LONG-TERM IN VITRO DYNAMIC CULTURE OF BOVINE OVARIAN CORTICAL TISSUE (BOCT) INCREASES FOLLICLE GROWTH, VIABILITY AND AMH SECRETION

Short-term ovarian tissue culture is the first step of in vitro folliculogenesis procedures aimed to obtain healthy secondary follicles necessary for further growth and generation of mature oocytes.1 However, the yield and health of secondary follicles remain limited. We previously reported that dynamic in vitro culture of ovarian cortical tissue in a novel perifusion bioreactor (PB) (patent 102020000027290) enhances follicle growth and health compared to conventional static culture (CD).2 In the present study, longterm culture of BOCT was attempted to preserve tissue health and obtain more competent secondary follicles. BOCT slices (1x1x0.5 mm) were cultured in group of 10 for 14 days in PB and CD, monitoring whole tissue viability (LDH) and AMH secretion in spent media every 2 days. Follicle stages and quality were assessed through histology and live-dead confocal analysis, respectively at the end of culture. Data (939 follicles) indicated that culture in PB vs CD increases the secondary follicle rate (21.2 vs 8.2%, p<0.05), and follicle quality and viability (grade I/II, 70 vs 18.9%, grade III, 30 vs 81.1%; live follicles 62.6 vs 35.3%, p< p0.01). LDH activity in PB was markedly lower than in CD at all culture times (PB14d 150.5 nmol vs CD14d 233.1 nmol). AMH secretion continuously increases in PB whereas in CD it markedly decreases (1700 vs 67 picomol/Vtot). Findings indicate that the increased transport of solutes and dissolved oxygen, and the biomechanical stimulation in our novel dynamic PB play a key role during BOCT culture. Analysis of LDH and AMH levels in spent media confirm that dynamic PB better maintains general tissue health and promote a better functionality of growing follicles.