Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation

Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaec ological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiat e the growth of the primordial and primary follicles, It was then divided i nto two parts, one of which was cultured further as slices, and the other o ne used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles, The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After similar to 2 weeks in culture, some oocytes beg an to extrude from the follicles, which were usually at the secondary stage . They were small, 20-80 mu m in diameter, and had a thin or absent zona, P olar bodies and meiotic chromosomes could be seen in these naked oocytes. T his premature extrusion probably resulted from sub-optimal culture conditio ns. It occurred sooner in follicles that had been partially isolated using collagenase, Histologically, larger numbers of oocytes were observed in non -isolated slice cultures than in the partially isolated cultures. Initiatio n of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isol ation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atret ic follicles increased during all cultures, and it was significantly higher after partial isolation, Because partial isolation did not improve the sur vival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.