In silico analysis identifies a novel role for androgens in the regulation of human endometrial apoptosis

Abstract

Context: The endometrium is a multicellular, steroid-responsive tissue that undergoes dynamic remodeling every menstrual cycle in preparation for implantation and, in absence of pregnancy, menstruation. Androgen receptors are present in the endometrium.

Objective: The objective of the study was to investigate the impact of androgens on human endometrial stromal cells (hESC).

Design: Bioinformatics was used to identify an androgen-regulated gene set and processes associated with their function. Regulation of target genes and impact of androgens on cell function were validated using primary hESC.

Setting: The study was conducted at the University Research Institute.

Patients: Endometrium was collected from women with regular menses; tissues were used for recovery of cells, total mRNA, or protein and for immunohistochemistry.

Results: A new endometrial androgen target gene set (n = 15) was identified. Bioinformatics revealed 12 of these genes interacted in one pathway and identified an association with control of cell survival. Dynamic androgen-dependent changes in expression of the gene set were detected in hESC with nine significantly down-regulated at 2 and/or 8 h. Treatment of hESC with dihydrotestosterone reduced staurosporine-induced apoptosis and cell migration/proliferation.

Conclusions: Rigorous in silico analysis resulted in identification of a group of androgen-regulated genes expressed in human endometrium. Pathway analysis and functional assays suggest androgen-dependent changes in gene expression may have a significant impact on stromal cell proliferation, migration, and survival. These data provide the platform for further studies on the role of circulatory or local androgens in the regulation of endometrial function and identify androgens as candidates in the pathogenesis of common endometrial disorders including polycystic ovarian syndrome, cancer, and endometriosis.

Fig. 1.

Expression of candidate androgen-regulated gene mRNA in total tissue extracts from human endometrium and decidua as determined by quantitative RT-PCR. Samples were homogenized from functional endometrium recovered during the proliferative (P) and midsecretory phases (MS) as well as from first-trimester decidua (Dec). Concentrations displayed relative to those in proliferative phase in each case (n = 4–6 per group).

Fig. 2.

Time-dependent changes in androgen-regulated gene expression in primary hESC. Cells were incubated with vehicle (gray bars) or 10⁻⁸ m DHT (black bars) for 2, 8, and 24 h (n = 6 each time point). Concentration of mRNA was quantified by quantitative RT-PCR and expressed as fold change compared with time-matched vehicle-treated hESC. Note that with the exception of CD44, incubation of cells with DHT resulted in either a significant reduction in concentration of mRNA or no significant change but with a trend to a reduction. For most genes, changes in mRNA expression were time dependent with the most striking change in total concentrations at 2 h for eight of 12 of the genes. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

Pathway analysis of endometrial androgen target gene set. Note 12 of 15 androgen-regulated genes identified by in silico screening (indicated with multicolored circles) were predicted to interact in a single Metacore pathway centered on AR; intermediate molecules are indicated to show putative intermediate signaling molecules. Key shows the functional classification of the target genes and the arrows indicate predicted regulation (red, negative; green, positive).

Fig. 4.

Treatment of hESC with DHT alters apoptosis and proliferation. A, Apoptosis as measured by caspase-3/7 assay in cells treated with staurosporine. Note addition of DHT had a significant impact (*, P < 0.05) on the rate of apoptosis when compared with controls but that the impact of DHT was blunted in the presence of E2 (n = 4). B, Wound-healing assay. Addition of DHT had a significant impact (*, P < 0.05) compared with its vehicle control (ethanol), whereas flutamide alone had no impact compared with vehicle (methanol) and pretreatment of cells with flutamide blocked the DHT-dependent reduction in wound closure. DHT, 10⁻⁸ m; flutamide, 10⁻⁵ m; n = 6.