In vitro growth and development of ovarian follicles for fertility preservation

A physiological culture system that supports the in vitro growth (IVG) and development of oocytes would be an invaluable tool for the development and optimisation of fertility preservation techniques. Markers of oocyte, follicle and ovarian stromal tissue normality need to be established to ensure in vitro-derived oocytes are healthy and developmentally competent. A 2-step, physiological IVG system was developed that supported (1) activation, growth and development of ovine primordial follicles in situ over 16-23 days; yielding 37 secondary follicles over 31 repeat cultures and (2) development of in vivo- (n=85) and in vitro-derived secondary follicles to the early antral stage, 25% and 19%, respectively. Step (1) was compared to an accelerated system (n=24), which, unlike the physiological system, resulted in a significant increase (p<0.05) in the proportion of degenerating follicles and decrease (p<0.05) in stromal tissue integrity following 6 days culture compared to day 0 control tissue. In addition a significantly higher yield of transitional (p<0.01) and secondary (p<0.05) follicles resulted from the physiological vs. the accelerated system. The expression patterns of 20 genes key to oogenesis and folliculogenesis were established in vivo and compared to stage-matched samples derived using the physiological IVG system, revealing significant changes (p<0.05) in the expression of AMH, IGF1, INHα, INHβA, FST, ZP2, GTSF1, BMP6, BMP15 and MEST. Step (1) was used to evaluate damage to oocyte and ovarian tissue health following the perfusion of 48 ovaries, with either 1.5M dimethyl sulphoxide (DMSO) or L-15 control medium, in combination with the use of NMR spectroscopy to determine the level of DMSO permeation. Perfusion times of 10 and 60 minutes were required permeate the pedicle and cortex tissue, respectively, however, 60 minutes perfusion resulted in a significant decrease in follicle number (p<0.01) and stromal tissue integrity (p<0.05). The overall results indicate that the IVG of oocytes is a suitable tool to both assess the patency of fertility preservation systems and as a means to restore female fertility.