P-374 In vitro folliculogenesis of bovine ovarian tissue strips: co-culture with endothelial cells stimulates vessel formation and follicular growth

Abstract Study question Does bovine ovarian tissue strips (BOTS) and endothelial cells (ECs) co-culture promote vessel formation and follicle growth? Summary answer ECs co-cultured with BOTS drive vessels-like network formation and improve follicle growth. What is known already Over the past decades, a multistep approach to in vitro folliculogenesis has emerged as a promising method for obtaining mature oocytes. In the first step, the activated pool of dormant primordial follicles is stimulated to progress to the secondary stage. However, due to suboptimal static culture conditions - such as low oxygenation, nutrient depletion, and catabolite accumulation - only a small percentage of secondary follicles are successfully obtained. In vivo, perifollicular vessels produce angiocrine factors, including VEGF, which promote neo-angiogenesis, stromal cell differentiation, and follicular development. Study design, size, duration The viability of ECs incubated with different ratios of ECs and BOTS culture media was assessed up to 72h (n = 3). Follicle growth, health, and overall tissue viability (n = 3) were evaluated in BOTS (1 × 1×0.5mm) cultured for 7 days in gas-permeable dishes (PD) using BOTS, ECs, and BOTS:ECs (1:1) culture media. The optimal medium, BOTS:ECs (1:1) was used for a 7-day BOTS culture or BOTS-ECs co-culture in PD to assess vascular formation, follicular development, and viability (n = 3). Participants/materials, setting, methods Bovine ovaries (8–24 months) were collected from a slaughterhouse, while ECs were commercially sourced. The compatibility of different media ratios was assessed in ECs using the MTT assay. Media compatibility for BOTS was evaluated through histology, confocal microscopy (follicle viability), and an LDH assay on spent media. Vessel-like structure formation in BOTS co-cultured with ECs was analyzed using Hoechst/UEA I staining, while follicle progression and viability were assessed through a live/dead assay under confocal microscopy. Main results and the role of chance The viability of ECs cultured in a BOTS:ECs (1:1) medium was not significantly different from that of ECs cultured in ECs medium alone. Similarly, BOTS cultured 7 days in either BOTS:ECs (1:1) or BOTS medium alone showed comparable follicle stage distribution, grading, viability (n = 1774 histology; n = 2728 viability), and overall tissue health. BOTS co-cultured 7 days and labeled with Hoechst/UEA I revealed a vessel-like network extending to a maximum depth of 80 µm on both the cortical and medullary sides of the tissue strip. Furthermore, BOTS co-cultured with ECs (n = 1976 follicles) exhibited a significantly higher percentage of secondary follicles compared to BOTS cultured without ECs (15.8 vs. 9.1%; p < 0.01). However, follicle viability did not differ significantly between BOTS cultured with or without ECs (54.5 vs. 58.4%). These findings highlight the potential of BOTS-ECs co-culture, using a 1:1 media ratio, to promote neo-vascularization and support follicular development, providing valuable insights for optimizing ovarian tissue culture. Limitations, reasons for caution Although the bovine model is widely accepted for reproductive studies, validating these findings in human ovarian tissue remains crucial. Further research is needed to assess the perfusion of the vessel-like network formed, as well as its long-term remodeling and impact on follicle growth. Wider implications of the findings Rebuilding a vascular network in ovarian tissue in vitro could enhance follicle growth during in vitro folliculogenesis. In the future, this approach, using autologous endothelial cells, may help mitigate ischemia-reperfusion injury following ovarian tissue transplantation to rescue the fertility of oncological patients. Trial registration number No