Molecular evidence for differences in endometrium in severe versus mild endometriosis

Abstract

Women with stage III/IV versus stage I/II endometriosis have lower implantation and pregnancy rates in natural and assisted reproduction cycles. To elucidate potential molecular mechanisms underlying these clinical observations, herein we investigated the transcriptome of eutopic endometrium across the menstrual cycle in the setting of severe versus mild endometriosis. Proliferative (PE), early secretory (ESE), and mid-secretory (MSE) endometrial tissues were obtained from 63 participants with endometriosis (19 mild and 44 severe). Purified RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway Analysis and subsequently validated. Comparison of differentially regulated genes, analyzed by cycle phase, revealed dysregulation of progesterone and/or cyclic adenosine monophosphate (cAMP)-regulated genes and genes related to thyroid hormone action and metabolism. Also, members of the epidermal growth factor receptor (EGFR) signaling pathway were observed, with the greatest upregulation of EGFR in severe versus mild disease during the early secretory phase. The extracellular matrix proteoglycan versican (VCAN), which regulates cell proliferation and apoptosis, was the most highly expressed gene in severe versus mild disease. Upregulation of microRNA 21 (MIR21) and DICER1 transcripts suggests roles for microRNAs (miRNAs) in the pathogenesis of severe versus mild endometriosis, potentially through regulation of gene silencing and epigenetic mechanisms. These observed differences in transcriptomic signatures and signaling pathways may result in poorly programmed endometrium during the cycle, contributing to lower implantation and pregnancy rates in women with severe versus mild endometriosis.

Conflict of interest statement

The authors declared no potential conflicts of interests with respect to the authorship and/or publication of this article.

Figures

Figure 1.

Clustering analyses of samples from participants with mild and severe endometriosis. Panels A and B, Principal component analysis (PCA) of samples. A, Analyzed by menstrual cycle phase; and B, Analyzed by disease stage. PCA was applied to all endometrial samples that were characterized by the gene expression of all probes on the Affymetrix Gene 1.0 ST platform. C, Hierarchical clustering analysis of no-endometriosis and mild and severe endometriosis samples throughout the menstrual cycle, using the profiles of significantly regulated genes. PE indicates proliferative phase endometrium; ESE, early secretory phase endometrium; MSE, mid-secretory phase endometrium; m, mild endometriosis; s, severe endometriosis.

Figure 2.

Quantitative real-time reverse transcriptase−polymerase chain reaction (QPCR) validation of microarray data. Panels A, VCAN; B, EGFR; C, SLC1A1; D, IGFBP5; E, SST; F, TAGLN; G, DIO2 gene expression in severe endometriosis expressed as fold change compared to the expression in mild endometriosis, throughout the menstrual cycle. Microarray data are presented in the insert. *Statistically significant differences (P < .05) between the same cycle phases in severe vs mild endometriosis determined by the (Mann-Whitney test). Error bars represent ± SEM. PE indicates proliferative phase endometrium; ESE, early secretory phase endometrium; MSE, mid-secretory phase endometrium; SEM, standard error of the mean; VCAN, versican; EGFR, epidermal growth factor receptor; SLC1A1, solute carrier family 1, member 1; IGFBP5, insulin-like growth factor binding protein 5; SST, somatostatin; TAGLN, transgelin; DIO2, thyroxine deiodinase 2.

Figure 3.

Neuregulin pathway regulation in proliferative endometrium (PE) and mid-secretory endometrium (MSE) from participants with severe vs mild endometriosis analyzed by ingenuity pathway analysis (IPA). Red color indicates upregulation of a gene; green color, down-regulation.

Figure 4.

Epidermal growth factor receptor (EGFR) immunoreactivity in endometrial tissue from women without endometriosis (A-C), women with mild (D-F), and severe (G-I) endometriosis in the proliferative phase ([PE] n = 4, n = 4, and n = 5, respectively; A, D, G), early secretory phase ([ESE] n = 4, n = 3, and n = 7, respectively; B, E, H), and mid-secretory phase ([MSE] n = 4, n = 3, and n = 5, respectively; C, F, I) of the cycle. Human myometrial tissue was used as an internal positive control (on the same slide as endometrium, adjacent to the basalis endometrium, J). Human 12-week gestation placental tissue (K) was used as an additional positive control. L indicates negative control, nonimmune IgG-treated human endometrium; Le, luminal epithelium; ge, glandular epithelium; st, stroma; myo, myometrium; IgG, immunoglobulin G. Magnification ×200.

Figure 5.

Versican (VCAN) immunoreactivity in endometrial tissue from women without endometriosis (A-C), and women with mild (D-F), and severe (G-I) endometriosis in the proliferative ([PE] n = 4, n = 4, and n = 5, respectively; A, D, G), early secretory ([ESE]; n = 4, n = 3, and n = 7, respectively; B, E, H), and mid-secretory ([MSE]; n = 4, n = 3, and n = 5, respectively; C, F, I) phases of the menstrual cycle. Mouse lung tissue (J) and mouse ovary (K) were used as positive controls. L indicates negative control, nonimmune IgG-treated human endometrium; Le, luminal epithelium; ge, glandular epithelium; st, stroma; v, blood vessel. Magnification ×200.