{"paper_id":"087cf86d-f7d1-4f67-8eee-f3323019f1be","body_text":"Endometriosis is an inflammatory and oestrogen-dependent disease with a prevalence of 6% to 10% in reproductive age women. It refers to the presence of endometrial tissue outside the uterine cavity ( 1 ). Although\nendometriosis is not an autoimmune disease, its inflammatory nature causes disorders in immune responses and\ncan reduce fertility in affected patients ( 2 ). One of the\naccepted theories of endometriosis is Sampson’s theory\nof retrograde menstruation ( 3 ). Although most women\nexperience retrograde menstruation, only 1 in 10 women\ndevelop endometriosis and the role of the immune factors\nin this disease is undeniable.\nThe immune system is believed to play an important\nrole in the aetiology, pathophysiology, pain, infertility,\nand poor outcomes of pregnancy in women with endometriosis ( 2 ). In particular, immune cells in the form of\ninnate and acquired immunity seem to play a key role in\nrejection or implantation of endometriotic cells in the peritoneal cavity ( 4 ). Under normal conditions, the immune\nsystem focuses on targeting endometrial cells that have\nbeen shed, even those coming from retrograde menstruation, to prevent these cells from implanting and causing\nendometriosis. Regulatory T cells (Tregs) are the main\nregulators responsible for managing this immune response ( 5 ). Treg cells are a subset of T cells that play an\nimportant role in homeostasis and immune tolerance.\nNumerous studies have been performed on the immunological aspects of endometriosis and there are evidences that show the relationship between Treg cells and\nendometriosis. The main marker of these cells is forkhead\nbox P3  (FOXP3) , a master transcription factor, which has\na special responsibility for the development and function\nof Treg cells ( 6 ). The expression level of  FOXP3  in Treg\ncells is very important for regulatory function of these\ncells. Decreased  FOXP3  levels lead to defective regulatory function of Treg cells ( 7 ), and mutations of this gene\ncauses autoimmune diseases in mice ( 8 ,  9 ) and humans\n( 10 ). A reduction in  FOXP3+  Tregs can induce autoinflammation ( 11 ,  12 ). These findings prove that regulation of  FOXP3  expression to control immune responses\nis critical.\nObviously, epigenetic factors control  FOXP3  transcription and regulate its expression profile. Histone modifications play key roles in regulating chromatin structure and\nnuclear processes, and can be passed as epigenetic markers during cell division ( 13 ). Different histone modifica\ntions, such as methylation and acetylation, act in combination and regulate nuclear events and can incorporate different signalling pathways at the chromatin level ( 14 ).\nThese mechanisms are a key focus in today’s biomedical\nresearch. “Epigenetics” involves changes in gene activity without altering the DNA sequence. It is well-known\nthat changes in epigenetics are a major biological factor\nthat cause issues with genes and lead to diseases. Covalent\nmodifications of histones, including acetylation and methylation, also play crucial roles in shaping genome organisation and influencing gene expression. Endometriosis is\nconsidered an epigenetic disease because it is difficult to\nexplain its inherited patterns due to genetic expression patterns alone ( 15 ). Knowing the relationship of the  FOXP3 \ngene with endometriosis is important because of its crucial\nrole in immune responses. For this aim, researchers have\nfocused on examining this gene’s expression pattern ( 16 ,\n 17 ) and polymorphism change in endometriosis ( 18 ). However, evaluation of  FOXP3  from an epigenetic perspective\nin endometriosis has not been thoroughly studied. In this\nstudy, we investigated the expression of  FOXP3  gene in\nnormal and endometriotic tissues. Then, for the first time,\nwe measured the correlation of two specific histone markers,  H3K9ac  (gene activating) and  H3K9me2  (gene repressive), in the promoter of the  FOXP3  gene, as a regulatory\nregion, and evaluated its relationship with the expression of\nthis gene in endometriosis.\n\nThis case-control study was approved by the Research\nEthics Committee of Royan Institute, Tehran, Iran (IR.\nACECR.ROYAN.REC.1397.189) and performed in accordance with the approved guidelines ( 19 ). Consent was\nobtained from all participants according to the guidelines\nof the Declaration of Helsinki (2000 revision), after which\nendometrial tissue samples were collected.\nThis study enrolled 40 convenient women of childbearing\nage (20 to 45 years old) with regular menstrual cycles. The\ncase group consisted of 20 women diagnosed with stages 3\nand 4 endometriosis according to the American Society for\nReproductive Medicine (ASRM-1997) classification. The\ncontrol group comprised 20 women with at least one child\nfrom a natural pregnancy who underwent laparoscopic sur\ngery for ovarian cystectomy or tubal surgery, and in whom\nthe absence of endometriosis was confirmed.\nIn the endometriosis group, ectopic tissues were ob\ntained by a laparoscopic procedure and eutopic endo\nmetrial tissues were obtained using a pipelle by a skilled\ngynecologist at the Royan Institute for Reproductive Bio\nmedicine. Biopsies were confirmed by pathological ex\namination for endometriosis. The endometrial biopsy in\ncontrol group was also obtained by pipelle sampling. All\ntissue samples were rapidly divided into sections of about\n50 mg, placed in RNAlater solution, and stored at -80°C\nuntil use. Additionally, six samples were selected from\neach tissue group for epigenetic studies to explore pos\nsible modifications associated with endometriosis.\nExclusion criteria included the use of hormonal treat\nment during the last three months before tissue sampling,\nirregular menstrual cycles, cancer, inflammatory and\nautoimmune diseases, endometrial hyperplasia, asthma,\nglomerulonephritis, osteoporosis, leukaemia, or benign\nuterine masses such as fibroids and polyps. All study par\nticipants completed a clinical questionnaire and signed a\nwritten consent form.\nEndometrial tissues were removed from RNAlater solution and homogenised using a scalpel blade and glass\nhomogeniser. Total RNA was extracted using the TRIzol\nreagent (cat: 15596026, Invitrogen, USA) according to\nthe standard protocol of the manufacturer. Digestion was\nperformed with DNase1 (cat: 2270, Takara, USA) to remove genomic DNA contamination from the RNA sample. cDNA synthesis was done using a Takara kit (cat:\nRR037A Takara Bio, Japan).\nFOXP3 mRNA expression was assessed by quantitative real-time polymerase chain reaction (PCR) using a\nStep One Plus™ Real-time PCR System (Applied Biosystems, USA). All cDNAs were co-amplified with endogenous glyceraldehyde-3-phosphate dehydrogenase\n (GAPDH)  as the control gene and by using specific\nprimers. The reaction program was set at 95°C for 4\nminutes, 40 cycles at 95°C for 15 seconds, and 60°C for\n1 minute. All primers were designed using PerlPrimer\nsoftware (version 1.1.21) and confirmed by Gene Runner software (version 3.05). Eventually the specificity\nof the primer sequences was checked by using BLAST\nsoftware ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) and\nthe UCSC genome browser ( http://www.genome.ucsc.\nedu ). Table 1 lists the primer sequences for  FOXP3  and\n GAPDH . Gene expression data were analysed using\nthe ΔΔCt quantitative method to estimate relative fold\nchange values.\nPrimers used in this study\nThe chromatin immunoprecipitation (ChIP) method\nwas performed as previously described ( 20 ). In order\nto evaluate epigenetic alterations, homogenised endometrial tissues were washed three times with phosphate\nbuffer saline and cross-linked with 1% formaldehyde.\nThen, by using a sonicator system (UCD200 Bioruptor\nsonication system, Diagenode, Belgium), we obtained\na soluble chromatin that contained 500 to 1000 bp of\nDNA fragments. After 5 minutes of centrifugation at\n4°C and 14 000 g, the supernatant was aliquoted into\nthree parts - one part for the input control, and the other\ntwo parts were incubated overnight at 4°C on a rotator\nwith anti- H3K9ac  (cat: ab1220, Abcam, UK) and anti\n- H3K9me2  (cat: ab4441, Abcam, UK). Incubation with\nprotein A-sepharose CL-4B beads (cat: 17-0780-01,\nGE Healthcare, Sweden) was performed for 2 hours at\n4°C to precipitate the immune-selected complexes. The\nbeads were washed with different washing buffers, then\ndecrosslinking of the antibody/beads was accomplished\nby heating the samples at 65°C for 4 hours. The purified DNA was obtained using a DNA purification kit\n(cat: 112-102, GeneAll, South Korea) and quantified by\nreal-time PCR using a Step One Plus™ Real-time PCR\nsystem (Applied Biosystems, USA). The PCR conditions were: 95°C for 3 minutes, 40 cycles at 95°C for\n15 seconds, and 60°C for 1 minute. As described previously ( 21 ), the data were expressed as fold enrichment\nof DNA associated with the different immunoprecipitated epigenetic markers relative to a 1/100 dilution of\ninput chromatin. Table 1 lists the primer sequences for\nthe  FOXP3  promoter.\nAll data analyses were performed using SPSS software\n(version 22.0) and one-way ANOVA followed by the\npost-hoc Tukey test to compare the differences between\neutopic, ectopic, and control tissues. P<0.05 were considered statistically significant.\n\nFOXP3  gene expression showed significantly reduced\nexpressions of this gene in ectopic and eutopic tissues of\nwomen with endometriosis compared to the control group\n(P=0.001). No significant difference in  FOXP3  gene expression was observed between ectopic and eutopic tissues in the endometriosis group ( Fig .1 ).\nFOXP3  expression in control, eutopic, and ectopic tissues. Relative\nmRNA expression of  FOXP3  in eutopic and ectopic tissues of 20 patients\nwith endometriosis compared to the 20 women in the control group.\nComparisons were made by ANOVA. Mean significant changes at the *;\nP<0.05 level and Ns; Not significant.\nAs expected, incorporation of the gene activating histone marker  H3K9ac  into the  FOXP3  promoter region\nshowed a significant decrease in both eutopic and ectopic\nendometrial tissues compared to normal endometrium.\nThere was no significant difference in the binding level of\nthis factor in eutopic and ectopic tissues ( Fig .2 ).\nIncorporation of  H3K9me2 , a gene silencing histone marker, was also assessed. As shown in Figure 2,\n H3K9me2  was significantly increased in eutopic endometrial tissue compared to normal endometrium (P=0.016).\nIn addition, this epigenetic marker was more prevalent in\nectopic tissue compared to normal endometrial tissue, but\nthis increase was not statistically significant.\nThese histone modification data paralleled the expression profile of the  FOXP3  gene in endometriotic tissues\ncompared to normal endometrium ( Fig .1 ).\nEpigenetic marker ( H3K9ac  and  H3K9me2 ) enrichment at the  FOXP3 \npromoter. Incorporation of H3K9ac and H3K9me2 histone markers into\nthe promoter of FOXP3 in eutopic and ectopic tissues of six patients with\nendometriosis compared to six women in the control group. Comparisons\nwere made by ANOVA. Mean significant changes at the *; P<0.05 level and\nNs; Not significant.\n\nVarious studies have reported aberrant function of the\nimmune system in endometriosis. This highly prevalent\ndisease is initiated and progresses by numerous genetic\nand epigenetic modifications that affect the immune system. Evidence exists that show the relationship between\nTreg cells and endometriosis. Tregs may play an important role in reducing the ability of newly recruited immune cells to target sloughed endometrial cells and, in\nturn, increase the survival and implantation of the endometriotic cells ( 22 ). Our findings have shown that the\nmRNA level of the  FOXP3  gene, as a master transcription\nfactor of Treg cells, significantly decreased in the eutopic\nand ectopic lesions of endometriosis compared to the control group. The epigenetic data in this study revealed a\nsignificant decrease in  H3K9ac  and a significant increase\nin  H3K9me2  of the  FOXP3  promoter in the eutopic and\nectopic tissues of endometriosis patients compared to the\ncontrol group. These epigenetic changes were aligned\nwith a decreased  FOXP3  gene expression profile in endometriosis. In addition, it seems that epigenetic modifications of the  H3K9ac/me2  in the  FOXP3  promoter can\naffect expression of this gene in endometriosis, which is\nexpected.\nUnder healthy conditions, some shed endometrial cells\nmay be transferred to regional lymph nodes for destruction, but a defective immune system may help the survival\nof these fragments and permit implantation at an ectopic\nsite ( 22 ). In addition, dysregulated immune responses and\ninflammatory conditions, influenced by Treg cells, play\na significant role in endometriosis progression ( 23 ). The\nbalance between phenotypic plasticity and stability of\nTreg cells is defined by the accurate regulation of transcriptional and epigenetic events required to ensure stable\nexpression of the  FOXP3  gene in Treg cells ( 24 ). Expression of this gene at the appropriate time and place is very\nimportant for inhibitory function of Tregs, which guarantees cleansing of the body from sloughed endometriotic\ncells. On the other side, decreased levels of FOXP3 can\nlead to defective regulatory function of Treg cells, autoimmune and auto inflammation ( 8 - 12 ). Our findings of\n FOXP3  gene expression supported previous studies reported by Koval et al. ( 25 ), which showed significantly\nless FOXP3 mRNA in eutopic endometrium samples\nfrom infertile women with endometriosis compared to a\ncontrol group. Additionally, studies showed a reduction\nin expression of this gene in hormone-sensitive cancerous tissues, such as breast, in 70% of cases ( 26 ). Some\nstudies reported that  FOXP3  gene expression levels in the\nendometrial tissues of infertile women were twice as low\nas those in healthy women ( 27 ). It is hypothesised that a\ndecrease in  FOXP3  gene expression in the eutopic and\nectopic endometrial tissues of women with endometriosis\nmay reflect an impaired function or altered phenotype of\nTreg cells. This dysfunction may lead to failure in maintaining proper immunological tolerance, and result in inadequate immune surveillance and allow growth of ectopic endometrial lesions, which would contribute to endometriosis. In contrast, some studies reported an increase\nin mRNA levels in the  FOXP3  gene in endometrial samples of women with endometriosis compared to a control\ngroup. Notably, the menstrual phase of the uterus affects\nthe results during sample collection; the abovementioned\nstudy was performed under different conditions (preimplantation phase) ( 28 ).  FOXP3  is a key factor in Treg cell\ndevelopment; for these purposes, simultaneous  FOXP3 \ngene expression and Treg-specific epigenetic changes are\ncritical ( 29 ). We selected  H3K9ac/me2  as the epigenetic\nmarker at the FOXP3 promoter because they represent\ntwo opposing chromatin states and have well-established\nroles in regulating gene expression. Since  FOXP3  plays a\ncritical role in Treg cell function and its expression must\nbe tightly controlled, understanding the balance between\nthese two epigenetic marks provides meaningful insights\ninto regulation of this gene. As expected, the gene expression results were in line with other studies, although the\nmain purpose of our study was to evaluate the epigenetic modifications of this gene in endometriotic tissues in\ncomparison with normal endometrial tissues. This is an\ninnovative part of our research. Various data suggest that\nTreg cells are influenced by epigenetic mechanisms that\nmodulate expression of the  FOXP3  gene and its associated epigenetic modifications ( 30 ).\nOur epigenetic assessment of the  FOXP3  gene promoter indicated that changes in the  H3K9me2  modification,\nwhich serves as a repressive histone marker, and in the\n H3K9ac  modification, which acts as an activating histone\nmarker, align with the observed gene expression results.\nIn other words, increased  H3K9me2  is associated with\ndecreased  FOXP3  gene expression, whereas increased\n H3K9ac  leads to enhanced  FOXP3  gene expression.\nIn the present study, the  H3K9ac  histone mark was\nsignificantly reduced in ectopic and eutopic tissues compared to the control tissues, the epigenetic modification\nwhich is in accordance to the decreased expression profile\nof  FOXP3  gene in endometriotic tissues.\nOn the other hand, the overall level of  H3K9me2  in the\npromoter region of the  FOXP3  gene was elevated in both\nthe eutopic and ectopic groups compared to the control\nsamples. The increase was statistically significant in the\neutopic group, but not in the ectopic group, which was\npossibly due to the sample size. Of note, the elevated\nlevel of this epigenetic marker,  H3K9me2 , in the endometriotic groups supported the findings related to the gene\nexpression.\nThe results of one study showed significantly lower total  H3K9ac  levels in the ectopic group compared to endometrial tissue of the normal and endometriosis groups\n( 31 ). Besides, the overall mean of  H3K9me2  in the endometrium of the control group was significantly lower than\nthe ectopic and eutopic tissues of patients with endometriosis. The overall level of H3 histone acetylation in endometriotic lesions was lower than the endometrial tissue\nof the control group, which supported our study results.\nReduced  FOXP3  expression may be one of the factors involved in endometriosis. The results of epigenetic\nstudies show that  FOXP3  gene expression appears to be\nregulated by  H3K9me2  and  H3K9ac  histone markers.\nUnderstanding the molecular setting of the  FOXP3  gene\nand Treg cell stability will shed light on their pathological\ndysregulation and determine novel therapeutic strategies.\nNumerous studies indicate that mutations in the  FOXP3 \ngene are associated with carcinogenesis and its expression level may serve as a prognostic indicator in the oncological context.  FOXP3  is an important factor in the\npathomechanism where the tumour escapes the immune\nsystem response ( 32 ). Endometriosis increases the risk\nof ovarian cancer ( 33 ), and it is hypothesised that studying the impact of epi-drugs in endometriosis patients may\nprovide novel options for control of this disease. In addition, histone acetylation is vital for the activation of key\ngenes related to endometriosis, which makes it a significant target for potential therapies. Adjusting histone acetylation could help reduce the symptoms of endometriosis. However, the complex interaction between different\nepigenetic markers suggests that acetylation might also\nregulate other molecular activities. For example, histone\ndeacetylase (HDAC) inhibitors increase histone acetylation, which can suppress cell division and influence DNA\ndamage repair mechanisms. These processes highlight\nthe impact of histone acetylation on cellular functions and\nprovide a basis for exploring new treatment avenues for\nendometriosis by modulating acetylation levels. It is essential to conduct comprehensive research to optimize the\ntherapeutic benefits while minimising adverse effects before employing HDAC inhibitors for endometriosis treatment ( 34 ).\nFinally, limitations of the current research included the\nsmall sample size and sample heterogeneity. It is essential to emphasise that the analysis was based on samples\nrepresentative of entire tissues, which includes stromal,\nepithelial, and inflammatory cells. This cellular mixture\ncan result in different epigenetic profiles between the\neutopic and ectopic endometrium from the same patient.\nMoreover, in this study, we did not consider the phases\nof the menstrual cycle. Another limitation of this study is\nthat some eutopic and ectopic tissue samples originated\nfrom the same patients, whereas others were obtained\nfrom different individuals.\n\nThe discovery of epigenetic settings of Treg cells creates a new perspective for understanding the role of these\nregulatory cells in disease and health. A set of precise\ntranscriptional and epigenetic adjustments is required for\nstable expression of the  FOXP3  gene and proper function\nof Treg cells. The data reported in this study contribute to\na better understanding of  FOXP3  expression and epigenetic alteration in endometriosis, and suggests the use of\nnew drugs and epi-drugs for this disease.","source_license":"CC0","license_restricted":false}