Isolation of pre-antral follicles from human ovarian medulla tissue

Abstract

Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content. Medulla was collected from 40 girls/women aged 3-35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated. NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P< 0.001) in the 3-9-year age group when compared with older groups (10-35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P< 0.0001 and <0.0007). Discarded medulla contained a considerable pool of pre-antral follicles, especially in young girls. Our new method allowed the isolation of viable pre-antral follicles from human ovarian medulla and provides a unique opportunity for basic scientific studies and for culture and grafting purposes.