DNA methylation is an epigenetic chromatin mark that allows heterochromatin formation, gene silencing, and regulates alternative splicing. Exons have higher levels of DNA methylation compared to flanking introns. Around 22% of alternative exons splicing is regulated by DNA methylation (Lev Maor et al., 2015). Two mechanisms use DNA methylation to regulate alternative splicing. The first one involves modulation of the elongation rate of RNA polymerase II, and the second one involves heterochromatin protein 1 protein, a fundamental unit of heterochromatin packaging, that recruits splicing factors onto transcribed alternative exons (Pappalardo and Barra, 2021).

DNA methylation is one of the most common epigenetic modifications, regulating gene expression by recruiting repressive proteins or through the inhibition of transcription factor binding (Moore et al., 2012). It consists of adding a methyl group to the fifth position of cytosine in CpG sites (Moore et al., 2012). DNA methylation can occur in different genomic regions namely, Intergenic Regions, Promoters, Gene Body and Enhancers (Moore et al., 2012; Kreibich et al., 2023). DNA methyltransferases (DNMTs) carry out this process, by using S-adenosylmethionine as the methyl donor to catalyze the addition of the methyl group to the cytosine ring to generate methyl cytosine (Gao et al., 2018) DNA methylation is a dynamic process that requires de novo DNA methyltransferases DNMT3A and DNMT3B, involved in adding methyl groups to cytosine at unmethylated DNA (Tóth et al., 2025). The next step consists of preserving the novel methylation patterns by DNMT1 (Tóth et al., 2025). During DNA replication, DNMT1 enzyme is recruited to ensure the inherence of the parental methylation pattern in the newly synthesized strands (Xu et al., 2025). The silencing achieved through the methylation at CpG sites can directly block transcription factor binding due to the methylation of response elements (Moore et al., 2012). Furthermore, another mechanism of gene regulation by DNA methylation involves the methyl-CpG-binding domain (MBD) protein MeCP2, MBD1, MBD2, and MBD3 (Wood and Zhou, 2016). These proteins bind to methylated DNA and recruit corepressor complexes, including histone deacetylases (HDACs), making the DNA less accessible for transcription (Newell-Price et al., 2000; Javaid and Choi, 2017). This leads to a stable transcriptional repression of the target genes (Miller and Grant, 2013). Beyond the classical dogma, there is growing evidence of a more complex effect of DNA hypermethylation on gene expression depending of the biological context. For instance, the expression of hypermethylated genes can be unaffected or even upregulated. Furthermore, some transcription factors tend to bind methylated rather than unmethylated CpGs (Rauluseviciute et al., 2020).

In endometriosis, there are more than 40,000 CpG sites, distal to classical CpG islands, differentially methylated (Dyson et al., 2014; Gerkowicz et al., 2020; Zubrzycka et al., 2020; Adamczyk et al., 2022). Furthermore, it has been proven that the expression patterns of DNMTs in endometriotic tissue differ from those of normal endometrium (Wu et al., 2007; Hsiao et al., 2015; Zubrzycka et al., 2020). Regarding DNA methylation, altered expression of DNMT1, DNMT3A and DNMT3B was shown in ectopic endometrium, compared to normal controls and eutopic endometrium of women with endometriosis (Wu et al., 2007).

Histones are proteins that play a crucial role in DNA compaction (Zhang et al., 2021). These proteins support the formation of DNA-protein complex, around 2 m of DNA is packed inside the nucleus (Janna et al., 2020). The nucleosome is the fundamental unit of chromatin, consisting of 4 central histones H2A, H2B, H3 and H4 (Janna et al., 2020). The H1 connects the nucleosomes to form a chromosome (Pathak et al., 2018). Histones have protruding tails that undergo post-translational modifications, namely, acetylation, phosphorylation, methylation, ubiquitylation and sumoylation (Bannister and Kouzarides, 2011; Keck and Pemberton, 2012). Most of these modifications are reversible, making it possible to develop new histone-targeting therapeutic strategies (Lu et al., 2025). It is well established that gene expression and chromatin remodeling depend on DNA methylation and histone modifications (Gagnidze and Pfaff, 2021). However, the mechanisms underlying histone modifications are still not completely understood (Gagnidze and Pfaff, 2021). The most widely recognized histone modifications are acetylation and methylation (Bannister and Kouzarides, 2011; Nasu et al., 2011; Bulun et al., 2019). In endometriosis, current findings have demonstrated the involvement of histone modifications in the pathogenesis of the disease, even if the mechanism is not fully understood (Psilopatis et al., 2023; Bedrick et al., 2024). Moreover, the impact of histone modifications on infertility related to endometriosis is still a subject of research (Psilopatis et al., 2023).

Histones acetylation is one of the first modifications revealed by Allfrey et al. (1964). It involves adding acetyl groups to the N-terminal tails of amino acids such as lysine and arginine, as well as serine, threonine and tyrosine in H3 and H4 molecules (Allfrey et al., 1964). It is regulated by 2 enzymes; Histone Acetyl Transferase (HAT) and Histone Deacetylase (HDAC) (Yang and Seto, 2007). These enzymes influence the binding of histones to DNA, resulting in condensation or decondensation of chromatin, and subsequently gene expression modulation (Bannister and Kouzarides, 2011). In patients with endometriosis, the levels of HDAC1 and HDAC2, the most abundant HDACs in human cells, were found deregulated in endometriotic stromal cells (Hsiao et al., 2017). Several studies have shown that in endometriotic stromal cells, HDAC1 and HDAC2 are upregulated (Colón-Díaz et al., 2012; Samartzis et al., 2013; Xiaomeng et al., 2013). In early proliferative phase, histone acetylation levels are globally increased and progressively decrease during the late proliferative phase until ovulation (Munro et al., 2010). Several studies have shown that overall histone acetylation profiles, particularly H3 and H4 are hypoacetylated in endometriotic stromal cells compared with normal endometrium (Xiaomeng et al., 2013; Monteiro et al., 2014). Furthermore, increased HDAC activity in endometriotic cells leaves promoter regions hypoacetylated, resulting in cell cycle induction and proliferation (Koike et al., 2015). In endometrial epithelial cells, Estradiol and Progesterone significantly downregulated HDAC1 expression (Colón-Díaz et al., 2012). However, in endometrial stromal cells, HDAC2 expression levels were upregulated by Estradiol and downregulated by Estradiol plus Progesterone treatment (Colón-Díaz et al., 2012; Hsiao et al., 2017). This pattern of HDAC1/2 hormonal regulation is lost in the endometriotic cell line, which can be explained by progesterone resistance due to an overall reduction in progesterone receptor levels in endometriotic stromal cells (Bulun et al., 2010).

Among the crucial components of epigenetic regulation are non-coding RNAs (Liao et al., 2025). They are essential in fundamental biological processes, namely, transcription, genome imprinting, and chromatin remodeling (Liao et al., 2025). They have the particularity of not undergoing the translation process of protein synthesis (Liao et al., 2025). Non coding RNAs can be classified into two categories according to their size, structure, and regulatory properties. Hence, small RNAs refer to RNAs under 200 nucleotides, and long RNAs with more than 200 nucleotides (Chen and Kim, 2024). Over the last two decades, several types of small non-coding RNAs, such as MicroRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), endogenous small interfering RNA (siRNAs), and Small nucleolar RNAs (snoRNAs) have been identified through genetic mapping (Huang Z.hao et al., 2022; Chen and Kim, 2024). With the development of deep sequencing technologies, a new world of small RNA has emerged (Brosnan and Voinnet, 2009). MiRNAs and piRNAs play an pivotal role in germline and somatic cells, respectively through RNA silencing and transposon activity reduction (Saini et al., 2007; Weick and Miska, 2014). On the other hand, long non-coding RNAs modulate the transcriptional and post-translational levels of gene expression (Mattick et al., 2023).

These modulatory RNAs are core elements of cellular machinery that function at several levels to control cellular fate (Yao et al., 2019). In endometriosis, several experiments demonstrated that non-coding RNAs contribute to the pathogenesis of endometriosis (Tables 2–5).

The Epigenetic modifications have a significant role in modulating the endometriosis immune microenvironment (Szukiewicz, 2022; Abbaszadeh et al., 2023; Shi et al., 2025). The immune system holds remarkable potential to recognize and eliminate endometrial implants in the peritoneal cavity (Suszczyk et al., 2024). However, in endometriosis, inflammation and altered immune system, including impaired natural killer (NK) and macrophages activity, T-helper1 (Th1)/T-helper2 (Th2) imbalance, and elimination of the regulatory function of T cells, reduce the clearance of regurgitated endometrial cells and elicits the oxidative stress response and inflammation (Szukiewicz, 2022; Abbaszadeh et al., 2023). The dysregulation of Th1/Th2 and Th17/Treg balances were associated with endometriotic lesions progression, through the abnormal cytokine secretion and enhanced inflammation (Le Menn et al., 2022; Szukiewicz, 2022). T cells dysfunction, including impaired cell proliferation, inflammation, immunogenicity of endometriotic stromal cells, angiogenesis, and sex steroid hormone responsiveness, are relevant mechanisms underlying the pathophysiology of endometriosis (Szukiewicz, 2022). The immune landscape-endometriosis crosstalk involves an interplay between T cells, prostaglandins (PGE2), metalloproteinases (MMP-2, -3, -9), cytokines (TNFα, IL-1β, IL-8, IFNγ, MCP-1, and MIF) and adhesive molecules (VCAM-1, ICAM-1) (Figure 4) (Chopyak et al., 2022). Shifting the Th1/Th2 balance to favor the Th2 phenotype is one of the most critical immunological features of endometriosis. Furthermore, accumulating data suggests that Th17 and Treg play a significant role in clearing refluxed endometrial tissue. IL-17a, inflammatory mediator, associated with TNFa, boost the secretion of IL-8 and COX-2 in a p38 MAPK, p42/44 MAPK, and stress-activated c-Jun N-terminal kinase dependent manner (Hirata et al., 2008). Interestingly, debris clearance is more effective when the Th17/Treg balance tips in favor of Th17, associated with IL-6 and IL-17 inducing inflammation (Hirata et al., 2008; Gogacz et al., 2016; Tanaka et al., 2017).

It is worth emphasizing that epigenetic modifications are among the factors modulating the immune landscape of endometriosis (Shi et al., 2025). Epigenetic modifications can directly modulate the immune microenvironment. Abnormal epigenetic regulation is closely associated with the occurrence and development of many diseases, with DNA methylation and post-translational modifications (PTMs) are the most common abnormal epigenetic mechanisms strongly associated with various disorders (Tsankova et al., 2007; Orioli and Dellambra, 2018; Lu et al., 2020). Accumulating evidence has shown the contribution of PTMs including phosphorylation, methylation, acetylation, glycosylation, lipidation, ubiquitination, and SUMOylation in Th1/Th2 and Th17/Treg imbalances through the key molecules involved in their differentiation and function (Le Menn et al., 2022; Riaz et al., 2023). In instance, the major regulatory transcription factors such as RORγt (retinoic acid-related orphan receptor gamma t) and Foxp3 (forkhead box P3) are directly regulated by PTMs (Le Menn et al., 2022; Szukiewicz, 2022; Le Menn et al., 2022; Szukiewicz, 2022). Regarding inflammation, non-coding RNAs play pivotal role in inflammatory responses and during activation of inflammasomes (Abbaszadeh et al., 2023).

Findings have indicated that miRNAs in endometrial tissue play a key role in modulating the expression of inflammatory mediators. It has been reported that miR-199a was linked to the inhibition of paramount regulator of inflammation NF-κB through the downregulation of inhibitor of nuclear factor kappa B (IκBα) (Abbaszadeh et al., 2023). miR-182 has also the potential of inhibiting NF-κB pathway by targeting one of its related transcriptional factors p65, inducing inflammation and promoting the establishment of endometriotic lesions (Abbaszadeh et al., 2023).

Aberrant function of almost all types of immune actors has been reported in endometriosis, including altered T-cell and NK cytotoxicity function, polyclonal B cells activation, enhanced peritoneal macrophages recruitment, and inflammation (Osuga et al., 2011; Ahn et al., 2015; de Barros et al., 2017; Riccio et al., 2018; Agostinis et al., 2021; Szukiewicz, 2022). Epigenetic reprogramming of T cells in endometriosis has now been well recognized. In endometriosis, a significant decrease in cytotoxic T cells frequency associated with impaired function has been demonstrated. In T cells, the altered apoptotic pathways have been suggested to be linked to DNA hypermethylation and chromatin structure changes in the perforin gene regulatory elements (Lu et al., 2003; Szukiewicz, 2022). In the other hand, IL-6, upregulated in endometriotic stromal cells, plays a significant role in Th2 differentiation. Recent findings show the IL-6 pathway regulation by DNA methylation, miRNAs, and posttranslational modifications (Candido et al., 2021; Lamprianidou et al., 2021).

According to Lin et al., endometriotic lesions show higher miR-20a levels, with the potential of enhancing PGE2 production, and thereby contributing to inflammation (Lin et al., 2012; Szukiewicz, 2022). It is noteworthy that PGE2 plays a significant role on immune cell functions, such as macrophages and NK cells (Hsiao et al., 2014; Mei et al., 2018). In endometriosis, Inflammation exacerbation can result from low levels of some miRNAs, such let-7b and miR-215-5p and high levels of some others, such as miR-20a and miR-125-5p, individuals compared to the control group (Abbaszadeh et al., 2023).

The polarization of the macrophages into M2, through PI3K signaling pathway, is another hallmark of endometriotic immune microenvironment. In endometriosis, peritoneal fluid or medium from cultured peritoneal macrophages exhibit higher IL-10 levels (Ramírez-Pavez et al., 2021). It is well established that miR-301a-3p and miR-887-5p promote M2 polarization and IL10 secretion (Suen et al., 2014; Huang et al., 2022a; 2022b).

Zheying Liu et al. have shown that along with reduced lncRNA H19 levels, miR-342-3p show higher serum expression level. Furthermore, miR-342-3p binds to the 3′UTR of Immediate early response gene (IER3) to suppress its expression, ending up with high level of TGF-β and RORγt, a master regulator of the Th17 cell lineage (Liu et al., 2019; Ghafouri-Fard et al., 2020).

Supplementary Material summarizes differential expressed miRNA and LncRNAs having roles in immune system response in endometriosis. Future studies are required to analyze how aberrant epigenetic modifications, notably PTMs can be a potential for failure of immune system in clearing endometriotic cells.

Compared to transcriptomic biomarkers, epigenetic biomarkers present several advantages namely, a high stability in multiple biological samples including fluids (plasma, serum, urine, saliva, semen, and vaginal secretion), and tissues (fresh, frozen, and FFPE tissues) (García-Giménez et al., 2017). Epigenetic biomarkers offer information on disease progression, making them valuable as biological fingerprints. Moreover, epigenetic biomarkers can reflect environmental and lifestyle influences (García-Giménez et al., 2017) (Anastasiu et al., 2020) (Toiyama et al., 2014; Taryma-Leśniak et al., 2020).

Currently, a significant body of research is dedicated to DNA methylation and miRNAs (Toiyama et al., 2014). Regarding DNA methylation, studies have reported that the methylome of cancer cells are distinct from healthy cells. Given the tissue-specific DNA methylation patterns, methylome can be used to distinguish between various cancer types (Rendek et al., 2024). Its note emphasizing that the methylome of cancer can be analyzed in different body fluids, mainly blood liquid biopsy (Wang and Valent, 2009). Besides its lower cost and minimally invasive nature, liquid biopsies exhibit the ability to track the progression of malignant tumors, whether primary or metastatic, and recognize the tumor recurrence (García-Saenz et al., 2017; Insua et al., 2017). Furthermore, the DNA methylation Profile is transmitted with high fidelity to daughter cells, making it advantageous for in vitro diagnostic tests (Taryma-Leśniak et al., 2020). Furthermore, DNA methylation is preserved despite variations in clinical sample handling procedure and storage (Kristensen et al., 2009). Thus, these features underscore the potential use of DNA methylation as IVD assays in cancer. Currently, available methylation-based liquid biopsy tests are designed for single cancer detection, applied for colorectal cancer, lung cancer, bladder cancer and liver cancer, or multi-cancer detection. Concerning colorectal cancer, almost commercially available tests use stool samples as source of DNA, namely, Colovantage (Warren et al., 2011), CologuardTM (Warren et al., 2011; Onieva-García et al., 2015), and ColoSureTM (Ned et al., 2011). Few blood-based tests are available on the market, limited to Epi proColon 2.0 CE, COLVERA and Nu.Q™ (Rendek et al., 2024). In colorectal cancer, a study performed with 9,989 subjects have demonstrated that Cologuard^® has a sensitivity and a specificity for colorectal cancer detection of 92.3% and 86.6%, respectively (Imperiale et al., 2014). The Nu.Q^® assay has also demonstrated a sensitivity of 91.2% for Colorectal cancer and 83.0% for high risk adenoma (Herzog et al., 2017).

In lung cancer the available validated epigenetic biomarker tests are EarlyTect^® and Epi ProLung. Regarding multiple cancer screening, Galleri^® test, PanSeer, IvyGene and CancerRadar are developped (Rendek et al., 2024). More than 30 DNA methylation-based assays to aid clinical decision making in cancer have reached the market, an unequivocal indicator of DNA methylation-based test growing market size (Davalos and Esteller, 2023).

In the other hand, miRNAs stand out as a great candidate for diagnostic applications. miRNAs have the particularity to be protected from degradation by exosomes during migration out of cells and into body fluids (Spada, 2021). Moreover, accessing sequencing data from circulation is relatively simple, making the detection of differentially expressed miRNAs, and correlation with therapeutic response establishment possible (Condrat et al., 2020; Hussen et al., 2021). Liquid biopsy markers include circulating tumor cells, ctDNA, exosomes, free miRNA, lncRNA, circRNA, proteins, and so on (Ma et al., 2024). MiRNAs have been proven to be a good noninvasive cancer biomarkers, namely, due to their enhanced expression levels in patients and ease of detection. Furthermore, expression levels can reflect treatment response and predict prognosis. In blood of cancer patients, exosomal miRNAs are stable and correspond closely to the expression profile in the tumor. It has been shown that material extracted from liquid biopsy often carry higher quality than that from a tissue biopsy. Hence, blood liquid biopsy emerges as a potential diagnosis and prognosis tool, easy to perform, minimally invasive, and can be repeated multiple times (Heidrich et al., 2021; Jung et al., 2021; Bagheri et al., 2024).

Epigenetic biomarker development faces many challenges for clinical application. One of these challenges is to discover potential candidates with rigorous evaluation of their specificity and sensibility in large scale validation trials (Wu et al., 2021). MiRNAs use as biomarkers experience challenges linked to several factors such as, appropriate control groups, sample sizes, sample collection and processing methods, independent validation, post-analysis of candidate biomarkers, and studies of differential miRNA expression in body fluids. The potential confounding effects of background factors needs to be taken into account (Takizawa et al., 2022).

Moreover, the extraction and purification of miRNAs are essential steps to accurately identify miRNAs (García-Giménez et al., 2017). Thus, the standardization of these methods is a critical step for the reproducibility and the replicability of studies. The use of endogenous reference miRNAs in RT-qPCR to normalize Cq values and minimize technical variations is an important step (Faraldi et al., 2019).

Despite the extensive research in epigenetic modifications, especially DNA methylation and miRNAs, only a small number of biomarkers have reached clinical application. This points to a critical need to enhance efforts toward their clinical implementation. Thus, the standardization of pre-analytical techniques, improved assay methodologies, and an enhanced understanding of the biological mechanisms underlying epigenetic patterns efforts are fundamental to resolving current challenges and advancing both foundational and translational research.

DNA methylation stands as the most frequent epigenetic modification in the endometrium (Adamczyk et al., 2022). The SF-1 gene promoter in endometriosis is specifically hypomethylated in peritoneal endometriosis (Annisa et al., 2018). Meanwhile, in a previous study realized by Noël et al., it has been shown that the SF-1 protein expression was undetectable in all type of endometriosis; peritoneal, ovarian, or deep infiltrating endometriosis (Noël et al., 2011). The GATA6 alone is essential in endometriosis pathogenesis but not sufficient to confer an endometriosis phenotype (Bernardi et al., 2019). In the same line, the cooperation between GATA6 and SF-1 is reported to be sufficient for endometriosis development and persistence (Bernardi et al., 2019). In endometriotic cells, Izawa et al. identified a specific region in GATA6 gene body with hypomethylated CpGs (Izawa et al., 2019).

Concerning hypermethylation, the most studied gene is HOXA10 and several studies have shown the link between its hypermethylation and endometriosis (Ji et al., 2017; Elias et al., 2023). Patients with endometriosis have decreased expression of HOXA10 in the eutopic endometrium during the secretory phase (Samadieh et al., 2019). The genes mentioned in Table 1 (SF-1, GATA6, COX-2, ESR-2, HOXA10 and PR-B) are the most likely to account for endometriosis onset and development. A recent study by Lei and his colleagues, based on NGS profiling, have identified 1,837 differentially expressed genes, including 1,079 upregulated genes and 758 downregulated genes in the ectopic groups (Lei et al., 2023). Additional confirmation of the highest-ranked genes involved in differential methylation revealed that Transmembrane Protein 184A (TMEM184A), Stratifin (SFN), Killer Cell Immunoglobulin Like Receptor three Ig Domains X1 (KIR3DX1), Estrogen Receptor 1 (ESR1), Phosphatidylinositol-4,5-bisphosphate 3-kinase Catalytic subunit Gamma (PIK3CG) and Ribonuclease A family member 1, pancreatic (RNASE1) were relevant candidate genes in ovarian endometriosis (Lei et al., 2023). Furthermore, this study stands out for having established a link between infection with the human papillomavirus (HPV) and endometriosis. The study revealed that hypermethylated and hypomethylated genes in ectopic environments were enriched in HPV infected tissue (Lei et al., 2023).

Histone modifications and their contribution in the endometriosis are still unclear (Psilopatis et al., 2023). This gap in knowledge results of the restricted set of research that have worked on this component. In endometriosis, The most reported histone modifications are acetylation and methylation (Bedrick et al., 2024). However, histone phosphorylation and ubiquitination studies are still lacking. One of the pioneer studies reported in endometriosis histone modifications profiling was conducted in 2013 by Xiaomeng et al. (Xiaomeng et al., 2013). First, they revealed low levels of histone H4 acetylation in eutopic and ectopic endometrial tissues. Furthermore, the ectopic endometrium showed a notable decrease in HDAC1 mRNA levels, while in eutopic endometrial tissue, HDAC2 mRNA expression was significantly increased (Xiaomeng et al., 2013). In 2019, Kim et al. found that infertile women with endometriosis had reduced levels of HDAC3 in their eutopic endometrium (Kim et al., 2019). In 2022, the same research team studied the role of NAD + dependent class III HDAC Sirtuin 1, a stress-response and chromatin-silencing factor, showing notable increase in Sirtuin 1 expression in epithelial and stromal cells from endometriosis patients (Kim et al., 2022). Furthermore, high levels of Sirtuin 1 in endometriosis lesions appeared to cause further aggravation of endometriosis symptoms (Kim et al., 2022). As a final consideration, histone modifications seem to have a greater significance in endometriosis pathogenesis (Psilopatis et al., 2023). However, future studies should improve methodology and investigate the specific mechanisms by which histone modifications influence endometriosis pathogenesis.

Several studies using NGS technologies have recently attempted to identify non-coding RNAs differentially expressed in endometriosis, not only for their potential clinical application as diagnostic or prognostic biomarkers of the disease, but also to better understand the pathogenesis of endometriosis (Hudson et al., 2021). To date, several studies have demonstrated the role of non-coding RNAs in the pathogenesis of endometriosis, especially miRNAs and lncRNAs (Maier and Maier, 2021; Bendifallah et al., 2022a; Liang et al., 2022; Abbaszadeh et al., 2023; Ravaggi et al., 2024; Oghenemaro et al., 2025). lncRNA and miRNA expression profiles have been investigated in various samples, endometrial tissue, blood and saliva, collected from patients with endometriosis (Petracco et al., 2019; Cui et al., 2021; Bendifallah et al., 2022a; 2022b; Cai and Lang, 2022) it is noteworthy that functional interactions exist between these two sets of transcripts, miRNAs and lncRNAs, with a number of miRNAs being inhibited by lncRNAs (Meng et al., 2021; Gao et al., 2025). In instance, it has been demonstrated that the lncRNA H19 acts as a molecular sponge and reduces the availability of let-7 miRNA (Ghazal et al., 2015). This let-7 downregulation increases the proliferation of endometrial stromal cells through Insulin-like Growth Factor 1 Receptor (IGF1R) overexpression (Ghazal et al., 2015). Evaluation of the RNA interaction network in endometriosis has revealed the role of miRNAs and lncRNAs associated with growth and apoptosis genes regulation in endometrial stromal cells, namely, Cyclin-Dependent Kinase 1 (CDK1) and Proliferating Cell Nuclear Antigen (PCNA) (Zhang M. et al., 2020). In the microenvironment level, another research group reported the importance of the H19/miR-342-3p/IER3 pathway in reducing the risk of endometriosis through suppressing Th17 cell differentiation (Liu et al., 2019). In ovarian endometriosis, it has been shown that CDKN2B antisense RNA 1 (CDKN2B-AS1) regulates AKT serine/threonine kinase 3 (AKT3) expression by sponging miR- 424-5p (Wang S. et al., 2021).

The interplay between environmental factors and epigenetics is increasingly established in endometriosis. Given that endometriosis is an epigenetic disease, the influence of lifestyle factors such as smoking, alcohol, dietary factors, phytoestrogens, physical activity, stress, and infections, remains an area of ongoing investigation, with findings varying depending on study populations and methodologies (Hemmert et al., 2018; Coiplet et al., 2022). It has been demonstrated that perinatal and childhood environmental exposures are positively linked to endometriosis, including intrauterine tobacco exposure, low birth weight, and pet exposure during childhood (Amazouz et al., 2025). A recent umbrella review meta-analysis of 354 observational studies with a population of over 5 million, has provided a detailed review and critical analysis of environmental risk factors associated with endometriosis (Zhang and Ma, 2021). In this study, a total of 40 risk factors, including lifestyle, reproductive factors, early life factors, race and ethnicity, and others were assessed for their association with endometriosis (Zhang and Ma, 2021). Among these factors, only alcohol intake and exposure to endocrine disrupting chemicals showed a strong link to endometriosis (Zhang and Ma, 2021).

In the same line, endocrine disrupting chemicals, namely, benzophenone and paraben families, harmful chemicals often found in cosmetics and personal care products, have been linked to heightened risk of endometriosis (Peinado et al., 2021).

In the current state of the fight against endometriosis in the European Union, the European Parliament emphasized the high-risk association of pollutants, namely, polychlorinated biphenyls, organochlorine pesticides and dioxins with endometriosis (Parliamentary question, 2023). Pointing that exposure to polychlorinated biphenyls is associated with 70% increased risk of developing endometriosis. Similarly, exposure to dioxins raises the risk by 65%, while exposure to organochlorine pesticides is linked to a 23% increase in risk (Parliamentary question, 2023). The complex nature of these chemicals co-existing as mixtures in the environment makes risk evaluation difficult (Bruner-Tran and Osteen, 2010; Yao et al., 2017). Few Epigenetic studies carried out on the interplay between environmental factors and epigenetic modifications, highlighting the need for more well-designed, sufficiently powered studies.

The expanding volume of epigenomic data calls for advanced database that can store, standardize, and facilitate the exploration of epigenomic patterns, namely, DNA methylation, histone modifications, and non-coding RNAs. Among the key databases used in epigenetic research is EpiFactors (http://epifactors.autosome.org), a manually curated database, offering information about epigenetic regulators, their molecular complexes, targets and products (Marakulina et al., 2023). The latest version of EpiFactors includes data on 902 proteins, comprising 101 histones and protamines, along with a newly compiled collection of 124 lncRNAs (Marakulina et al., 2023). Besides EpiFactors, various open-access databases field are available. Regarding DNA methylation, there are several databases that offer data on methylation patterns obtained across normal and pathological conditions, such as methDB (http://www.methdb.net/), NGSmethDB (http://bioinfo2.ugr.es/NGSmethDB), MethBank (https://ngdc.cncb.ac.cn/methbank/), MethHC (http://methhc.mbc.nctu.edu.tw), and The Cancer Genome Atlas (TCGA) (Ghai et al., 2020; Shang et al., 2022; Ragini et al., 2023; Zhang et al., 2023). Nevertheless, a gap remains in detailed knowledge about the proteins involved in establishing or performing active DNA demethylation, especially when linked to their expression in different cell types and conditions (Medvedeva et al., 2015). Concerning histone modifications, database such as Histone Modification Database (HHMD), Histone Database, and HIstome database are used (Medvedeva et al., 2015). About miRNAs, the identification of miRNAs-target interaction (MTI) is crucial for biological processes annotation and therapeutic strategies development (Cui et al., 2023). Numerous databases of miRNAs are available, namely, HMDD (Human MicroRNA Disease Database), which is a continuously updated by the integration of experimentally verified miRNA–disease associations (http://www.cuilab.cn/hmdd). Compiled from biomedical literature, HMDD features 53,530 documented association between 1871 miRNAs and 2,360 distinct diseases (Cui et al., 2023; Cui et al., 2024). Expanded miRNATissueAtlas2, is a database that compiles miRNAs expression atlas based on 46,997 human tissue samples from 74 different organs, including physiological tissues, cell lines and extracellular vesicles (Rishik et al., 2025). A recent comprehensive database, TheMarker contains diverse types of biomarkers used for therapy and monitoring, including miRNAs (Cui et al., 2024; Zhang et al., 2024b). Launched in 2011, miRTarBase a database of experimentally validated MTIs has been manually curated and updated ten times (Cui et al., 2024). In its latest update, miRTarBase extends its scope by integrating miRNA regulatory networks associated with diseases, along with data on miRNA biomarkers, drug resistance, miRNA-targeted small molecule inhibitors, and miRNA oxidation, providing an integrative multidimensional database (Cui et al., 2024). With this update, miRTarBase now features upwards of 3,817 550 validated MTIs from 13,690 studies, representing a notable increase in data volume and improvements in curation workflow (Cui et al., 2024).

The adoption of high-throughput transcriptome sequencing technology, has made the identification of differentially expressed genes in diseases easy, allowing to gain better understanding of disease onset and guiding therapeutic decisions. The use of different bioinformatic analysis approaches, including HMDD and miRtarbase, have provided unique insights into the underlying mechanisms of endometriosis. Based on HMDD, 150 miRNAs have been found associated with endometriosis (Ye et al., 2022). Furthermore, the mechanisms of endometriosis-induced repeated pregnancy loss were discovered to be connected to the PI3K/AKT signaling pathway and platelet activation (Ye et al., 2022). Thus, miRNAs databases seem to be valuable in constructing miRNAs-mRNAs regulatory networks associated with different conditions, allowing precise targeting of the transcriptome and epigenome.

While previously cited studies have provided valuable insights, the reproducibility remains a major hindering and limiting factor in endometriosis research. Discrepancies in results need to be treated with caution since the majority of studies have limitations. Study weaknesses include monocentricity of the majority of studies, small sample size, and high heterogeneity in samples collected during different phases of the menstrual cycle. The intra-lesion heterogeneity is an additional limiting factor whose importance is generally underestimated. Study designs overlook key aspects such as (1) endometriosis type and severity; superficial peritoneal endometriosis, ovarian endometrioma and deep infiltrating endometriosis, (2) endometriosis stages; minimal, mild, moderate, and severe, and (3) anatomic distribution of endometriotic lesions; utero sacral ligaments, pouch of Douglas, ovarian fossa. Regarding in vitro models, they have severe limitations. Primary cells used in endometriosis research lack purity and are not phenotypically characterized, and cell lines are not genotypically authenticated (Romano et al., 2020). The analysis of sparse and incomplete medical data is a significant challenge in endometriosis research. Complete medical and clinical history, especially hormonal treatment, other illness conditions, past surgical history, and family history of endometriosis, are often missing which introduce biases and affect the generalizability of research findings. Finally, clinical trial landscape in endometriosis is advancing on multiple fronts, with numerous epigenetic focused ongoing trials, namely, micro-RNAs; RC 2.6.2022 (NCT05680350), ADOmiARN (NCT05928442), ENDOmiARN (NCT04728152), FR-21-001 (NCT05244668), STUDY00009584 (NCT05331053), ENDOmiRNA (NCT06414720), EMPOWER (NCT04598698), ENDMET (NCT06168097), 35,617/8/22 (NCT05556213), Pro00009633 (NCT02253251). However, due to epigenetic clinical trials are challenging, no epigenetic blockbuster drug for endometriosis seems to be on the horizon yet. One of the challenges that needs to be overcome is associated with the precise localization and targeted activity of epigenetics-targeted drugs. For instance, histone-modifying enzymes are found both in the nucleus and the cytoplasm. On the other hand, the function of ncRNAs depends on their localization and distribution within intracellular compartments. Hence, a deeper understanding of the intracellular trafficking of epigenetic modifiers is warranted. It is noteworthy that resistance to epigenetic drugs is another limiting factor for epigenetic drug application (Dai et al., 2024). Continued research into the biological and pathological roles of targets for epigenetic drugs is essential.