H3K27me3-mediated epigenetic regulation of TET1 in the eutopic endometrium of women with endometriosis and infertility

We analyzed H3K27me3 levels in three TET1 promoter regions (1–3), comparing all endometriosis patients to controls, with and without ChIP efficiency. Without considering ChIP efficiency, no significant H3K27me3 differences were found between groups in any TET1 promoter region ( p  = 0.76, 0.25, 0.90 for regions 1–3; Fig.  2 ; Table  1 ). Fig. 2 H3K27me3 levels in three TET1 genomic regions in endometriosis patients and controls (test power = 0.05). H3K27me3 levels in three TET1 genomic regions in endometriosis patients and controls (test power = 0.05). Table 1 Comparison of H3K27me3 levels across three TET1 promoter regions in endometriosis patients and controls – analysis with and without stratification by chip efficiency. Endometriosis and controls TET1 promoter regions Endometriosis ( n  = 18) vs. Controls ( n  = 7) Good ChIP efficiency a ( n  = 5) vs. Controls ( n  = 5) Very good ChIP efficiency a ( n  = 3) TET1 reg 1 p  = 0.76, power = 0.05 p  = 0.51, power = 0.05 N/A TET1 reg 2 p  = 0.25, power = 0.05 p  = 0.04, power = 0.20 N/A TET1 reg 3 p  = 0.90, power = 0.05 p  = 0.38, power = 0.05 N/A a The promoter region of MYT1 gene was used as a positive control to validate pulldown efficiency. A sample with IgG immunoprecipitation was used as a negative control for all pairs of starters. N/A: not applicable due to insufficient data. Comparison of H3K27me3 levels across three TET1 promoter regions in endometriosis patients and controls – analysis with and without stratification by chip efficiency. a The promoter region of MYT1 gene was used as a positive control to validate pulldown efficiency. A sample with IgG immunoprecipitation was used as a negative control for all pairs of starters. N/A: not applicable due to insufficient data. Stratification by ChIP efficiency showed a significant difference in region 2 with good ChIP efficiency ( p  = 0.04), indicating higher H3K27me3 in endometriosis (Fig.  3 ). Regions 1 and 3 showed no differences ( p  = 0.51, 0.38). Analysis in the very good ChIP efficiency subgroup was not possible due to small sample size (Table  1 ). Fig. 3 High-efficiency ChIP analysis of H3K27me3 levels at TET1 promoter region 2 in endometriosis and controls (test power = 0.20). Statements and Declarations. High-efficiency ChIP analysis of H3K27me3 levels at TET1 promoter region 2 in endometriosis and controls (test power = 0.20). Statements and Declarations. We separately compared infertile and fertile endometriosis patients to controls. Without considering cycle phase, no significant H3K27me3 differences were found among infertile endometriosis ( n  = 11), fertile endometriosis ( n  = 7), and controls ( n  = 7) ( p  = 0.67, 0.28, 0.21 for regions 1–3; Table  2 ). Table 2 Comparison of H3K27me3 levels in three TET1 promoter regions among infertile, fertile endometriosis patients and controls. TET1 promoter regions Infertile endometriosis ( n  = 11) vs. Fertile endometriosis ( n  = 7) vs. Controls ( n  = 7) a TET1 reg 1 p  = 0.67, power = 0.05 TET1 reg 2 p  = 0.28, power = 0.05 TET1 reg 3 p  = 0.21, power = 0.05 a Kruscal-Wallis rank test with multiple comparison. Comparison of H3K27me3 levels in three TET1 promoter regions among infertile, fertile endometriosis patients and controls. a Kruscal-Wallis rank test with multiple comparison. During the secretory phase, no significant differences were found among infertile endometriosis, fertile endometriosis, and controls ( p  = 0.17, 0.54, 0.57 for regions 1–3; Table  3 ). Combined endometriosis groups also showed no differences vs. controls ( p  = 0.91, 0.38, 0.51; Table  3 ). Table 3 Comparison of H3K27me3 levels in three TET1 promoter regions among infertile, fertile, and all endometriosis patients vs. controls (secretory phase). TET1 promoter regions Infertile ( n  = 6) vs. Fertile endometriosis ( n  = 5) vs. Controls ( n  = 5) a All endometriosis patients ( n  = 11) vs. Controls ( n  = 5) b TET1 reg 1 p  = 0.17, power = 0.05 p = 0.91, power = 0.05 TET1 reg 2 p  = 0.54, power = 0.05 p = 0.38, power = 0.05 TET1 reg 3 p  = 0.57, power = 0.05 p = 0.51, power = 0.05 a Kruscal-Wallis rank test with multiple comparison. b Mann-Whitney U test comparison. Comparison of H3K27me3 levels in three TET1 promoter regions among infertile, fertile, and all endometriosis patients vs. controls (secretory phase). a Kruscal-Wallis rank test with multiple comparison. b Mann-Whitney U test comparison.

The study included 25 women aged 18–40 years and was approved by the local Bioethics Committee (no. 879/18). Exclusion criteria were comorbidities and recent hormonal treatment (contraception or intrauterine devices) within six months before surgery. All patients underwent laparoscopy; infertile women also had hysteroscopy. Eutopic endometrium was obtained by biopsy: pipelle aspiration (Rovers, France) in fertile women and hysteroscopic biopsy in infertile women. Participants were grouped by laparoscopy-confirmed endometriosis status. Eighteen had endometriosis and seven did not. Women with endometriosis were further stratified by fertility status. Among women with endometriosis, 11 were infertile and 7 fertile. Final groups: infertile women with endometriosis, fertile women with endometriosis, and fertile controls without endometriosis. Endometriosis was diagnosed by visible lesions or histopathological confirmation of ectopic tissue. Infertility was defined per Polish Society of Gynecologists and Obstetricians guidelines after a full reproductive history. No identifiable infertility cause was found in the infertile group. Tubal hydrosalpinx and intrauterine pathology (polyps or submucosal fibroids) were excluded. Before surgery, these women had documented ovulation (mid-luteal progesterone), normal HSG (patent tubes, normal cavity), normal endocrine tests (thyroid, prolactin), and partners’ semen within fertility standards. Fertile women had regular cycles and conceived naturally, giving birth within two years. Only women without prior assisted reproduction (ovulation induction or IVF) were included in fertile groups. Fertile women underwent laparoscopy to treat benign ovarian lesions detected by ultrasound. Biopsies were divided: one part fixed in formalin for histology, with cycle phase determined by H + E (Noyes and Hertig criteria 20 . The second part was snap-frozen at − 80 °C for H3K27me3 analysis in three TET1 promoter regions (within/outside CpG islands) using chromatin immunoprecipitation. Frozen tissues were pulverized in liquid nitrogen with a mortar and pestle. ~ 50 µg of tissue was dissolved in PBS with protease inhibitors and homogenized with a Dounce homogenizer (DWK Life Sciences, USA). Samples were cross-linked with formaldehyde and quenched after 10 min with glycine. DNA (100–300 bp) was generated using Diagenode EasyShear Kit (C01020010) and Bioruptor Plus sonicator. Chromatin was pre-cleared with non-specific IgG and magnetic A/G beads (Thermo Fisher, 10003D). After removing beads with IgG and non-specific chromatin, samples were divided: 5% input, H3K27me3 antibody (Diagenode, C15410195), and non-specific IgG (R&D Systems, AB-105-C). Samples were incubated overnight at 4 °C with rotation, then 30 µl Dynabeads ® were incubated for 3 h at 4 °C to bind antibodies. Beads were washed: twice with low-salt buffer, twice with high-salt buffer, once with LiCl wash buffer, and twice with TE buffer. Samples were eluted, decrosslinked (10% SDS, 1 M NaHCO3), and treated with RNase A, NaCl, and proteinase K at 45 °C for 2 h. DNA was purified (Active Motif kit), and H3K27me3 in TET1 promoter regions quantified with SYBR ® Green Supermix (Bio-Rad). The MYT1 promoter served as a positive ChIP control, and IgG as a negative control. ChIP efficiency was categorized as good (10–100) or very good (> 100). Three TET1 primer pairs amplified promoter regions (Fig.  1 , Supplementary Table 1). Results were normalized to non-specific IgG 21 . Fig. 1 Localization of three TET1 regulatory regions within the TET1 gene promoter. Localization of three TET1 regulatory regions within the TET1 gene promoter. Statistical analysis was performed in SigmaStat 3.5. Normality was tested with Shapiro-Wilk and Kolmogorov-Smirnov tests. Group comparisons used Mann-Whitney U and Kruskal-Wallis tests with multiple-comparison correction. A p-value < 0.05 (Bonferroni-corrected) was considered significant.

This study aimed to evaluate H3K27me3 levels in the TET1 promoter in eutopic endometrium of infertile and fertile women with endometriosis and fertile controls.

Our main finding was no significant H3K27me3 differences in any TET1 promoter region between endometriosis and controls, regardless of ChIP efficiency. This contrasts with our hypothesis, suggesting a more complex H3K27me3–TET1 relationship in endometriosis. The absence of differences across TET1 promoter regions, even after stratifying by fertility and cycle phase, suggests H3K27me3 changes may not drive endometriosis-related infertility. However, region 2 showed higher H3K27me3 in endometriosis ( p  = 0.04) in the good ChIP efficiency subgroup. This highlights the importance of considering technical factors such as ChIP efficiency when interpreting epigenetic data. The region 2 difference, limited to the good efficiency subgroup, warrants further study. This region may be sensitive to technical variation or reflect a real difference masked by technical noise. The low test power increases the risk of type II error, suggesting the effect may be too small to detect in this diverse sample. This may suggest that the effect is too small to detect in this diverse sample, highlighting the need for stricter analysis and larger groups 22 , 23 . This study included fertile endometriosis patients to explore H3K27me3 in infertility but was limited by whole-tissue analysis. Stem cell theory suggests circulating progenitors form stroma and glands at ectopic sites, creating endometriotic lesions. Noë et al. showed ectopic epithelial and stromal cells develop clonally and independently, forming distinct compartments 14 . Mahajan et al. found estrogen-dependent TET1 expression in eutopic endometrium is mainly in stromal cells 15 . Roca et al. examined which compartment mainly drives TET expression in endometriosis-affected endometrium 24 . Epithelial cells expressed TET transcripts 10–30 times higher than stromal cells, while stromal fibroblasts showed significantly reduced TET mRNA vs. controls. These differences may reflect stromal cell enrichment, which largely drives altered TET expression. Thus, stromal TET expression seems key in defective TET regulation. Future epigenetic studies should analyze specific cell compartments, not whole tissue, to avoid misinterpretation 24 – 27 . Future studies should use laser capture microdissection or cell-type-specific chromatin profiling. ChIP-seq of decidual vs. myometrial stromal cells in mice revealed > 800 differential peaks, highlighting the need for cell-type-specific analysis 28 . The lack of significant overall H3K27me3 differences may have several causes. Endometriosis-related infertility involves many pathways, and H3K27me3 changes may be just one piece of this puzzle. Other epigenetic or genetic changes may be more important. Our small sample size may have limited power to detect subtle H3K27me3 changes. Environmental, genetic, and hormonal factors affecting epigenetics also warrant consideration. H3K27me3 and H3K9me3 are key repressive marks in promoters of silenced genes, including TET1 . The balance between H3K27ac and H3K27me3 further complicates TET1 regulation. These interactions highlight the need for genome-wide methylation analysis with H3K27me3 profiling to understand endometriosis epigenetics 29 . EZH2, a PRC2 methyltransferase, catalyzes H3K27 methylation 30 . EZH2 gain-of-function mutations raise H3K27me3 and suppress differentiation 31 , 32 . EZH2 overexpression promotes proliferation, epithelial–mesenchymal transition, and invasiveness 33 . Endometriotic epithelial cells show higher progesterone-regulated EZH2 than non-endometriotic cells 11 . Elevated EZH2 in endometriotic tissue suggests a role in pathogenesis 11 , 34 . The EZH2–PRC2–H3K27me3 axis regulates endometrial differentiation and implantation receptivity 35 . Inhibiting histone methyltransferase activity reduces endometriotic cell growth and invasiveness, supporting EZH2 as a therapeutic target 11 . We did not assess EZH2 expression. Further research should examine EZH2 expression and its link to H3K27me3 in endometriosis. Few studies examined H3K27me3 in the TET1 promoter, mostly in cancers with low TET1, similar to endometriosis 36 , 37 . These studies suggest EZH2 may regulate TET1 , warranting studies on EZH2 expression and H3K27me3 in eutopic endometrium. Other studies linked active histone marks, such as H3K4me3, to higher TET1 expression 25 , 38 . No studies examined H3K27me3 in TET1 in eutopic or ectopic endometrium in endometriosis. H3K27me3 represses developmental genes (e.g., HOX ) and COX-2 , lowering PGE2 39 , 40 . Because these genes are involved in endometriosis, further research into H3K27me3 is warranted. Epigenetic changes are reversible but harder to reverse as they accumulate. Epigenetic mechanisms may offer non-hormonal therapies for endometriosis. Identifying gene expression patterns in eutopic and ectopic endometrium may enable therapies targeting epigenetic regulators in key promoters. In conclusion, we found no overall H3K27me3 differences in the TET1 promoter between endometriosis and controls, except for one significant difference in region 2 ( p  = 0.04) with good ChIP efficiency, confirmed after multiple testing correction. This finding should be interpreted cautiously due to the small sample size. The lack of consistent subgroup differences limits biological conclusions. The complex histone interactions, multifactorial infertility, and methodological issues highlight the need for larger cohorts, comprehensive epigenetic profiling, and optimized methods. Cell-type-specific analyses are likely to be especially informative. Such studies are essential to clarify the role of epigenetics in endometriosis and infertility. The data are available from the corresponding author upon request.

Endometriosis is a common gynecological disorder in reproductive-age women, characterized by endometrial-like tissue outside the uterus. Although its cause remains unclear, evidence suggests a complex interplay of genetic and epigenetic factors 1 , 2 . Recent molecular studies revealed genetic and epigenetic heterogeneity in histologically similar tissues, highlighting the complexity of endometriosis pathogenesis 3 , 4 . This heterogeneity may explain differences in pain severity and treatment response in endometriosis 5 – 7 . Therefore, studying specific epigenetic changes in eutopic and ectopic endometrium is crucial to understanding the disease. Endometriosis is strongly associated with infertility, often requiring assisted reproductive technologies (ART) 8 , 9 . Physical factors such as tubal obstruction, anatomical distortion, and pelvic inflammation contribute to infertility, but its persistence without these factors suggests additional, less obvious mechanisms. Inherited genetic and epigenetic changes may predispose to endometriosis, linking it to infertility through shared heritable factors 2 . Epigenetic modifications regulate gene expression and are involved in many diseases, including endometriosis 10 . Evidence supports a role for epigenetic dysregulation in endometriosis-related infertility 11 , 12 . Epigenetic mechanisms modify gene expression without altering DNA sequence, including DNA methylation, histone modifications (e.g., H3K27me3), and non-coding RNA activity. In endometriosis, epigenetic abnormalities in eutopic endometrium may impair endometrial receptivity, embryo implantation, and early development, leading to infertility. One frequent epigenetic change in eutopic endometrium of women with endometriosis and infertility is dysregulated ten-eleven translocation ( TET ) genes compared to healthy fertile women 8 , 9 . TET dioxygenases initiate DNA demethylation by oxidizing 5-methylcytosine (5mC), causing CpG demethylation in promoters and activating genes 13 , 14 . TET gene expression varies during the menstrual cycle; TET1 is higher in the secretory than proliferative phase 8 , 15 . Unlike healthy women, who show high TET1 expression and optimal receptivity in the secretory phase, women with endometriosis and infertility have reduced TET1 expression 8 , 9 . TET gene expression is regulated at multiple levels, but the role of histone modifications is unclear. Histone methylation, a key epigenetic regulator, is crucial for cellular processes. Aberrant histone methylation, especially H3K27me3 (a gene repression marker), is linked to several diseases, including endometriosis 11 , 12 , 16 . Higher H3K27me3 in endometriotic glands and stroma versus eutopic endometrium underscores its role in pathogenesis 12 , 16 , 17 . We hypothesize that altered H3K27me3 levels in the TET1 promoter CpG island are critical in endometriosis pathogenesis. TET1 promoter CpG hypermethylation has been reported in endometriosis 8 , 9 , and H3K27me3 is strongly linked to promoter CpG islands 18 . High H3K27me3 may silence genes essential for endometrial function, while reduced TET1 activity further limits gene activation 18 . A delicate balance of histone modifications is crucial for cell function 17 , 19 . The opposing roles of H3K27me3 (repression) and TET1 (activation) are increasingly recognized in various diseases 11 . This interaction is affected by cyclical H3K27me3 changes in healthy endometrium driven by hormonal fluctuations 11 . Higher H3K27me3 in the secretory vs. proliferative phase in both patients and controls suggests hormonal regulation 11 .

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1