A Novel Mouse Model for Investigating the Effects of Gender-affirming Hormone Therapy on Surgical Healing

Abstract

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.

Fig. 1.

A, Average serum testosterone levels by experimental group. Groups with no exogenous T showed negligible testosterone levels, whereas groups with exogenous T showed a dose-dependent cyclic T serum level. B, Macroscopic wound pictures taken at POD5 showed a delayed wound healing rate in the mice exposed to exogenous testosterone therapy (ie, OVX/+T and OVX/+2T). C, Wound planimetry data. Pairwise comparisons revealed 2T injections led to 24.9% and 24.7% increases in macroscopic wound area relative to SHAM and OVX/-T, respectively (P = 0.004; P = 0.001).

Fig. 2.

Murine wound tissue analysis. A, qPCR gene expression profiles of mouse wound tissue. Relative expression levels of cytokine (Tumor Necrosis Factor alfa, Vascular Endothelial Growth Factor A, Transforming Growth Factor beta-1, Macrophage Migration Inhibitory Factor, Interferon gamma, Interleukin 6) extracellular matrix (Collagen 1, Col 3, Smooth Muscle alpha-actin, Fibronectin-1), cell phenotype (F4/80, Arginase-1, Nitric Oxide Synthase-2, FOXP3, CD4), and androgen receptor (AR)-related genes (n = 12; P < 0.05). B, ELISA of TNFα in mouse skin wound tissue (n = 6; P < 0.05). *indicates significance of P < 0.05.