Anti-Mullerian hormone (AMH) protects ovarian follicle loss by downregulating granulosa cell function in in vitro and in vivo models

Purpose: AMH inhibits hormone production in luteinized granulosa cells (GCs) and stalls ovarian follicle development in vitro and in vivo. We sought to confirm AMH’s mechanism of action through SMAD activation and investigate AMH inhibition of follicle development and function, in vitro and in vivo. Methods: A primary culture of GCs isolated from follicular fluid was used, and cells were treated with recombinant AMH (rAMH) or placebo for 24 h. For the mouse model, 18-weeks old C57BL female mice were either euthanized at the beginning or treated with rAMH or normal saline for 3 weeks. Primordial (PDF), primary follicle (PRF), secondary (SEF), and tertiary follicles (TEF) were calculated. Real-time RT-PCR and ELISA were performed to quantify GC gene expression and protein translation of human SMAD 1, 5, and 8, FSH-R and mouse FSH-R, inhibin B, caspase 3, Ki67, BMP15, GDF9, and the epigenetic regulators miRNAa and b. Results: In vitro, rAMH-treated GC showed activation of the SMAD 1, 5 and downregulation of SMAD 8, with greater magnitude at increasing rAMH doses (p < 0.04) and consequential control of downstream regulators. In vivo, the rAMH-treated mice showed increased SEFs and decreased PRFs while PDFs, TEFs, were unchanged compared with baseline. Compared with Placebo, the rAMH group showed increased PDFs, while PRFs, and TEFs were significantly decreased, and SEFs were unchanged. Conclusions: AMH caused SMAD activation in a dose-dependent manner, with downstream downregulation of cell function and replication, also through activation of miRNAs. These mechanisms were confirmed by the in vivo findings with ultimate downregulation of follicular development and preservation of the ovarian follicle number. Counteracting follicular depletion, AMH could be used to protect the ovarian follicle reservoir.