**4.6 Epigenetics in endometriosis: methylation pattern of sexual steroid hormone receptors**

There is accumulating evidence that various epigenetic aberrations exist in endometriosis. In the last 10 years, evidence from reviewed and retrieved studies has emerged that endometriosis may be an epigenetic disease [153], epigenetics appearing to have a better explanatory power than genetics, and to be a common denominator for hormonal and immunological aberrations in the puzzle of endometriosis. Genomic imprinting; DNA methylation; histone modifications with different nominations (acetylation/ histone phosphorylation/histone ubiquitylation/ histone sumoylation); microRNAs [183] and recently discovered, stranded, noncoding RNAs (ncRNAs) [168]; transcription factor network; and chromatin remodeling are known to regulate transcription of target genes. Genomic imprinting is an epigenetic phenomenon known to regulate DNA methylation of either maternal or paternal alleles [184], and the male and female germ lines guide the allele-specific DNA methylation marking and histone modification onto specific gene regions of parental alleles [185]. The epigenome, the collection of DNA methylation, and the histone modifications can be influenced by environmental factors. Exposure to xenobiotics, chronic inflammation, and transient hypoxia are associated with DNA hypomethylation of stromal endometrial stem cells through the destabilization of *DNMT1* mRNA; *DNMT1*, *DNMT3A*, and *DNMT3B* are overexpressed in the epithelial component of endometriotic implants as compared to normal controls or in the

*Molecular Bases of Endometriosis - The Integration Between Research and Clinical Practice*

**in endometriosis**

enhance translation in certain biological scenarios.

**4.5 MicroRNA (miRNA) dysregulation of steroid hormone receptor expression** 

The receptors of sexual steroid hormones (ERs, PRs) are involved in endometriosis, and correlated to them is the exploration of the correlation between microRNA (miRNA) and ER/PR in eutopic and ectopic endometrium. There are recently discovered stranded noncoding RNAs (ncRNAs) [168]. MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules with approximately 22 nucleotides in length, and they control posttranscriptional gene regulation, which were proposed to contribute to human reproductive physiology; their abnormal expression was involved in the pathogenesis of many diseases of female reproductive tract, including endometriosis. While the majority of the literature supports the notion that miRNAs inhibit translation, there is some evidence that miRNAs can actually

miRNAs are postulated to play a role in normal biological processes, to be critical regulators of cellular development and physiology, while their mis-expression has been associated with numerous diseases [169]. A recently published paper [170] at the Department of Molecular and Integrative Physiology, at the University of Kansas Medical Center (USA) tries to determine if the "mis-expressed" endometriotic tissue is a cause (driver of the disease) or a result of endometriosis (passenger). More than 10 years ago, there are comparative microanalyses of genetic expressions—the miRNAs in women with ectopic endometrial cells and eutopic endometrium—that have proven an alternative pattern in the two groups [171–173], as well as the differences of ill women versus disease-free women [172–175]. miRNA regarding ER shows a ratio of 100:1 in *ESR2* (RE*-*β) to *ESR1* (RE-α) in endometriomas vs. superficial endometriotic lesions and eutopic endometrium [112, 113].

Different miRNAs are identified by microarray with real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and they were done in paired eutopic/ectopic endometrium from the same patients. In the published papers, there are identified upregulated (over expressed) and downregulated (underexpressed) miRNA expressions in eutopic and different ectopic lesion locations (peritoneal, ovarian), and some are "mis-expressed." Using the Ingenuity Pathway Analysis (IPA) software, potential molecular pathways were assessed and determined to involve c-Jun, CREB-binding protein, protein kinase B (AKT), and cyclin D1 (CCND1) signaling, all of which have previously been associated with endometriosis pathogenesis [136]. According to the involvement in endometriosis, the molecules directly involved in endometriosis (cytokines, enzymes, growth factors, kinases, ion channels, ligand-dependent nuclear receptors, peptidases, phosphatases) are assessed. There are differences between authors; there were conflicting reports on whether or not miRNA expression was influenced by the stage of the menstrual cycle (proliferative [136] or secretory [176]), the type of endometrial cell (miRNA of endometrial stromal cells was for the first time assessed by Hawkins et al. [177]), the type of miRNA, and the level in eutopic and ectopic tissue [miRNA from *miR-29* family (was evaluated using primary human endometrial stromal cells in vitro)], which had different levels—(high for [136] and [176] and decreased for [178], who compared it to disease-free women). The role of *miR-29c* in endometrial cell proliferation, invasion, and apoptosis in vitro was examined. *miR-29c* suppressed endometrial cell proliferation and invasion, promoting cell apoptosis. The conclusion was that *miR-29c* exhibits inhibitory action on endometrial cell proliferation and invasion by inhibiting the expression of *c-Jun*. Another studied miRNA—the stromal cell *miR-183*, was examined in response to ovarian steroids (17β-estradiol and P4), and inflammatory cytokines (TNF-α) were concluded from this study that repressed levels of *miR-183* may modulate the growth and invasive potential of

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eutopic endometrium of women with endometriosis [186], but fetal programming postulates that chronic adult-onset diseases with an epigenetic component originate in utero when the early embryo is exposed to factors that permanently shape its epigenetic mark, a fact presented in the previous subchapter.

Aberrant DNA methylation represents a possible mechanism, linking gene expression alterations observed in endometriosis with hormonal and environmental factors. Methylation is one of the most important epigenetic functions that implies the addition of methyl group at the DNA dinucleotides in the position 5 of the cytosine of the "promoter" zone and induces silencing of the gene under DNA methyltransferase action [68, 187].

Endometriotic lesions have altered methylation patterns of ER-β, and the ER may mediate regulation of one another [188]. The authors consider that aberrant DNA hypomethylation of ER may favor the progression to cancer of old ectopic endometrial lesions.

DNA methylation makes possible the process "epithelial to mesenchymal transition" (EMT), in which the epithelial cells lose polarized organization of the cytoskeleton and cell-to-cell contacts, acquiring the high motility of mesenchymal cells. It is supposed that two stimulating signals, hypoxia and estrogen, can activate the EMT process in endometriosis through different pathways. The pathways involve many cellular factors such as TGF-β and Wnt, ultimately leading to cell proliferation and migration, and the changes of epithelial cells are thought to be prerequisites for the original establishment of ectopic endometriosis lesions [189].

The reasons for non-response to progestins in endometriosis are not entirely clear, studies point the possible epigenetic silencing of the PR gene, without knowledge of causes for the epigenetic silencing of PRs. DNA methylation of the ER and PR promoter has been demonstrated in endometriosis; inflammation and oxidative stress can be involved in epigenetic changes in DNA and chromatin remodeling proteins [190**–**192]. ER-β promoter is hypomethylated in endometriotic cells, which accounts for its overexpression [193]. The promoter of PR-B hypermethylation is concomitant to reduction of PR-B, a fact explaining progesterone resistance in endometriosis [11, 114].

Ectopic endometrial stromal cells are hypomethylated, and are different from normal endometrial stromal cells which are hypermethylated [194].

The hypermethylation of *HOXA 10* promoter reduces HOXA 10 expression in induced endometriosis of stromal endometrial cells of mice [195].
