Effects of noncavity-distorting fibroids on endometrial gene expression and function

Abstract

Uterine fibroids are a common finding in infertility patients. Impaired implantation and decidualization have been proposed to contribute to compromised fertility. Data are limited on the endometrial transcriptome from subjects with uterine fibroids, as well as endometrial receptivity and decidualization potential of endometrial stromal fibroblasts (eSF) from women with fibroids. Our objective was to investigate the endometrial transcriptome of women with noncavity-distorting intramural fibroids and compare them to control subjects with no uterine pathology throughout the menstrual cycle. We also evaluated endometrial receptivity gene expression and basic endometrial functions such as decidualization, proliferation, and apoptosis in women with fibroid uterus. Results showed that large numbers of transcripts were significantly dysregulated throughout the menstrual cycle in fibroid subjects compared to controls. However, there were essentially no differences in the expression of receptivity markers at the tissue level, as well as decidualization markers in tissue and eSF in subjects with fibroids compared to controls. However, eSF from women with a fibroid uterus exhibited decreased proliferation potential and increased apoptosis upon decidualization. These data indicate preserved implantation and decidualization potential despite observed gene expression changes in endometrium from women with noncavity-distorting fibroids compared to controls. How this phenomenon and altered proliferation/apoptosis may contribute to impairment of endometrial function in subfertile patients warrants further investigation.

Keywords: apoptosis; decidualization; endometrial fibroblasts; noncavity-distorting fibroids; proliferation; receptivity.

Figure 1.

(A) Principal component analysis of eutopic endometrium in patients with noncavity-distorting fibroids throughout the menstrual cycle in proliferative phase (PE, n = 7, gray), early secretory phase (ESE, n = 3, pink), and mid-secretory phase (MSE, n = 4, green), in comparison to controls with no uterine/pelvic pathology (PE, n = 20, burgundy), (ESE, n = 6, red), and (MSE, n = 8, blue). (B) Hierarchical cluster analysis of relative expression of genes in the endometrium of subjects with noncavity-distorting fibroids throughout the menstrual cycle in comparison to controls, using the profiles of statistically significantly differentially expressed genes. Yellow = no difference in gene expression, blue = downregulated genes, red = upregulated genes. Each horizontal row represents a single sample, and each vertical line represents a single gene. (C) Venn diagram of common and unique genes expressed in in the endometrium of subjects with noncavity-distorting fibroids throughout the menstrual cycle in comparison to controls with no uterine/pelvic pathology.

Figure 2.

Expression of mRNA for implantation and decidualization markers, steroidogenic enzymes, and steroid hormone receptors in human endometrial stromal fibroblasts (eSF) from subjects with noncavity-distorting fibroids throughout the menstrual cycle (n = 14) in comparison to controls with no uterine/pelvic pathology (n = 6), treated with (A) 10 nmol/L estradiol (E₂) alone or E₂ (10 nmol/L) plus progesterone (P₄, 1 μmol/L) (E₂/P₄) for 14 days, expressed as fold change to the expression in 14 days no-treatment controls, (B) 0.5 mM with 8Br-cAMP for 96 h or vehicle, expressed as fold change to the expression in 96 h no-treatment controls, as revealed by real-time RT-PCR. *Statistical significance accepted as P ≤ 0.05. Error bars represent means ± SD. (C) IGFBP1 and PRL protein secretion in culture media from eSF treated with and without E2 and P4 for 14 days or cAMP for 96 h, normalized to the total RNA level. *Statistical significance accepted as P ≤ 0.05. Error bars represent means ± SD.

Figure 3.

Proliferation and apoptosis in endometrial stromal fibroblasts (eSF) from women with and without intramural fibroids. (A) WST-1 proliferation assay in eSF (n = 5 for subjects with and without fibroids each, in duplicate), cultured for up to 5 days in 96-well plates and treated with WST-1 for 4 h prior to analysis. Negative control wells had no cells and were treated with WST1. Asterisks indicate statistically significant differences at P < 0.05. Error bars represent means ± SD. (B) BrdU incorporation in eSF (n = 3 for subjects with and without fibroids, in triplicates) treated with (decidualized) and without cAMP for 96 h. Statistical significance accepted at P < 0.05. Error bars represent means ± SD. Same letters represent significantly different comparisons. (C) TUNEL apoptosis assay on eSF (n = 3 for subjects with and without fibroids, in triplicate) on tissue chamber slides incubated with (decidualized) and without cAMP for 96 h. The number of positively stained cells (images not shown) per 100 blue cells was counted within three different high-power magnification (×40) fields for each subject and each treatment group. Values represent the mean percentage of positively stained cells ± SD. Statistical significance accepted at P < 0.05. Same letters represent significantly different comparisons.