Single cell RNA-sequencing reveals an association between testosterone treatment and reduced hormone signaling in the human mammary gland

Testosterone is the most studied androgen in hormone replacement therapy in postmenopausal women. Many transgender men also receive long-term testosterone replacement therapy (TRT) and have high serum testosterone levels compared to the low levels seen in cisgender women. Compared to other sex hormones such as estrogen and progesterone, the effects of testosterone on the mammary gland have been relatively understudied and there is little data regarding the long term safety of this treatment. Comparison of mammary glands from transgender men on TRT and cisgender women can reveal the effects of testosterone treatment on mammary gland biology and provide critical information regarding the long-term effects of TRT on patient health and disease outcomes. In this study, we performed single-cell RNA sequencing of breast tissues from a demographics-matched cohort of cisgender women and transgender men on TRT. Surprisingly, participants on TRT had unchanged serum levels of estradiol compared to controls. Among the observed transcriptional differences for participants on TRT were a dramatically reduced expression of genes downstream of estrogen signaling pathways in hormone receptor positive (HR+) luminal epithelial cells, as well as a decreased overall menstrual cycle-related hormone signaling. We confirmed this finding experimentally by showing reduced expression of progesterone receptor a/b, a prominent marker of estrogen signaling, in donors on TRT. Our results support the hypothesis that high levels of testosterone in transgender men on TRT suppress sex hormone signaling in the breast as seen on their impact on HR+ mammary epithelial cells, with implications for TRT as an antagonist of estrogen signaling and protection against breast cancer.