Molecular biological markers of endometriosis

Introduction

Endometriosis is a chronic benign disease characterized by the proliferation of tissue, functionally and morphologically similar to the endometrium, beyond the uterine cavity. Endometriosis affects up to 10% of women of reproductive age and is of great social importance, as it significantly reduces the woman’s performance efficiency and quality of life. Clinically, this disease is quite multifaceted; its main symptoms include pain syndrome, dysmenorrhea, dyspareunia, abnormal uterine bleeding, heavy menstrual bleeding, decreased fertility, and infertility [1].

Diagnostics of endometriosis is based on the clinical picture, ultrasound examination, and data of magnetic resonance imaging but establishing diagnosis is often complicated by the lack of objective criteria. Moreover, the clinical picture of endometriosis often begins to manifest itself at a stage when the disease is difficult to detect instrumentally; as a result, women remain without a diagnosis for several years. Invasive methods such as hysteroscopy or laparoscopy with subsequent histological examination are the "gold standard" of diagnosis although they are involved exclusively in severe clinical cases and cannot be recommended for widespread use.

Despite the practical necessity, there are currently no minimally invasive molecular or genetic markers for the diagnosis of endometriosis. The findings of a large number of studies have shown that the disease arises from a complex combination of hormonal, immunological, inflammatory, genetic, and epigenetic factors. A detailed investigation of these factors should ultimately lead to the introduction of new approaches to effective and minimally invasive diagnostics of endometriosis into clinical practice. This review highlights some of the results of recent studies aimed at identifying features of the eutopic endometrium in endometriosis, which can ultimately be used for early diagnosis of the disease.

Modern data indicate that the eutopic endometrium in patients with endometriosis differs from that in healthy women by structure, proliferative activity, ability to invade through components of the proteolysis and angiogenesis systems, the state of the receptor apparatus, expression of various genes, and possesses the properties of autonomous growth that ensures its high level of survival [1].

Below we will highlight current data on the role of various molecules in the pathogenesis of endometriosis, as possible markers for diagnosing the disease.

The paper provides an overview of modern data on the results of the search for molecular biological markers of endometriosis in ectopic foci and eutopic endometrium, the features of their expression, and the possibility of using them in medical practice. The works, cited in this study, were selected using the keywords "endometriosis", "microRNA", "molecular markers of endometriosis", and "eutopic endometrium" in the PubMed and MedLine search engines. Publications had to meet the following criteria: publication dates no more than 5–7 years ago; publication languages were Russian and English; access to the full text of the publication was mandatory. Papers not indexed in medical databases were not subject to study. In addition, oncological studies, studies on extragenital forms of endometriosis, and investigations on methods of invasive diagnostics and surgical treatment were excluded from the analysis. From approximately 500 publications, 29 papers were selected that met these criteria and best reflected the current state of the issue of non-invasive and minimally invasive diagnostics of endometriosis.

Matrix metalloproteinases

One of the features of endometrioid lesions is the ability to invade surrounding tissues, while being a benign process. Invasion of ectopic endometrium into tissue occurs partly under the action of matrix metalloproteinases (MMPs) with subsequent cell proliferation and formation of endometrioid heterotopias under the effect of growth factors and steroid hormones. Membrane-type MMPs (MT-MMPs) are a subgroup of the MMP family and are key molecules in the degradation of the extracellular matrix. Regulation of membrane MMP type 1 (MT1-MMP, MMP14) is often disturbed in different cancer tissues and fluids in body of cancer patients. Considering the fact that endometriosis also involves invasion, albeit benign, into surrounding tissues it has been suggested that MMPs may also participate in the pathogenesis of this disease. This assumption was confirmed in the works by Maoga et al. The studies revealed that in the case of adenomyosis, MT1-MMP protein levels were significantly increased in ectopic foci compared to eutopic endometrium. Analysis of MT1-MMP levels assessed with enzyme immunoassay also revealed a significant increase in the concentration of MT1-MMP in the serum of patients with endometriosis compared to the control group [2]. Similar data were obtained by Muharam et al. who showed that the expression level of MMP type 3 (MMP3) tended to increase in both eutopic and ectopic endometrial tissues of women with endometriosis [3]. Various forms of adenomyosis are characterized by uneven boundaries of the endometrium and myometrium, which is stipulated by the presence of multiple foci of ingrowth of the basal layer of the endometrium through the terminal plate into the myometrium. In both diffuse adenomyosis and adenomyomas, the basal layers of the eutopic and ectopic endometrium differ by many (3–8.5) times, demonstrating a higher expression of enzymes (moesin, PAK 4, MMP 2 and MMP 9) in epithelial and stromal cells; that affects their invasive activity as well as an increase in the number of CD34 cells in the stroma [4].

HOXA10 gene

The HOXA10 gene is a regulator of morphogenesis and differentiation of embryonic tissues and affects the growth and differentiation of endometrial cells, receptivity, decidualization, and susceptibility to embryonic implantation. In healthy fertile women, the level of HOXA10 expression varies during the menstrual cycle. It is reduced in the follicular phase but by the middle of the secretory phase, gene expression increases, which coincides with the moment of embryo implantation. High levels of HOXA10 expression are necessary for normal decidualization of the endometrium. In women with endometriosis, the level of HOXA10 expression remains low during the secretory phase of the menstrual cycle, which ultimately leads to implantation failures and increases the ability to invade the eutopic endometrium through changing its receptivity. Some studies have demonstrated that this occurs as a result of aberrant hypermethylation of the HOXA10 gene due to increased levels of H3K9ac, H3K27me3, and H3K4me3 in the promoter region of the gene [5-7]. In other studies, high levels of HOXA10 DNA methylation were also revealed in endometrial tissue from women with endometriosis, which entailed its decreased expression [8-10]. Thus, HOXA10 may be proposed as one of the potential biomarkers for diagnosing endometriosis.

Telomerase activity

Interesting data were obtained in the study of the peculiarities of telomerase activity in the eutopic endometrium in patients with endometriosis. Telomeres are specialized nucleoprotein complexes at the ends of chromosomes, which contain repeating nucleotide sequences. They protect the genome from nucleolytic degradation, unwanted recombinations, repairs, and end-to-end fusion of chromosomes. Their activity is supported by a specialized enzyme, telomerase. The human telomerase enzyme consists of three main subunits including the telomerase RNA component (hTERC), telomerase reverse transcriptase (hTERT), which is the catalytic subunit of the enzyme, and the dyskerin protein.

During retrograde menstruation, elevated telomerase activity in the late secretory endometrium of women with endometriosis may result in the survival of endometrial cells in the peritoneal cavity and the development of ectopic lesions. This is consistent with the evidence that hTERC RNA levels are significantly higher in eutopic endometrium samples at the secretory phase in women with endometriosis, providing the increased proliferative potential of endometrial cells [11][12].

MicroRNA

However, microRNA appears to be the most promising candidate as a molecular genetic marker for endometriosis. MicroRNAs (miRNAs) are short non-coding RNA molecules, which post-transcriptionally regulate the expression of genes and are involved in certain biological processes such as cell proliferation, differentiation, and apoptosis. MicroRNA expression in normal endometrium varies throughout the menstrual cycle, and its involvement in non-pathological processes can be used to understand the pathogenesis of the disease [13]. In patients with endometriosis, miRNA profiling of eutopic endometrium and blood may provide useful information to confirm the diagnosis in women at different stages of endometriosis. Since miRNA acts as a regulator of gene transcription, one can assume that aberrant miRNA expression leads to dysregulation of gene expression in the eutopic endometrium. Thus, miRNA profiling of eutopic endometrium in patients with endometriosis could provide a kind of molecular fingerprint, which could be used for further comprehension of the disease pathogenesis and as potential minimally invasive markers of endometriosis. The analysis and search for the miRNA types that could best correspond to endometriosis markers is a current and relevant area of research; moreover, a lot of unique data has been collected about their involvement in the pathogenesis of the disease and the diagnostic potential of these molecules over the past few years.

Considering that endometriosis is an abnormal invasion, researchers, first of all, paid attention to those types of miRNAs that are involved in the pathogenesis of certain types of reproductive organ cancer. In this regard, microRNA-451 is of particular interest, since it acts as a tumor suppressor and is associated with poor prognosis of cancer. Aberrant expression of miR-451 was detected in eutopic and ectopic endometrial tissues. In particular, the miR-451 level was reduced in eutopic tissues as well as in cell lines obtained from patients with endometriosis compared to controls, although no dependence of a decrease in its level on the stage of the pathological process was found [13].

Not all microRNA types directly participate in the pathogenesis of endometriosis. Being non-specific regulators of the expression of other genes and proteins, they can indirectly affect the development of the disease. For instance, the studies by Muharam et al. showed that the decrease in the expression level of miR-93 coincided with the increases in the expression of MMP type 3 and the expression of vascular endothelial growth factor VEGFA. At the same time, the expression of miR-93 was significantly lower both in the eutopic endometrium (by 16.7 times) and in the ectopic endometrium (by 20 times) in women with endometriosis compared to the normal endometrium [3]. It is important to note that an elevated level of MMP3 is one of the factors in the invasion of ectopic foci as it was reported above.

Interesting data were obtained in the study on the Talin-1 protein microRNA expression in the pathogenesis of adenomyosis. Talin-1 is a protein located in the adhesion complex between cells and the extracellular matrix; it interacts with many adhesion molecules and activates cell-cell signaling [14]. Recent studies have shown that dysregulation of Talin-1 could lead to cell proliferation, migration, and survival; that has promoted extensive research into its role in cancer and other diseases including endometriosis [14-16]. The results obtained by Tang et al. showed that the expression of Talin-1 protein microRNA was significantly increased in both ectopic and eutopic endometrium in comparison with the healthy tissue. Concurrently, decreased Talin-1 levels did not affect the proliferation and apoptosis of endometrial stromal cells but resulted in inhibition of adhesion, invasion, and migration of endometrial stromal cells [17]. It is important to emphasize that in the initial stages of endometriosis, the crucial step is the attachment of retrograde endometrial tissues to the pelvic mesothelium. Some types of integrins, including αv, β3, β4, and β1, have been reported to mediate the attachment of endometrial cells to the mesothelium [18]. The expression of these integrins is tightly regulated by various molecules, including Talin-1. The results of the study by Tang et al. also showed that downregulation of Talin-1 expression affected the expression of integrin β3, indicating that Talin-1 may promote adhesion and migration through regulating integrin β3.

In addition to searching for microRNA types and identification of their involvement in the pathogenesis of endometriosis, active work is underway to develop the most sensitive methods for using the obtained data in diagnosing the disease. In the study by Borisov et al., the ratio of reciprocally dysregulated microRNA pairs was analyzed, which made it possible to diagnose adenomyosis with a sensitivity range from 65% to 74% and specificity from 72% to 86%. A statistically significant difference in the expression of the miR-181b/miR-10b pair in the endometrium of women with adenomyosis relative to the indicators in healthy women was revealed, specifically manifested in an increase in the level of miR-181b and a decrease in the level of miR-10b. These two miRNAs, which had a gradual increase and decrease in expression, may form a "mutual pair" with high diagnostic efficiency [19].

Some microRNA types and changes in their expression may be involved in the pathogenesis of pain syndrome in endometriosis. In particular, miR-146b was reported to have a high expression in patients with endometriosis complicated by pain syndrome [20]. Presumably, increased expression of miR-146b modulates the activity of tissue macrophages, and thereby increases the expression of proinflammatory cytokines, including TNF-α and MCP-1; that leads to chronic inflammation and the formation of pain syndrome. In addition, some patients have higher pain sensitivity, possibly due to the fact that they are carriers of the miR-146b rs1536309 CC/CT phenotype. Furthermore, miR-146b rs1536309 CC/CT phenotype was found to reduce miR-146b expression and, consequently, makes the immune system more prone to a pro-inflammatory state. These processes may be the main reason why patients with this phenotype have higher pain sensitivity [21].

Many recent scientific publications have in one way or another revealed the expression patterns of different types of miRNAs in the pathogenesis of endometriosis. For instance, levels of miR-34c-5p, miR-9, and miR-34b have been shown to be decreased in patients with disease relative to those in healthy women. In addition, miR-483-5p and miR-629-3p were found to be associated with angiogenesis, inflammation, cell proliferation, steroidogenesis, and other mechanisms involved in endometriosis [22]. miRNAs such as microRNA-21, microRNA-155, and microRNA-a125b have been shown to be involved in the regulation of apoptosis [23]. Besides, miR-191 was found to be overexpressed in endometrioma tissue and is a key regulator of the proliferation and invasive properties of endometriosis cells in vitro. Regulation of the migratory capacity of endometrial stromal cells is realized with the participation of MiR-181b [24]. Another study on endometriosis-associated miRNAs showed that lower levels of miR-126-5p facilitated migration and invasion of stromal cells [25]. Important information has been obtained from investigations of the miR-200 family, which regulates the process of epithelial-mesenchymal transition and is critical for the development of endometrioid lesions. In particular, miR-223 manifests decreased expression in both endometriomas and endometriotic lesions, while overexpression of miR-200b is associated with increased cell proliferation and enhanced mesenchymal-epithelial transition in the eutopic endometrium in patients with endometriosis. It should also be noted that downregulation of microRNA-34a-5p in endometrial stem cells leads to increased expression of VEGFA, which in turn promotes angiogenesis and the development and progression of endometriosis [26][28][29]. The role of microRNA, in particular miR-194-3p, in reducing fertility due to the formation of resistance to progesterone has also been proven [27].

Conclusion

An analysis of the available information makes it possible to conclude that all the studies described above are single, often pilot. Currently, there is no meta-analysis confirming the efficacy of any type of microRNA in diagnosing endometriosis. Nevertheless, the accumulated experience and continued research in this area opens up broad prospects for minimally invasive diagnostics, and subsequently, treatment of such a socially significant disease as endometriosis.