Impact of 6 month conjugated equine estrogen versus estradiol-treatment on biomarkers and enriched gene sets in healthy mammary tissue of non-human primates

PLoS One. 2022 Mar 17;17(3):e0264057. doi: 10.1371/journal.pone.0264057. eCollection 2022.


Objective: To identify distinctly regulated gene markers and enriched gene sets in breast tissue of cynomolgus monkeys (Macaca fascicularis) treated for six months with either conjugated equine estrogens (CEE) or estradiol (E2) by analysis of corresponding mRNA levels of genes associated with breast development, carcinogenesis, apoptosis and immune regulation. Additionally, translation of three nuclear markers was analyzed.

Methods: RNA from breast biopsies and necropsies was isolated from two independent study trials from Ethun et al. (CEE) and Foth et al. (E2) after 6 month of treatment duration. RNA was subjected to qRT-PCR and MicroArray analysis. Immunohistochemical stainings were performed for the estrogen receptor alpha subunit (ERa), the progesterone receptor (PGR) and the proliferation marker Ki67.

Results: We identified a total of 36 distinctly enriched gene sets. Thirty-one were found in the CEE treatment group and five were found in the E2 treatment group, with no overlap. Furthermore, two individual genes IGFBP1 and SGK493 were upregulated in CEE treated animals. Additional targeted qRT-PCR analysis of ten specific estrogen-related genes showed upregulation of three genes (TFF1, PGR and GREB1) after CEE treatment, respectively one gene (TFF1) after E2 treatment. Immunohistochemical stains of breast biopsies showed a significant increase in expression of the PGR marker after CEE treatment.

Conclusions: In this study we identified enriched gene sets possibly induced by CEE or E2 treatment in various processes associated with cancer biology and immunology. This preliminary translational data supports the concept that different estrogen types have different effects on healthy breast tissue and may help generate hypotheses for future research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Estradiol* / pharmacology
  • Estrogen Replacement Therapy
  • Estrogens / metabolism
  • Estrogens, Conjugated (USP)* / pharmacology
  • Female
  • Humans
  • Macaca fascicularis
  • RNA


  • Estrogens
  • Estrogens, Conjugated (USP)
  • Estradiol
  • RNA

Grants and funding

This study was funded by the Department of Pathology Research Endowment, Wake Forest School of Medicine through a grant awarded to JMC (2016). The study was also supported by Pfizer Inc. through a grant awarded to TBC (2007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.