P-391 Importance of oxygen tension in human ovarian tissue in vitro culture
Authors: Vitale F , Cacciottola L , Yu F , Barretta M , Hossay C , Donnez J , Dolmans M M 
Published: June 2023

Abstract

Study question
Is there any difference between 20% and 5% oxygen (O2) tension on the viability and quality of human follicles contained in cultured ovarian cortex?

Summary answer
5% O2 yields higher follicle viability and quality than does 20% O2 tension after 6 days of in vitro culture (IVC).

What is known already
The primordial follicle (PMF) pool resides within the ovarian cortex, where in vivo O2 tension ranges between 2% and 8%. However, IVC is commonly performed at atmospheric O2 tension (20% O2), yielding low follicle survival rates. Elevated O2 tension or insufficient antioxidant protection can cause excessive reactive oxygen species (ROS) accumulation, leading to DNA lesions, such as oxidative stress damage and double-strand breaks (DSB).

Study design, size, duration
This prospective experimental study included frozen-thawed ovarian cortex from 6 adult patients (mean age: 28.5 years; age range: 26–31 years) 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.

Participants/materials, setting, methods
Cortical fragments were analyzed according to: hematoxylin and eosin (H&E) staining for follicle count and classification; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine (8-OHdG) and gamma-H2AX (γH2AX) immunolabeling to detect oxidative stress damage and DNA DSBs; and β-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore gene expression of superoxide dismutase 2 (SOD2) from the antioxidant defense system, and cyclin-dependent kinase inhibitors (p21 and p16) as senescence-related genes.

Main results and the role of chance
Apoptosis (p = 0.002) and follicle senescence (p = 0.03) rates were significantly lower in the 5% O2 than the 20% O2 group. Moreover, granulosa cells (GCs) in follicles in the 20% O2 group exhibited significantly (p &lt; 0.001) higher oxidative stress damage rates than in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (p = 0.001) in the 20% O2 than the 5% O2 group. SOD2 values were 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 20% O2 (p = 0.03) and 5% O2 (p = 0.008) compared to no culture. 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 5% O2 and no culture.

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 O2 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% O2 tension culture is a promising step towards potentially solving the problem of poor follicle viability after IVC.

Trial registration number
N/A