Testosterone treatment in transmen is associated with reduced hormone signaling in the mammary gland

Many transgender men receiving long-term testosterone replacement therapy (TRT) have high serum levels of testosterone, 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. Comparison of mammary glands from transgender men on TRT and cisgender women can reveal the poorly understood consequences of testosterone expression on mammary gland biology and 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 cisgender women and transgender men on TRT. We identified that the donors on TRT had a decreased expression of genes downstream of estrogen signaling pathways in hormone receptor positive luminal epithelial, as well as a decrease in luteal phase gene activity in these donors. 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 suppresses hormone signaling in the breast through their impact on hormone receptor positive (HR+) mammary epithelial cells, with implications for TRT as an antagonist of estrogen receptor alpha (ER?) signaling. Overall design: To describe the effect of testosterone replacement therapy on the mammary gland in transgender men, we performed scRNAseq on healthy pre-menopausal and nulliparous breast tissues of 7 donors on testosterone and 7 not on testosterone, from either reduction mammoplasty or gender-affirming mastectomy surgeries. MULTI-seq barcoding and in silico SNP-based genotyping were used to multiplex samples in order to minimize batch effects. The pooled samples were selected for live (DAPI negative) and singlets by fluorescence activated cell sorting (FACS) prior to sequencing on a 10X Genomics platform.