In the past two decades, epigenetic mechanisms have been recognized as key factors in the development of complex human disorders, including cancer, and neurodegenerative, neurological and autoimmune diseases (1). Changes in chromatin conformation constitute the basis of epigenetic effects since they can alter gene expression without depending on DNA sequence changes. Chromatin epigenetic mechanisms include DNA methylation and post-translational covalent modifications of histones. These modifications comprise heritable alterations that regulate essential biological processes, such as gene and microRNA expression, cellular differentiation, X-chromosome inactivation and genomic imprinting. DNA methylation is the most widely studied and best characterized epigenetic modification of the human genome (2). This epigenetic modification is catalyzed by DNA methyltransferases (DNMTs), which add a methyl group to the carbon 5 of cytosines that are followed by a guanine. Thus, DNA methylation occurs almost exclusively in the context of a 5′-CpG-3′ dinucleotide, particularly in dense CpG regions termed CpG islands. Promoter-specific CpG island hypermethylation is known to be associated with gene silencing due to their transcription repression effect (3).

Recent evidence indicates that endometriosis, an enigmatic disease in which endometrial-like tissue is detected outside the uterus, is also an epigenetic disease (4–6). This hypothesis was based on the abnormal DNA methylation patterns observed in the promoter regions of specific genes (7–12) and the higher expression levels of DNMTs in endometriotic lesions in comparison with normal endometrium (13). The first evidence of increased levels of DNA methylation of a specific gene came from a pioneer study by Martini et al (7), which found aberrant DNA methylation in the tumor-suppressor genes, cyclin-dependent kinase inhibitor 2A (TP16) and mutL homolog 1, colon cancer, nonpolyposis type 2 (hMLH1) in one and four samples of endometriosis, respectively. Subsequently, by using a candidate gene approach, other studies have described aberrant methylation patterns in the promoter region of genes encoding the transcription factor homeobox 10A (HOXA10) (8), progesterone receptor isoform B (PGR) (9), estrogen receptor 2, ERβ (ESR2) (10), nuclear receptor subfamily 5, group A, member 1 (NR5A1, primarily named SF-1 or steroidogenic factor-1) (11) and cyclooxygenase-2 (COX-2) (12) in endometriotic lesions derived from different anatomical sites. Furthermore, endometriotic cells treated in vitro with the demethylating agent 5-aza-2′deoxycytidine showed an increase in the expression of aromatase mRNA, suggesting that DNA methylation of the CYP19A1 aromatase gene also occurs in stromal cells from endometriotic lesions (14).

Endometriosis is believed to be an estrogen-dependent disease characterized by the loss of progesterone-protective signaling in endometriotic cells. Xue et al (10) demonstrated that this effect could be related to hypomethylation of the ESR2 gene in stromal endometriotic cells, which could lead to an increase in the expression of estrogen receptor β (ERβ). These authors also demonstrated the presence of DNA methylation in a specific region of ESR2 that confers promoter activity and co-localizes with a CpG island: in vitro methylation was correlated with inactivation of this promoter. Subsequently, it was proposed that the increased expression of ERβ, due to the hypomethylation of its coding gene, could inhibit the expression of the PGR gene (15). In addition, hypermethylation of the PGR gene, particularly at promoter B, was also observed in the epithelial component of endometriotic lesions (9), with consequent decrease in the expression of its transcript.

Overall, steroid hormone receptor genes show a complex pattern of regulation involving multiple promoters and alternative mRNA isoforms. The ESR1 gene has two proximal promoters (transcripts A and B) located within ~2 kb of the translation start site and an upstream promoter C (16,17). Similarly, the PGR gene has two alternative transcripts regulated by two promoter-specific regions for the PGRA and PGRB isoforms, which are associated with well-characterized CpG islands (18). The ESR2 gene also presents alternatively spliced transcript variants and distinct promoters regulated by DNA methylation (19), although not associated with the presence of classic CpG islands.

Potentially, aberrant DNA methylation can disrupt the effects of hormone steroids due to changes in the expression levels of its receptors and may influence the origin and progression of endometriosis. Thus, the present study was conducted to investigate the methylation patterns of the alternative promoter regions of the ESR1, ESR2 and PGR genes in intestinal deep endometriosis, which accounts for 8–12% of all endometriosis. This condition is characterized by multifocal infiltrating and aggressive lesions associated with pelvic pain and infertility (20).

The set of biological samples investigated in the present study was comprised of 44 cases of intestinal deep endometriosis. In this group of women, the most frequent symptoms were moderate to severe dysmenorrhea (n=39, 89%), dyspareunia (n=29, 66%) and infertility (n=24, 54%). Moreover, the majority of the endometriotic specimens were classified as advanced stages III and IV (n=31, 70%). The histological classification was able to be assessed in 36 cases indicating a predominance of glandular and stromal pattern (n=26, 72%) and an undifferentiated glandular pattern in 9 cases (25%). In addition, the menstrual cycle stage was determined as proliferative in almost all of the tissue samples evaluated for this parameter (34/39 cases, 87%).

As expected, the presence of methylation as determined by the MS-PCR assays was inversely correlated with the expression at the protein level in the three breast cell lines investigated. Methylated alleles for PGRA, PGRB, ESR1A and ESR1B were detected in the MDA-MB-231 cell line (ER- and PgR-negative) and only unmethylated alleles were found in the T47D breast cancer cells (ER- and PgR-positive). Similarly, in the MCF-7 cells (ER- and PgR-positive) only unmethylated alleles were detected in the ESR1A, PGRA and PGRB promoter regions, while both methylated and unmethylated alleles were found in the ESR1B promoter. These methylation pattens were also confirmed in the Cancer Methylome System, which present the methylation profile generated by immunoprecipitation using the methyl-CpG binding domain of the MBD2 protein followed by high-throughput sequencing (27) (available at http://cbbiweb.uthscsa.edu/KMethylomes/) (data not shown).

Subsequently, the PGRB and ESR2 promoter methylation patterns were obtained in the 44 cases of infiltrating intestinal deep endometriosis. Unmethylated and methylated alleles were observed for the ESR2 gene in all samples analyzed. PGRB promoter methylation was observed in 39% (17/44 cases) of the endometriotic lesions. In 7 of the 44 samples, it was also possible to determine the methylation pattern in matched endometrium obtained from the same patient. No difference was observed in the ESR2 methylation pattern in these cases; however, only unmethylated alleles were observed in the PGRB promoter region. Thus, direct comparison of the PGRB promoter region methylation between matched endometrium and endometriotic samples revealed a differential pattern in 4 pairs since hypermethylated alleles were specifically detected in the endometriotic tissue (Fig. 1).

In an attempt to remove the adjacent intestinal tissue in the fragments containing the endometriotic infiltrating lesions, macrodissection of the frozen tissue specimens was performed, which resulted in a limited quantity of DNA for the multiple MS-PCR tests. Thus, the MS-PCR analysis of the ESR1A, ESR1B and PGRA promoters was possible in a subset of only 37 out of 44 endometriotic lesions. While methylated and unmethylated alleles were simultaneously detected for both ESR1 promoters in all of these samples, PGRA methylated alleles were only detected in 7 lesions (19%) (Fig. 1).

Differences in the DNA methylation frequencies in endometriotic lesions were observed for the two promoter regions of the PGR gene: 14 samples were methylated at the PGRB promoter, 7 were methylated at the PGRA promoter, and 3 were methylated at both promoters (cases 11, 13 and 14). However, no statistically significant differences were observed between the presence/absence of DNA methylation of the A and B promoter regions of the PGR gene and the stage of the lesions, histological classification, or menstrual cycle phase (Table III).

The immunostaining of the ERα, ERβ and PgR proteins in the endometriotic lesions exhibited a heterogeneous pattern with the presence of both positive and negative epithelial and stromal cells (Fig. 2). Of note, some endometriotic samples showed positive staining for PgR only in the stromal component, while the epithelial component were negative (Fig. 1, case 3 and Fig. 2H). In these cases, both methylated and unmethylated alleles were identified, probably due to the epigenetic silencing mediated by DNA methylation of the PGR gene in the epithelial fraction of the endometriotic lesions.

The major finding of our study was that promoter region B of the PGR gene differed in regards to the methylation status between eutopic endometrium and deep endometriosis compromising the rectum: methylated alleles were specifically detected in the endometriotic lesions, while endometrium samples showed only unmethylated alleles. These data suggest that this epigenetic alteration may be a possible biomarker for endometriosis. Using the same MS-PCR primer set, Wu et al (9) reported the presence of partial methylation in this promoter in the epithelial component of peritoneal and ovarian endometriotic implants, but not in the promoter region that controls the expression of the A isoform. In addition, the authors reported low levels of PGRB expression in endometriotic epithelial cells. In the present study, we demonstrated that both A and B promoter regions of the PGR genes were methylated in a subset of infiltrating intestinal endometriosis.

It has been proposed that the PGRB isoform is crucial for mediating the inhibitory effects of progesterone on cell growth and invasion, while PGRA has a suppressor function (28,29). Thus, the results of the present study indicates the importance of DNA methylation alterations of the PGR gene in a subset of infiltrative deep endometriosis, which is characterized by multifocal lesion sites, increased aggressiveness and severe clinical symptoms as compared to pelvic endometriosis, which was the type commonly investigated in previous epigenetic studies (7,9–11,14).

Collectively, these data support the observation that endometriotic lesions exhibit a trend of partial methylation of the PGR gene, which does not completely inactivate gene expression in the entire lesion focus, yet to some extent, is responsible for a decrease in the expression of the PgR receptor restricted to various areas or specific cells. These findings were validated in the present study by the heterogeneous pattern observed upon PgR immunohistochemistry (Fig. 2H), which showed positive and negative staining for the PgR receptor coexisting in the same endometriotic focus. This observation may explain the lower expression level of progesterone receptors in endometriosis and may be associated with the fact that approximately 9% of women with endometriosis are not responsive to treatment with progestin (5).

At present, endometriotic lesions are described as estrogen-dependent endometrium-like tissue consisting of glands and stroma. This abnormal tissue exhibits a unique expression profile of steroid hormone receptors as compared to matched eutopic endometrium or endometrium from women without endometriosis. Previous studies have detected lower levels of ERα and higher levels of ERβ in endometriosis (30,31). ERα appears to be the primary mediator of estradiol-induced progesterone action in this tissue (32), and progesterone exerts its functions in the endometrium by binding to the nuclear receptors PgRA and PgRB.

Epigenetic mechanisms modulate the dynamic regulation of the estrogen receptor genes and their functions (32). Promoter-specific DNA methylation may be causally related to the differential expression of the ERα and ERβ receptors in endometriosis. These receptors have differential affinity for ligands, are differentially expressed in a tissue-specific manner and may function antagonistically (33). Both ER isoforms exist as several splice variants. The alternative transcripts are ubiquitous, although their biological significance remains poorly understood (34). Furthermore, alternative promoter usage leads to pre-mRNAs with a variable length non-coding 5′ untranslated region (35–37).

In breast cancer cell lines, ESR1 expression was previously found to be suppressed by promoter DNA methylation (38,39) and histone hypoacetylation (40) and was derepressed by DNA methyltransferase (5-aza-2′deoxycytidine) and histone deacetylase (trichostatin A) inhibitors (41,42). Promoter region hypermethylation has also been shown to be inversely associated with ESR2 expression levels (43). In line with this, the methylation pattern of the ESR1 gene was investigated in endometriosis in the present study and methylated alleles at both promoters A (ESR1A) and B (ESR1B) were found. The consequences of DNA methylation in gene expression are best understood at CpG island-containing promoters, however, the two promoter regions investigated in the present study, ESR1B and ESR2, are CpG island-free (Fig. 1). However, this also represents a promising gene region for further DNA methylation studies in endometriosis, since this region has been confirmed to be differentially methylated in studies investigating other diseases, such as prostate or breast cancer (25,44), suggesting that the regulation of this gene by DNA methylation overlaps other complex molecular mechanisms as recently described (45,46).

To the best of our knowledge, only one study has evaluated the methylation status of the ESR2 gene in 8 ovarian endometriomas by bisulfite-modified DNA sequencing (10). The authors described high levels of ESR2 transcripts associated with hypomethylation of the respective promoter region in comparison with endometrium. In contrast, in the present study, MS-PCR analysis targeting a different region of an alternative promoter of ESR2 (Fig. 1) in matched samples of the endometria and intestinal deep endometriosis showed a more complex pattern, with methylated and unmethylated alleles in both tissues. Notwithstanding, the region investigated in this study represents the beginning of numerous new transcripts identified as expressed at premenopausal endometrium or endometriosis (GenBank, available at http://www.ncbi.nlm.nih.gov/nuccore, accession numbers AF074598 and AF074599, respectively). In this context, it is tempting to speculate whether the ESR2 gene generates multiple transcripts by triggering transcription initiation at alternative sites that could be inactivated by tissue-specific methylation. Therefore, interpretation of differential DNA methylation patterns has proven difficult, in part because the functional consequences depend on the genomic region involved, the specific CpG dinucleotides, and inter-tissue and intra-tissue heterogeneity. In line with this, the immunostaining performed in this study was able to indicate the heterogeneity inherent to endometriotic lesions, due to the different staining pattern between epithelial and stromal cells, or even within the same cellular component (stromal or epithelial). Furthermore, this heterogeneity was found to be increased due to the additional presence of other types of infiltrative cells, such as inflammatory cells (Fig. 2A and B).

From a translational research perspective, gene-specific methylation patterns may be useful biomarkers for detection since alterations in DNA methylation potentially provide a positive signal for endometriotic cell detection. MS-PCR is an extremely sensitive PCR-based method, and it has been used to detect hypermethylated genes in samples of limited quantity derived from patients with various types of malignancies, such as those obtained from fine needle aspiration, biopsies and corporal fluids (47). In this context, epigenetic studies may lead to the identification of the target genes of aberrant DNA methylation in endometriosis, thus representing a promising strategy for the monitoring of the onset and progression of the disease. In the present study, we showed that DNA methylation in the promoter of the PGRB gene is a potential biomarker for endometriotic lesions as compared to endometrium from the same patient. Moreover, aberrant PGRB methylation was able to be identified in the entire lesion focus, without the necessity of isolation of the glandular and stromal components. Thus, this epigenetic change has the potential to be considered as a useful biomarker for the pathogenesis of a subset of endometriosis consisting of intestinal deep endometriosis.

In summary, these data indicate that abnormal methylation patterns occur in deep endometriosis compromising the rectum. Further studies are clearly necessary to elucidate the possible relationship between DNA hypermethylation of the A and B promoter regions of the PGR gene and progesterone resistance. In addition, future epigenetic studies involving the evaluation of steroid receptor methylation in endometriosis should consider the histological pattern and each cellular component separately (stromal or epithelial) in order to verify the effect of DNA methylation on the expression of each alternative steroid receptor transcript in this complex disorder.