Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture

Abstract STUDY QUESTION Can human pre-antral follicles isolated enzymatically from surplus medulla tissue survive and grow in vitro during long-term 3D culture? SUMMARY ANSWER Secondary human follicles can develop to small antral follicles and remain hormonally active in an alginate-encapsulation culture system for more than 30 days. WHAT IS KNOWN ALREADY Ovarian tissue cryopreservation followed by transplantation is a promising fertility preservation approach for cancer patients. However, transplantation of cryopreserved tissue to patients may carry the risk of re-implanting malignant cells. Grafting of follicles enzymatically isolated from ovarian tissue or developing a method for follicular culture and maturation in vitro may provide fertility to such patients without the risk of reintroducing the malignancy. However, the growth of pre-antral follicles isolated by enzymatic digestion from medulla tissue during long-term culture has received only little attention. STUDY DESIGN, SIZE, DURATION Two to ten human pre-antral follicles were encapsulated together within an alginate bead and cultured with or without ovarian interstitial tissue for either 7 days or >30 days. Follicles were cultured in either 20% oxygen or 5% oxygen or encapsulated in a lower concentration of alginate together with a lower concentration of FSH in high oxygen. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 395 pre-antral follicles from 16 cancer patients, aged 9–37 years, were co-cultured for either 7 days or >30 days. A proportion of follicle (64) were removed from culture on Day 7 and assessed for viability using confocal fluorescence microscopy following calcein-AM and ethidium homodimer-1 staining or histology. The remaining follicles (331) were continued in culture for >30 days then assessed for survival and growth. Anti-Müllerian hormone (AMH) and estradiol levels were quantified in the medium. MAIN RESULTS AND THE ROLE OF CHANCE An optimized protocol for isolation of intact healthy pre-antral follicles from ovarian medulla was developed. After 7 days of culture, secondary follicles had a significantly higher survival rates compared with primary and primordial follicles (70 versus