{"paper_id":"56f41fc9-46c5-490f-95bc-9ce0e7ca9354","body_text":"Endometriosis is an endometrial inflammatory disease \nthat affects 5 to 10% of women in reproductive age. Its \nmain feature is the presence of endometrium-like tissue \nin sites other than the uterus. These extra uterine tissues \nappear mostly on the ovaries and peritoneum. The main \nclinical symptoms are chronic pelvic pain, dyspareunia, \nand infertility. Although the exact mechanism and \npathology of the disease remains unclear, there are distinct \nmolecular and immunologic differences between normal \nendometrium and eutopic or ectopic endometriosis tissue. \nOverproduction of estrogen and cytokines, progesterone \nresistance, and most importantly epigenetic deregulation \nof gene expression in endometriotic tissue are recently \ninvestigated. Gene-expression profiling of endometrium \nfrom women with endometriosis has revealed the \ncandidate genes related to implantation failure, infertility, \nand progesterone resistance as compared to endometriosisfree \nwomen ( 1 ,  2 ).\nSeveral studies have failed to show a major pure \ngenetic contribution in endometriosis. However, a \nnumber of studies have emphasized on epigenetics as a\nmajor underlying pathogenic mechanism. For example,\ngenome-wide methylation analysis has indicated that in\nendometriosis, there is DNA methylation abnormalities, \nlike expression of clusters of genes involved in \ndifferentiation through GATA family transcription factors \n( 3 ), which are the proteins that bind to DNA and regulate \nmany functions, like differentiation. Progesterone \nresistance and abnormal decidualization present in \nendometrial tissues are the other supporting evidences \nfor differentiation defect in endometriosis ( 4 ). These \nfindings have indicated that epigenetic deregulation \nmight be involved in the main biological processes found \nin endometriosis ( 5 ).\nEndometriosis is also known as a stem cell disorder \nthat is the underlying mechanism in extra endometrium \nimplantation. Although the exact stem cell pathology \nand its mechanisms are still unclear, abnormalities in \ncell motility and invasive capacity have been reported \nin endometriosis ( 6 ). Furthermore, it has been shown \nthat decreased level of proteins expression involved \nin cell adhesion and cytoskeleton and lower level \nof proteolytic activity promote endometriotic lesion \ngrowth in endometriosis ( 7 - 11 ). These abnormalities \nin stem cells might be in part mediated by microRNAs \n(miRNAs) deregulations. It has been mentioned that an \nabnormal decrease or increase in miRNAs expression\nin endometriotic stromal cells lead to up-regulation of\nmiR-503 expression, down-regulation of microRNA, \ninhibition of proliferation, as well as promotion of \napoptosis ( 12 ). However, proliferation of endometrial \nstromal cells (ESC), cell invasiveness and motility \nare known to be increased in endometriosis, mostly by \nepigenetic mechanisms and miRNAs ( 13 ).\nmiRNAs are non-coding RNAs of 18-25 nucleotide \nlong that regulate post-transcriptional gene expression \nby hybridizing to the complementary regions of target \nmRNA, leading to inhibition of translation with or without \ndegradation of the mRNA level. Among miRNAs, miR31 \nis a key player in spermatogenesis, implantation, and \nembryo development ( 14 ). Furthermore, miR-31 controls \nseveral vital processes. In breast cancer and liver cancer, \ndown-regulated miR-31 acts as a tumor suppressor, while \nin colorectal cancer, cervical cancer and lung cancer, up-\nregulated miR-31 is considered as oncomir. In addition, \nmiR-31 acts as a prognostic biomarker. High expression \nlevel of miR-31 is correlated to shorter survival in patients \nwith malignant pleural mesothelioma, whereas normal/ \nlow expression of miR-31 is associated with longer \nsurvival in this patients ( 15 ). miR-31 also plays important \nroles in fertility and pathogenesis of endometriosis through \ndown-regulating FOXP3 ( 16 ). Despite relevant strong \nsupporting evidences, the role of miR-31 in endometriosis \nhas not yet been studied.\nOn the other hand, miR-145 is shown to be a regulator\nof endothelial cell function and increase cell proliferation\nand invasiveness in tumors stem cells ( 17 - 22 ). Wang et \nal. ( 23 ) have found that in the serum of patients with \nendometriosis, miR-145 is down-regulated using \ncirculating miRNA array in comparison to the control \nwomen. Therefore, their results have suggested that \nmicroRNAmay be a potential biomarker of endometriosis. \nIn addition, miR-145 down-regulates posttranscriptionally \nthe pluripotency factor SOX2 and stemness-associated \nMusashi RNA binding protein 2 (MSI2).\nIt seems that miR-31 with its wide and complex functions \nin apoptosis, differentiation, proliferation and invasiveness, \nas well as miR-145 with its proved influence on local \ninvasiveness, proliferation and stemness of endometriotic \ncells may be involved in the pathogenesis of endometriosis. \nConsidering this hypothesis, we tried to analyze the plasma \nlevel of these two miRNAs in patients with endometriosis \nto have a clear understanding of the pathogenesis of \nendometriosis and find potential functional biomarkers.\n\nIn this case-control study approved by the Tarbiat \nModares University Ethics Committee, based on revised \nAmerican Society of Reproductive Medicine (rASRM) \nguidelines ( 24 ), we recruited 34 patients with histologically\nproven endometriosis stage 3 or 4 (moderate or severe forms, \nrespectively) and 21 patients with stage 1 or 2 (minimal and \nmild forms, respectively). There was also a control group \nincluding 23 endometriosis-free patients who underwent \nlaparoscopic examination for other indications than infertility, \nlike prolapsed uterus, ovarian cyst, or urinary incontinence. \nExclusion criteria of the control group were presence of other \nendometrial pathologies like myoma or fibroma. Exclusion\ncriteria of the patients groups were the presence of signs and\nsymptoms of endometriosis, like infertility and dysmenorrhea. \nExclusion criteria for both patient and control groups were \npresence of systematic inflammation diseases and infections, \nongoing pregnancy, history of pregnancy in the last 3 months, \ncancer, and other major systemic diseases. The mean age of \npatients was 28 ( 23 - 34 ) years. All subjects were enrolled in\nthis study after a standard genetic counseling and signing an \ninformed consent form.\nTo investigate the circulating miRNAs in plasma from \npatients and controls, 5 ml peripheral blood samples \nwere collected in heparinized tubes before laparoscopy. \nThen, tubes were centrifuged at 1900 rpm for 10 \nminutes at room temperature. Plasma was collected in \na sterile 1.5-ml microcentrifuge tubes RNase free and \ncryopreserved at -80°C. To evaluate the expression of \nhsa-mir-145 and hsa-mir-31 in endometriosis, miRNAs \nwere isolated from samples using miRCURY RNA \nIsolation Kits-Biofluids (Exiqon, Denmark) according to \nmanufacturers’ instructions. miRNAs were eluted in 50µl \nof nuclease free water. For first-strand cDNA synthesis \nreaction, miRCURY LNA™ Universal RT miRNA \npolymerase chain reaction (PCR), polyadenylation \nand cDNA synthesis kit (Exiqon, Denmark) were used. \nBriefly, each reverse transcription reaction consisted of 2 \nµl 5X reaction buffer, 1 µl enzyme mix, 0.5 µl synthetic \nRNA spike-ins, 4.5 µl nuclease free water, and a final \nconcentration of 100 ng/µl of total RNA. RT reaction was \nperformed using an Applied BiosystemsVeriti™ thermal \ncycler (Life Technologies, USA) at 42°C for 10 minutes, \nfollowed by heat-inactivation at 95°C for 5 minutes, and \nstored at 4°C. Real-time PCR was carried out using an \nApplied Biosystems StepOne Real-Time PCR System \n(Life Technologies, USA). For quantitative PCR (qPCR), \n10 µl PCR reaction mixture was prepared using ExiLENT \nSYBR® Green PCR master mix (Exiqon, Denmark) as \nrecommended by manufacturer. Briefly, 5 µl of 2XSYBR \nGreen master mix, 4 µl of diluted cDNA (1:4) and 1 µl of \nLNA primer mix were added. Then, has-mir-103-3p was \nused as the endogenous control. qPCR was performed at \n95°C for 10 minutes for polymerase activation followed by \n45 cycles of 95°C for 10 seconds and 60°C for 1 minutes. \nFinally, melting curve analysis was performed based of \nthe dissociation characteristics of double stranded DNA \nduring cycles with increasing denaturing temperature.\n\nAll results were expressed as the mean ± SE. Statistical \nanalysis was performed using GraphPad Prism 5 software \n(GraphPad, USA). One-way analysis of variance (ANOVA) \nwas applied for comparison of the differences between\ngroups. REST Software was also used to calculate relative \nquantity (RQ) of the expressions using Pffafl formula.\n\nPatients presented with infertility and dysmenorrhea in \n38 and 29%, respectively.\nTo explore miRNAs profiling from plasma samples, \nthe expression levels of miR-145 and miR-31 were \nevaluated by qPCR in endometriosis plasma as compared \nto the control group. The efficiency of qPCR reactions for \nmiR-31 and miR-145 were 97.3 and 101.8, respectively \n( Fig .1 ). Melting curves showed no nonspecific or primer \ndimer peaks ( Fig .2 ). We showed that the expression \nlevels of miR-31 in stage 3 or 4 and stage 1 or 2 were \nsignificantly down-regulated (less than 0.01-fold, \nP<0.05), whereas the expression level of miR-145 was \nup-regulated in endometriosis women in stage 1 or 2. The \nexpression level of miR-145 in stage 3 or 4 in the patient \ngroup was up-regulated more than 1.4-fold, indicating \nthat it did not reach to a significance level. miR-145 was \nsignificantly up-regulated in stage 1 or 2 more than 6.7(P<0.05,  Fig .3 ).\nStandard curves. A. miR-31, B. miR-145, and C. miR-103-3p.\nEfficiencies of amplifications are 97.3 (R 2 =0.98), 101.8 (R 2 =0.974) and \n100.958 (R 2 =0.999), respectively. R 2 ; The coefficient of correlation and Ct; \nCycle threshold.\nMelting curves of quantitative polymerase chain reactions (qPCR) of\nmiR-31 and miR145. There is no nonspecific or significant dimmer primer \npeak confirming the specificity of reactions.\nExpression analysis of miR-31 and miR-145 in patients with endometriosisstage 1 or 2 and stage 3 or 4. Relative expressions of different groups are shownin comparison with normal controls. A. The relative expression of miR-31 in stages1 or 2 and stages 3 or 4 compared with Normal and B. The relative expression ofmiR-145 in stages 1 or 2 and stages 3 or 4 compared with normal. In stage 1 or2, the expression level of miR-145 was increased by 6.7-fold (P<0.05), whereas theexpression level of miR-31 was decreased [relative quantity (RQ)<0.01, P<0.05)]. Instage 3 or 4, the expression level of miR-145 was increased by 1.4-fold, suggestingthis change did not reach to a statistical significance level. The expression level of\nmiR-31 was decreased as compared to normal controls (RQ<0.01, P<0.05).\n\nEndometriosis is a hormone dependent inflammatory \ndisease, in which the role of pure genetic variations \nis minor, while environmental and epigenetic factors \nare mainly involved. Many cellular functions like \ndifferentiation, proliferation, mitochondria, reactive \noxygen species (ROS) production, invasion and \ndecidualization are deregulated in this disease. miRNAs, \nincluding miR-31 and miR-145, regulate many of these \nbiological processes in different pathologies, especially \nin cancers. Clinical signs and symptoms are insufficient to \nhelp an early diagnosis Therefore, there is an urgent need \nto find biomarkers for early selection of patients with \nhigh risk of endometriosis and confirmatory diagnosis by \nlaparoscopy and pathological examination.\nWe found that in patients with stage 3 or 4 endometriosis, \nmiR-31 was down-regulated as compared to normal \nwomen. In addition, our results demonstrated down-\nregulation of miR-31 and significant up-regulation of \nmiR-145 in patients with stage 1 or 2 endometriosis. Our \nfindings also indicated that although several miRNAs \nwere deregulated in endometriosis ( 25 ), miR-31 and miR145 \nmay be considered as potential biomarkers for noninvasive \ndiagnosis of patients with endometriosis.\nInfertility in patients with endometriosis is due to \nmechanical as well as functional abnormalities of \nendometrium. Decidualization defect and abnormal \nreceptivity are the main mechanisms that can explain \ninfertility in endometriosis ( 5 ). In concordance, our \nfindings showed that miR-31 as one of the factors \ninfluencing decidualization was down-regulated in \npatients with endometriosis in all stages as compared \nto endometrium of normal controls. miR-31 is a highly \nconserved miRNA in evolution that acts as a vital factor \nin embryonic implantation and development, as well \nas immune system balance. Deregulation of miR-31 is \ncorrelated to some cancers and autoimmune diseases \npartly by epigenetic modifications, like methylation and \nacetylation ( 14 ). Although many biomarkers in plasma are \nnot functional and as surrogate biomarkers show no role \nin pathology, some others are involved in controlling the \npathogenic processes ( 25 ). Due to the regulatory role of \nmiR-31on biological and vital processes in endometriosis, \nits down-regulation might be an underlying epigenetic \nmechanism in the pathogenesis.\nCosar et al. ( 26 ) have showed that miR-145 is up-\nregulated 10-fold higher in patients with endometriosis. \nHowever, we found that an increase in miR-145 is mostly \nin stage 1 or 2, whereas there was a relative decrease in \nthe expression of miR-145 in stage 3 or 4. Adammek et \nal. ( 27 ) have indicated that miR-145 inhibits proliferation, \nwhile its over-expression can inhibit proliferation rates \nup to 45%. In addition, invasiveness was decreased by \n80%. A relative decrease in the expression of miR-145 \nin our patients with moderate to severe endometriosis \nmight promote an increase in proliferation that was seen \nin more advanced stages of the disease. Although the\npathogenesis of endometriosis is unknown, it is believed \nthat endometriosis is a disease of stem cells. Stemness \nhas been studied in endometriosis. There is an overall \nincrease in expression of genes involved in stemness in\nectopic as compared to eutopic tissues of patients with\nendometriosis ( 28 ). More specifically, the genes  UTF1, \nTCL1  and  ZFP42  show a trend for higher expression in \nendometriosis than in normal endometrium. However, \nexpressions of other genes involved in stemness are \nnot significantly different between endometriosis and \nnormal endometrium. miR-31 is directly correlated to \nstemness in some cancers. Its expression increases the \nexpression of Nanog/Sox2/oct4 in cancers ( 29 ). On the \nother hand, stemness seems to be de-regulated in eutopic\nendometrium of patients with endometriosis as compared\nto normal endometrium. Overexpression of miR-200b\nwhich is down-regulated in endometriosis can increase\nstemness-associated side population phenotype ( 13 ).\nmiR-145 is also a regulator of stemness. Down-\nregulation of miR-145 in patients with stage 3 or 4 as \ncompared with patients in stage 1 or 2 indicates the roles \nof miR-145 in down-regulation of the pluripotency factors \nand MSI2. A decrease in miR-145 relative expression \nin more advanced stages might be the underlying cause \nof increased invasiveness and proliferation, as well as \nincreased expression of stemness related genes, which are \nfound to be up-regulated in endometriosis ( 13 ).\nRelative down-regulation of miR-145 may promote\nthe progression of the disease from milder stages to\nmore severe stages. In other words, increased expression \nof miR-145 may inhibit proliferation and promote \ndifferentiation ( 30 ) in stage 1 or 2, while the disease is \nstill mild. However, when the disease progress to more \nsevere clinical forms, stage 3 or 4, the expression level \nof miR-145 decreases. It has been shown that increased \nexpression level of miR-145 is involved in infertility \nand repeated implantation failure ( 31 ), which is also the\ncommon features of endometriosis.\nIt is noteworthy that migration and invasion are \ncontrolled by miR-31 in cancer cells. Over-expression \nof miR-31 inhibits MDA-MB-231 cell migration and \ninvasion, while down-regulation of miR-31 promotes \nMCF-7 cell migration and invasion ( 32 ). In addition \nto invasion inhibition, miR-31 is a proapoptotic agent. \n Histone deacetylase inhibitors  (HDACIs) can induce \napoptosis through miR-31 induction ( 33 ). Endometriosis \nconsists primarily of stromal cells with low rate of \napoptosis, little differentiation and more invasiveness \n( 34 ). Nasu et al. ( 35 ) have showed that in endometriosis, \nthere is a resistance to apoptosis. Lack of normal immune \nresponses to endometrial cells, in addition to a decrease \nin apoptosis, facilitate proliferation and the implantation \nof ectopic endometrial tissues ( 36 ). A decrease in the \nexpression of miR-31 may induce resistance in apoptosis \nand increase proliferation found in endometriosis. miR31 \nthat is increased during the window of implantation is \nknown as a potential biomarker of optimum receptivity \n( 37 ). Our findings showed that in the plasma of patients \nwith endometriosis, there is a decrease of miR31 \nexpression level, suggesting that it was a sign of \nlower receptivity found in endometriosis in previous \nstudies ( 5 ,  38 ). Increased production of estrogens and \nprostaglandins, as well as progesterone resistance are\ncorrelated to the pathogenesis of endometriosis in eutopic \ntissues of patients with endometriosis as compared to\nwomen without endometriosis ( 39 ). Progesterone as\na differentiating agent is necessary in implantation.\nDecidualization, which is defected in endometriosis, is \nunder the control of progesterone. A significant decrease \nin miR-31 that is present in patients with endometriosis \ncould be the underlying cause. As shown in cancers, \nmiRNA has a p53 related anti proliferative activity ( 40 ), \nand p53 has a balancing role between differentiation and \nproliferation ( 41 ).\nAlthough the pathways are still unclear in endometriosis, \nrecent findings have strongly supported an epigenetic role \nin the underlying pathology of this disease, especially \nin miRNAs. Mostly, several miRNAs are important in \nthe pathogenesis and treatment of endometriosis. Since \nmiRNAs expressions are highly specific in tissues and \ntumors, they may be the potential biomarkers of early \ndiagnosis for the new treatment strategies.\n\nOverall, we found that miR-31 was under-expressed in \npatients with endometriosis, while miR-145 is over-expressed \nin stage 1 or 2 and relatively under-expressed in more severe \nforms of the disease. It means that deregulated expression of \nmiR-31 may explain the dysfunctions related to this disease. \nFurthermore, our findings indicated that expression analysis \nof these miRNAs in the plasma may serve as potential \nbiomarkers in noninvasive diagnosis of endometriosis, while \ntheir deregulated expression provides an understanding of the \nmechanism of pathogenesis of endometriosis.","source_license":"CC0","license_restricted":false}