Summary: Wound healing problems are a major cause of morbidity for gender-affirming surgery (GAS) patients. Prior studies have shown sex differences in wound healing may exist. We hypothesized exogenous testosterone supplementation may impair post-GAS wound healing and developed a model to investigate this phenomenon. Mice were randomized by hormone regimen and gonadectomy (OVX). Gonadectomy or sham occurred on day 0 and mice were assigned to no testosterone (-T), mono- or bi-weekly (T/2T) testosterone groups. Dorsal splinted wounding occurred on day 14 and harvest on day 21. Serum testosterone levels were quantified with mass spectrometry. Tissue underwent analysis with planimetry, qPCR, ELISA, and immunofluorescence. Mean testosterone trough levels for bi-weekly regimen were higher compared to mono-weekly (397 versus 272 ng/dL; P = 0.027). At POD5, 2T injections led to 24.9% and 24.7% increases in mean wound size relative to SHAM and OVX/-T, respectively (P = 0.004; 0.001). Wounds in OVX/+2T mice demonstrated increased gene expression for inflammatory cytokines and macrophage marker F4/80 (P < 0.05). ELISA confirmed elevated wound TNFα levels (P < 0.05). Quantitative multiplex immunofluorescence with F4/80/NOS2/ARG1 showed significant increases in macrophage prevalence in OVX/+2T (P < 0.05). We developed a novel model of GAS hormonal milieu to study effects of exogenous testosterone on wound healing. Optimized twice-weekly dosing yielded serum levels comparable to clinical therapy. We showed exogenous testosterone administered to XX/OVX mice significantly impairs wound healing. A hyperinflammatory wound environment results in increased macrophage proliferation and elevated cytokines. Future efforts are directed toward mechanistic investigation and clinical validation.

with a decrease in epithelial gap, dermal gap, and granulation tissue area and thickness compared to the placebo group (p=0.002, p=0.039, p=0.012, respectively). Gene set analysis showed POD14 wounds exposed to T had upregulation of genes related to extracellular matrix degradation. Gene ontology analysis showed T was associated with an upregulation of genetic pathways associated with lymph vessel development and angiogenesis throughout the wound bed. In the mouse, comparison of POD5 wounds showed T injections led to 24.9% and 24.7% increase in mean wound size relative to sham and ovariectomy controls (p=0.004; p=0.001). Spatial transcriptomics demonstrated opposing T effects spatially: pathways such as epithelial-mesenchymal transition and angiogenesis were highly upregulated in granulation tissue but downregulated in neo-epidermis. CONCLUSIONS:Testosterone impairs wound healing in a preclinical model, potentially via altering the homeostatic balance of re-epithelialization and granulation. This was largely reversed by androgen receptor blockade. Future studies will characterize the molecular mechanisms driving these effects.

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