Enhancing oxygen availability in the dynamic culture of bovine ovarian cortical tissue improves the yield of secondary follicles

Study question Does enhancing oxygen availability during dynamic in vitro culture of bovine ovarian cortical tissue (BOCT) improve follicle growth and health? Summary answer Enhancing oxygen availability during dynamic in vitro culture of BOCT in perifusion bioreactors (PB) does improve follicle health and yield to secondary follicles What is known already Oxygen availability has been demonstrated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical tissue (HOCT) under static culture conditions. Disruption of solutes gradients and application of physiological fluid mechanical stress, through in vitro dynamic culture of HOCT in a newly designed perifusion bioreactor have been shown to further enhance follicle growth and health. As it shows striking similarities with human, bovine folliculogenesis is considered a valuable model to study follicle growth in vitro Study design, size, duration Bovine ovaries from animals aged 8-24 months were collected at slaughterhouse. In each experiment (n = 3), BOCT strips from the same ovary were cultured for 6 days in perifusion bioreactors (PB, dynamic culture) and conventional dishes (CD, static culture). Culture outcome in static culture was analysed and compared to two bioreactor configurations in which medium oxygenation was kept low by using a standard tube reservoir (StPB) or was enhanced by using a gas-permeable dish reservoir (PB+O2). Participants/materials, setting, methods Slices of BOCT 0.5mm thick were cut with a tissue slicer and chopped into 1x1mm strips. In each experiment, fresh (D0) and cultured tissue (groups of ten strips) were analyzed. Follicle stages and health were assessed by histology (hematoxylin-eosin staining). Follicle viability was estimated by labelling with live-dead far-red and propidium iodide followed by clearing before analysis at the confocal laser scanning microscope. Main results and the role of chance Overall, 2417 follicles were analyzed (histology, 1476; viability, 941). At day 0 most follicles were primordial (primordial, 88.7%; primary, 10.6%; secondary, 0.7%), and had good quality (grade 1-2, 92.2%; grade 3, 7.8%), and high viability (91.8%). At day 6, follicle growth and health in StPB was superior than in CD (StPB vs CD - staging: primordial, 6.8 vs 16.3, P < 0.01; primary, 70.7 vs 74.1, NS; secondary, 22.5 vs 9.6%, P < 0.01; grading: grade 1 + 2, 71.4 vs 44.8, P < 0.01; grade 3, 28.6 vs 55.2%, P < 0.01). Dynamic culture in StPB better-preserved follicle viability compared to static culture in CD (StPB vs CD: 77.75 vs 64.9%, P < 0.01). Enhancing oxygen availability during dynamic culture increased follicle progression and viability (PB+O2 vs StPB - staging: primordial, 5.1 vs 6.8, NS; primary, 65.4 vs 70.7, NS; secondary, 29.5 vs 22.5%, P < 0.05; viability - 92 vs 77.75, P < 0.01). Overall, the obtained results demonstrate that i) disruption of stagnant layers of medium and application of shear stress to BOCT through dynamic culture improves follicle activation, growth and health; ii) enhancing oxygen availability by means of a gas-permeable medium reservoir further increases follicle progression and viability. Limitations, reasons for caution Although the bovine is considered a reliable model for human folliculogenesis, the study should be validated on human ovarian tissue. Wider implications of the findings A limiting step in the in vitro production of mature oocytes starting from primordial follicles is the low yield of secondary follicles after organ culture. The adoption of a newly designed dynamic bioreactor and modulation of oxygen availability could represent a valuable tool for multistep in vitro folliculogenesis.