Endometrial profile of tamoxifen and low-dose estradiol combination therapy

Abstract

Purpose: Combination estrogen + progestin therapy has been associated with increased breast cancer risk in postmenopausal women. Selective estrogen receptor modulators (SERM) are potential alternatives to progestins, although the endometrial safety of estrogen + SERM co-therapies is not known. The goal of this study was to evaluate the endometrial profile of low-dose estradiol and the SERM tamoxifen alone and in combination.

Experimental design: Twenty-four postmenopausal female cynomolgus macaques were randomized by social group to receive placebo, low-dose micronized estradiol (E(2); 0.25 mg/1,800 kcal), the SERM tamoxifen (Tam; 20 mg/1,800 kcal), or E(2) + Tam for 4 months in a parallel-arm design.

Results: Tamoxifen alone resulted in overlapping but distinct effects compared with E(2). Both E(2) and Tam increased uterine weight and endometrial thickness, whereas only E(2) increased endometrial proliferation. Morphologic effects were similar for Tam and E(2) + Tam, which both induced stromal fibrosis and cystic change. Tamoxifen inhibited E(2)-induced proliferation and expression of genes related to cell cycle progression while exhibiting mixed agonist and antagonist effects on gene markers of estrogen receptor activity. The gene expression profile for E(2) + Tam was distinct from either E(2) or Tam alone but dominated by the Tam effect for estrogen-regulated genes. Tam also attenuated E(2) effects on both vaginal maturation and cervical epithelial height.

Conclusions: These findings characterize a novel phenotype resulting from estrogen + SERM co-therapy. The predominance of Tam effects on endometrial proliferation, morphology, and transcriptional profiles suggests that endometrial risks for E(2) + Tam may be similar to Tam alone.

Fig. 1

Tamoxifen (Tam) increases uterine weight and endometrial thickness while antagonizing estradiol (E2)-induced proliferation. A, Uterine weight and endometrial thickness were higher in all treatment groups. B-C, Endometrial proliferation, determined by immunolabeling and relative gene expression for the Ki67 marker, was greater only in the E2 group. The addition of Tam antagonized E2-induced proliferation (B). Images of Ki67 labeling (C) were taken at 200x magnification. Gene expression values were measured by qRT-PCR, corrected for internal control gene expression, and expressed relative to control group values. Vertical bars indicate 90% confidence intervals (gene expression) or standard errors (other measures). * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to respective control group values; ^# P < 0.05 compared to E2 group.

Fig. 2

Tamoxifen (Tam) and estradiol (E2) exhibit divergent effects on gene expression profiles in the endometrium related to cell proliferation and cell cycle. A, Venn diagram (upper) and hierarchical clustering dendrogram (lower) show greater percent overlap and tighter clustering between Tam and E2+Tam groups for all differentially expressed genes at fold-change (FC) >2. Dendrogram axis values represent Euclidean distances between groups. B, C, Principal component analyses (B) and heatmaps (C) of differentially regulated genes related to cell proliferation (n = 461) and cell cycle (n = 563) show a distinct pattern for E2 with close overlap of vectors for Tam and E2+Tam groups. D, Functional categories with significant overrepresentation of genes included cancer, cell growth and proliferation, and cell cycle. Treatment with E2 resulted in the largest number of differentially altered genes among these categories, while Tam and E2+Tam resulted in lesser number of genes represented. Diagrams in B and C correspond to significantly altered genes (ANOVA P < 0.05) within cell proliferation and cell cycle ontology categories. Diagrams in A and D correspond to the following gene filter: FC >2 in at least one group versus control, adjusted ANOVA P < 0.05, and quality > 2.

Fig. 3

Effects of tamoxifen (Tam) + estradiol (E2) on endometrial morphology are dominated by Tam. A, Increased endometrial fibrosis in Tam-treated groups corresponded with increased gene expression of the proteinase inhibitor ovostatin 2 (OVOS2). B, Representative images of superficial endometrium stained with Masson’s trichrome show increased stromal collagen (pale blue) in Tam and E2+Tam groups; images were taken at 100x magnification. C, Tam and E2+Tam groups showed greater luminal area within endometrial glands, indicative of cystic change noted on histology, while stromal edema was marginally higher only in the E2 group. Morphometric measures are expressed as percent total endometrial area measured. Vertical lines indicate 90% confidence intervals (gene expression) or standard errors (other measures). ** P < 0.01, and *** P < 0.001 compared to respective control group values; ^## P < 0.01 and ^### P < 0.001 compared to E2 group.

Fig. 4

Tamoxifen (Tam) antagonizes estradiol (E2) effects on lower reproductive tract measures. A, Representative images of vaginal epithelium show increased overall epithelial maturation and keratinization (superficial laminar zone) following E2 and antagonism of this effect by Tam. Images were taken at 100x magnification; H&E stain. B-D, Tam fully antagonized E2 effects on overall epithelial thickness (B) and keratin thickness (C) and partially antagonized stimulatory E2 effects on cervical glandular height (D). Vertical lines indicate standard errors. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to respective control group values; ^## P < 0.01 compared to E2 group.