.03) and 5% O2 (P = 0.008) groups compared to the non-cultured group. Moreover, the 20% O2 group showed significantly greater p16 expression (P = 0.04) than the non-cultured group, while no significant variation was observed between the 5% O2 and no culture groups. N/A. 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 O2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. Our findings suggest that 5% O2 tension culture is a promising step toward potentially solving the problem of poor follicle viability after IVC. 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.