Isolation and long-term culture of human preantral follicles*

Objective

To isolate intact preantral follicles from the human ovary for in vitro studies.

Procedure

Premenopausal human ovary was digested by a mixture of collagenase and deoxyribonuclease (DNase) for 1 hour at 37°C and for 36 hour at 4°C. Intact preantral follicles at classes 1 and 2 were isolated and cultured for up to 120 hours in a serum-free Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY) with ITS⁺ (insulin, transferrin, selenium, linoleic acid, and bovine serum albumin; Collaborative Research, Bedford, MA) with or without FSH. Follicular DNA synthesis was measured by [³H]thymidine incorporation, whereas steroidogenic capacity was assessed by P, androstenedione (A), and 17β-E₂ RIA. The morphology of long-term cultured follicles was studied by routine histologic procedure.

Results

A large number of intact, preantral follicles was efficiently dissociated from a portion of ovary. Morphologically, follicles were healthy and did not have any thecal layer. Follicle-stimulating hormone, in vitro, induced follicular DNA synthesis by 24 hours and antrum formation in class 2 follicles after 120 hours. Both class 1 and 2 follicles were able to produce a small basal amount of P and A, but they did not produce any measurable E₂. However, both classes synthesized increasing amounts of P, A, and E₂ after FSH exposure.

Conclusion

This is a first report of a successful enzymatic isolation of human preantral follicles and their long-term culture in vitro. The growth and differentiation of preantral follicles by FSH clearly indicate the importance of FSH in human follicular development. These results provide an opportunity for quantitative studies on factors regulating folliculogenesis in the human and a novel future direction for human IVF.