Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β superfamily. In females, AMH is produced by the granulosa cells of small growing ovarian follicles, where it suppresses several stages of folliculogenesis. As such, AMH is considered an attractive therapeutic to address diverse reproductive needs, including fertility preservation and/or contraception. We set out to characterise the molecular mechanisms that govern the synthesis and activity of human AMH, to then generate potent analogues. By introducing an optimised furin cleavage site, we enhanced processing of the AMH precursor from <10% to >90%, corresponding with a dramatic increase in signalling activity. Based on species differences across the AMH type II receptor-binding interface, we introduced a series of double mutations (Gln484Met/Leu535Thr or Gln484Met/Gly533Ser) into human AMH that enhanced potency 5- and 10-fold, respectively. Subsequently, we showed that similar mutations also enhanced the activity of murine and feline AMH.
As recent studies in mice and cats have indicated that AMH overexpression results in durable contraception; by preventing primordial follicle activation in mice and breeding-induced ovulation in cats, we envisaged that our potent AMH analogues would be more efficacious. Adeno-associated viral vector delivery to female cats increased serum AMH levels >1000-fold, with supraphysiological activity confirmed via ELISA and ex vivo signalling assays throughout 9 months of measurements. High serum AMH was associated with non-follicular ovarian cyst formation and a progressive decline in antral follicles, however, the few surviving large follicles continued to ovulate. As such, most cats conceived during a breeding trial, but none of the cats within the AMH overexpression group gave birth. Our findings highlight the complexity of AMH signalling on reproductive physiology.