A Three-Dimensional Culture Model Supporting Human Secondary-to-Antral Follicle Development In Vitro

Human ovarian folliculogenesis is a complex, tightly regulated process that is challenging to study directly in vivo. Although in vitro models are essential for mechanistic research, existing systems remain suboptimal because they cannot recapitulate the spatiotemporal dynamics of follicle development. This study presents a 3D culture model that supports human follicle development from the secondary to the antral stage. This model successfully recapitulates key in vivo morphological events, including sustained follicular growth, a distinct diameter expansion phase from day 10, and antral cavity formation around day 20. Importantly, this developmental progression culminated in the successful retrieval of viable oocytes at the germinal vesicle (GV) stage. Furthermore, immunofluorescence analysis revealed distinct expression patterns of gonadotropin receptors in somatic cells, consistent with granulosa and theca cell identity. Inner granulosa-like cells exhibited high follicle-stimulating hormone receptor (FSHR), whereas outer theca-like cells showed high luteinizing hormone receptor (LHR) expression. This model offers a valuable platform for studying human folliculogenesis and reproductive toxicology and provides a reference for optimizing in vitro follicle culture systems for secondary-to-antral stage development.