Hypo-glycosylated FSH enhances the ovarian microenvironment for follicular development compared to fully glycosylated FSH

Differential N-glycosylations of follicle-stimulating hormone (FSH) influence follicular development dynamics, significantly affecting assisted reproductive technology outcomes. However, the ovarian microenvironment induced by different FSH glycoforms remains incompletely understood. In this study, in vivo analyses using RT-qPCR, single-cell RNA sequencing, and bulk RNA sequencing demonstrated that FSH administration rapidly activated responses in both granulosa cells (GCs) and theca cells (TCs) within 2 h. Hypo-glycosylated FSH (hgFSH) initiated GCs and TCs differentiation more effectively than fully glycosylated FSH (fgFSH), and genes with higher expression in hgFSH were predominantly located in these cell types. HgFSH significantly upregulated 20.2% of total ovarian ligand genes, suggesting a more actively ovarian communicating microenvironment. In contrast, fgFSH inhibited the expression of oocyte-specific ligands such as Bmp15, Gdf9 and Oosp1, potentially disrupting oocytes-GCs communication. More importantly, hypo-glycosylated FSH upregulated genes significantly enriched in TCs. HgFSH activated key biological pathways in TCs such as lipid metabolic process and enhanced communication between GCs and TCs through more interactions than fgFSH. Our findings suggest that hgFSH fosters a more effective ovarian microenvironment for follicular development compared to fgFSH, highlighting the potential clinical benefits of hgFSH applications.