The new biocompatible material for mouse ovarian follicle development in three-dimensional in vitro culture systems

Abstract To establish a protocol of optimized three-dimensional (3D) culture of ovarian follicles, various biomaterials have been investigated with regard to their properties and functions on in vitro follicle growth. The present study aims to compare the new biomaterial, extracellular matrix-derived soft hydrogel (ES-hydrogel) and alginate, and evaluate the effects of biomaterials on further in vitro 3D culture growth of ovarian follicle and oocyte maturation. The isolated follicles from mouse ovaries were randomly divided into two-dimensional (2D) culture, alginate and ES-hydrogel, and just seeded on culture wells (2D culture) or encapsulated with alginate or ES-hydrogel (3D culture). Culture media from each group were collected on days 4, 8 and 10 or 11 for 17s-oestradiol (E2) and progesterone (P4) measurement. On day 10 of in vitro culture, follicular survival and pseudo-antrum formation rate were examined, and oocyte maturation was induced by adding human chorionic gonadotropin and epidermal growth factor. After 17 h, ovulated mature oocytes collected and analyzed for oocyte diameter, normal spindle and chromosome alignment configuration, reactive oxygen species (ROS) level, and mitochondrial membrane potential level. To compare mechanical properties of two biomaterials, storage modulus was measured with the advanced rheometric expansion system. Our results showed that follicles cultured in ES-hydrogel, were significantly superior to those cultured 2D or alginate in the pseudo-antrum formation rate, cumulus-oocyte complexes (COCs) rate, MII oocyte rate, normal spindle rate, and E2 production. The ES-hydrogel and alginate groups were not significantly different in follicle survival rate, oocyte diameter, P4 production, ROS, and mitochondrial membrane potential levels. The storage modulus of ES-hydrogel was lower than that of alginate, suggesting that the improved follicular physiology and oocyte maturation in the ES-hydrogel group was due to better hormone exchange through a less stiff encapsulating material. This study shows that 3D culture system using ES-hydrogel effectively improve the outcome of in vitro ovarian follicle culture, supporting follicle morphology and growth and enhancing oocyte maturation. It means one of the most important factors for 3D culture of ovarian follicle was the selection of appropriate and effective biomaterial that can preserve the structure and morphology of ovarian follicle and facilitate nutrition and hormone exchange.