Melatonin potentiates estrogen therapy to prevent bone loss in an orchiectomy transfeminine mouse model

Transfeminine individuals undergoing orchiectomy face an increased risk of early osteopenia and osteoporosis. Here, we investigated whether melatonin enhances estradiol (E2)-mediated skeletal protection after androgen deprivation using an orchiectomy plus E2 mouse model designed to mimic gender-affirming hormone therapy (GAHT). Bone architecture was assessed by micro-computed tomography (micro-CT) and histology. Enriched LepR+ bone marrow stromal cells (MSCs) were analyzed for cell viability and osteogenic mineralization. In parallel, publicly available single-cell RNA-seq datasets were reanalyzed to characterize sex-steroid and melatonin receptor expression across bone marrow stromal populations. Orchiectomy caused marked loss of bone mass, which high-dose E2 only partially rescued but failed to restore the LepR + MSC pool. Single-cell analyses identified LepR+ MSCs as a stromal subset enriched for Ar / Esr1 , with further upregulation during bone regeneration. These progenitors also expressed Mtnr1a / Mtnr1b , supporting coordinated sex-steroid and circadian regulation of skeletal progenitor function. Orchiectomy reduced pineal expression of melatonin synthesis enzymes ( Tph1 / Ddc ), and this deficit was only partially corrected by E2 treatment. Functionally, combined E2-melatonin treatment significantly improved LepR + MSC viability and mineralization in vitro. In vivo, co-treatment increased trabecular number, bone volume, and connectivity density beyond E2 alone. Collectively, these findings suggest that E2 maintains mineralization capacity but is insufficient to replenish skeletal progenitor reserves, whereas melatonin provides complementary progenitor support. Therefore, E2-melatonin co-therapy may represent a mechanism-based and low-risk strategy to mitigate post-orchiectomy GAHT-associated bone loss and warrants clinical evaluation.