GADD45A is Essential for Granulosa Cells Differentiation and Ovarian Reserve in Human and Mice

Abstract

Diminished ovarian reserve (DOR) poses significant challenges in reproductive health, with emerging evidence implicating DNA damage repair pathways. While GADD45A is a critical regulator of DNA repair, cell cycle and apoptosis, its role in DOR pathogenesis remains unexplored. We employed transcriptome sequencing, qPCR and Western Blot analyses to compare GADD45A expression in granulosa cells (GCs) between DOR patients and controls. Functional studies included GADD45A overexpression/knockdown in human granulosa cells (KGN line) and phenotypic characterisation of Gadd45a knockout (KO) mice. Ovarian reserve parameters (follicle counts, hormone levels, oestrus cyclicity) and in vitro fertilisation outcomes were systematically evaluated. DOR patients exhibited significant upregulation of GADD45A in GCs, concomitant with reduced FSHR and CYP19A1 expression. In vitro experiments revealed that GADD45A overexpression disrupted both proliferation (Cell cycle analysis and EdU staining) and differentiation (Reduced CYP19A1 and FSHR expression) in GCs, while knockdown specifically impaired differentiation (Elevated CYP19A1 and FSHR expression). Gadd45a KO mice displayed hallmark DOR features: irregular oestrus cycles (Shorter oestrus), ovarian volume reduction, ovarian hormones dysregulation and decreased ovarian reserve (reduced primordial follicles and antral follicles, and increased atretic follicles). We found GADD45A was robustly expressed in the ovarian stroma and GCs of atretic follicles. KO oocytes showed compromised developmental competence with decreased two-cell embryo rate in vitro fertilisation. Our findings establish GADD45A dysregulation as a mechanistic contributor to DOR through dual impacts on granulosa cell differentiation and follicle survival. The Gadd45a KO mouse recapitulates key clinical DOR phenotypes, providing a validated model for therapeutic discovery.