Distinct estrogenic transcription programs of estetrol and 17β-estradiol across mammary, uterine, and vaginal cell types of pseudomenopausal mice

Menopause results from a marked decline in estrogen production and leads to symptoms that impair quality of life. Menopausal hormone therapy effectively alleviates these symptoms but is associated with an increased risk of estrogen receptor-positive (ER + ) breast cancer. Estetrol (E4), a natural fetal estrogen produced by the liver, has emerged as a promising alternative because it relieves menopausal symptoms without promoting mammary tumor growth in preclinical mouse models. To further assess its potential, we compared the transcriptional effects of E4 and 17β-estradiol (E2) across three estrogen-responsive tissues: the mammary gland, uterus, and vagina. Ovariectomized adult mice, used as the standard preclinical model of menopause, were treated for two weeks with either a therapeutic dose of E4 (E4low), a tenfold higher supratherapeutic dose (E4hi), or E2. In mammary luminal cells, E2 induced a proliferative gene signature that was absent following E4 treatment. E4 did not elicit a distinct transcriptional profile, supporting the notion that it acts as a weaker estrogen in this tissue. In contrast, in the uterine and vaginal epithelium, E2, E4low, and E4hi similarly regulated most genes, and all treatments produced comparable morphological responses. However, in uterine and vaginal fibroblasts, E2 induced a distinct transcriptional signature that was not reproduced by E4. Overall, this study provides a comprehensive comparison of the transcriptional programs induced by E2 and E4 across estrogen-responsive tissues and support further investigation of E4 as a potentially safer option for menopausal hormone therapy.