Enhancing oxygen delivery to ovarian follicles by three different methods markedly improves growth in serum-containing culture medium

In vitro ovarian follicle culture systems are routinely used to study folliculogenesis and may provide solutions for infertility. Mouse follicles are typically cultured in standard gas-impermeable culture plates under gas phase oxygen concentrations of 5% or 20% (v/v). There is evidence that these conditions may not provide adequate oxygenation for follicles cultured as non-attached intact units in medium supplemented with serum and high levels of FSH. Three different methods of enhancing follicle oxygenation were investigated in this study: increasing the gas phase oxygen concentration, inverting the culture plates and using gas-permeable culture plates. Follicles cultured under 40% O2 were significantly larger (P < 0.01), had increased ovulation rates (P < 0.0001) and produced more oestradiol (P < 0.05) than follicles cultured under 20% O2. These effects were associated with reduced secretion of vascular endothelial growth factor (P < 0.05) and lactate (P < 0.05), and reduced expression of hypoxia-related genes. Increasing oxygen delivery with gas-permeable plates or by culture plate inversion also improved follicle growth (P < 0.01). An important aspect of enhancing oxygen delivery in this culture system is that it allows development of three-dimensional spherical mouse follicles over 6 days in serum- and FSH-supplemented medium to sizes comparable to in vivo-matured follicles (~500 µm in diameter). Such follicular development is not possible under hypoxic conditions.