Influence of strip thickness and FSH supplementation on follicle growth in the in vitro culture of strips of bovine ovarian cortical tissue in gas-permeable dishes

Study question: Do tissue thickness and FSH supplementation affect follicle growth and health in the vitro culture of bovine ovarian cortical strips (BOCS) in gas-permeable dishes? Summary answer: Culture of 0.5mm thin BOCS with 5ng/ml FSH does im- prove follicle health and yield to secondary follicles compared with 1mm thick BOCS What is known already: Oxygen availability inside tissue has been demon- strated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical strips (HOCS). Although, strip thickness can limit nutrients and gases diffusion in and out of the innermost tissue zone, the presence of the outer medulla in thick strips could positively affect follicle growth. The role of FSH on the progression of primordial to sec- ondary follicles in ovarian organ culture is still debated. Study design, size, duration: Bovine ovaries from animals aged 8-24 months were collected at a slaughterhouse. In each experiment (n ¼ 3), BOCS of varying thickness collected from the same ovary were cultured with or without FSH in gas-permeable dishes for 10 and 15 days. Participants/materials, setting, methods: Slices either 0.5mm or 1mm thick were cut with a tissue-slicer and were chopped into 1x1mm2 strips. BOCS were cultured for 10 or 15 days at the same tissue/medium volume ratio (groups of either five 1mm strips or ten 0.5mm strips in 5ml medium) with 0, 1, or 5ng/ml FSH. Follicle stages were assessed by histology. Follicle viability was estimated by labeling with live-dead far red and propidium iodide at the confocal laser scanning microscope. Main results and the role of chance: Overall, 2314 follicles were analyzed (histology, 998; viability, 1316). At day 0 most follicles were primordial (pri- mordial, 89.4%; primary, 8.7%; secondary, 1.9%), and had a high viability (94.69%). The best follicle growth and viability was observed in 0.5mm thin BOCS cultured with 5ng/ml FSH. In particular, when compared to 1mm thick BOCS cultured with 5ng/ml FSH, 0.5mm thin BOCS cultured with 5ng/ml FSH showed a higher and significant proportion of secondary follicles at day 10 (0.5 vs 1mm, % secondary follicles: 26.5 vs 10, P < 0.05) and a significantly higher proportion of viable follicles at day 15 (0.5 vs 1mm, % viable follicles: 89.4 vs 60.7, P < 0.01). These results demonstrate that smaller BOCS thickness and 5ng/ml FSH sup- plementation significantly improve the growth and health of secondary follicles. Limitations, reasons for caution: Although the bovine is considered a reli- able model for human folliculogenesis, the study should be validated on hu- man ovarian tissue. Wider implications of the findings: A limiting step in the production of mature oocytes starting from primordial follicles is the low yield to secondary follicles at the end of organ culture. Given the similarities between bovine and human folliculogenesis, the best culture conditions herein identified could con- tribute to the refinement of human in vitro folliculogenesis. Trial registration number: Not applicable