{"paper_id":"1d23d4a2-0fff-4c2d-9628-53d3f7f56d3d","body_text":"Expression analysis of \nplasma extracellular vesicle \nassociated candidate MiRNAs in \nendometriosis using integrative \nbioinformatics and experiential \ndata\nSamaneh Alsadat Sadati1,2, Zahra Chekini3, Faezeh Shekari4, Maryam Hafezi3, \nAzadeh Ghaheri5, Maryam Shahhoseini2, Ashraf Moini3,6, Reza Aflatonian3, \nMehdi Totonchi 2 & Parvaneh Afsharian1,2\nLaparoscopy is the gold standard for diagnosing endometriosis; however, it is an invasive and costly \nmethod. Recent studies offer a non-invasive approach based on extracellular vesicle miRNA. Despite \nthis, no consensus diagnostic biomarker has been identified to date. For addressing this gap, we \ndecided to investigate plasma derived extracellular vesicle associated candidate miRNAs. In order \nto identify candidate miRNAs, a comprehensive search was performed in PubMed database using \nthe search terms “micro-RNA” and “endometriosis”. Then, bioinformatics analysis was performed \nutilizing the miRTarBase database, Enrichr, and relevant software. During the experimental phase, the \npresence of candidate miRNAs was assessed in blood samples of 13 women with severe endometriosis, \nconfirmed through laparoscopy or doppler sonography, as well as in 11 endometriosis-free women, as \ncontrol group. After literature review of 405 articles published between 2007 and 2023, followed by \nbioinformatics analysis, were identified five miRNAs (miR-451a, 148a, 23b, 100, and 154) as candidate \nmiRNAs. Subsequently, the expression levels of miR-451a, 148a, 23b, and 100 found to exhibit \ndifferences between the case and control groups. Our study suggests to serve of these miRNAs as a \npotentially diagnostic biomarker panel for endometriosis, however it needs to be confirmed by future \nstudies with large diagnostic validation.\nKeywords Endometriosis, MiRNA, Extracellular vesicle\nEndometriosis is an inflammatory disorder that affects 10–15% of women of reproductive age and is \ncharacterized by the presence of endometrial tissue outside the uterus1. The symptoms are heterogeneous, with \nthe most common including pelvic pain, dysmenorrhea, and infertility 2. Often, Nonspecific symptoms may \nlead to misinterpretation, resulting in delays in diagnosis 3. Visualization of ovarian endometriotic cysts and \ndeep endometriotic lesions can be performed using ultrasonography and MRI; however, these methods are \nunable to identify peritoneal endometriosis or endometriosis-associated adhesions 4. The American Society for \nReproductive Medicine (ASRM) classification system categorizes endometriosis into four stages based on the \n1Department of Genetics, Faculty of Basic Sciences and New Biological Technologies, University of Science and \nCulture, ACECR, Tehran, Iran. 2Department of Genetics, Reproductive Biomedicine Research Center, Royan \nInstitute for Reproductive Biomedicine, ACECR, Tehran, Iran. 3Department of Endocrinology and Female Infertility, \nReproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, \nIran. 4Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for \nStem Cell Biology and Technology, ACECR, Tehran, Iran. 5Department of Basic and Population Based Studies \nin NCD, Reproductive Epidemiology Research Center, Royan Institute, ACECR, Tehran, Iran. 6Department of \nObstetrics and Gynecology, Arash Women’s Hospital, Tehran University of Medical Sciences, Tehran, Iran. email:  \nm.totonchi@royan-rc.ac.ir; p.afsharian@royan-rc.ac.ir\nOPEN\nScientific Reports |        (2025) 15:24970 1| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports\n\n\nnumber of lesions and the degree of infiltration. More advanced stages of endometriosis (III-IV) can lead to \ncomplications such as pelvic pain, which may lead to a hysterectomy. Moreover, Pearce et al. demonstrated \nthat malignant transformation of ovarian endometrioma into ovarian cancer, particularly the clear cell and \nendometrioid subtypes5.\nThe gold standard diagnosis of endometriosis is performed by laparoscopy with direct visualization. This \nprocedure is invasive, costly, and in endometrioma cases through the excision of ovaries can lead to subfertility \nor infertility in women since is associated with diminished ovarian reserve 6. Therefore, the need for a non-\ninvasive, biomarker-based approach is evident 7. To the best of our knowledge, reliable diagnostic biomarkers \nare still unavailable.\nExtracellular vesicles (EVs) refer to a diverse population of vesicles that vary in size and biogenesis \nmechanism, which are released into the extracellular space by various cell types 8. Exosomes and microvesicles \nare two main subtypes of EVs that can transfer signals to recipient cells, establishing a new communication \nmechanism between cells, which they are considered as a novel communication paradigm9. mRNA and miRNA \nderived from EVs are functional and can exchange genetic material among cells 10. Recently, there has been \nincreasing interest in utilizing the content of EVs—especially miRNA—as a diagnostic biomarker 11.\nmiRNAs are small non-coding RNA molecules, typically ranging from 20 to 24 nucleotides in length. It is \nestimated that approximately 30% of transcriptomes are regulated by miRNAs 12,13. A miRNA can be found \nbound to proteins, such as Argonaute proteins, sorted in EVs, or circulating freely in plasma/ serum. miRNAs \nplay vital functional roles in cells, such as differentiation, neoplastic transformation, regeneration, and cell \nreplication14. Several studies have demonstrated that the dysregulation of miRNAs is involved in endometriosis \nand may develop lesion implantation, angiogenesis, proliferation, adhesion, and increased levels of cytokines, \nsuch as migration inhibitory factor (MIF) 15. In a study conducted by Caroline Frisendahl et al., it was shown \nthat the downregulation of miR-193 and miR-374 promotes cell migration and may serve as therapeutic targets \nfor this condition16. Research has also evaluated the expression of miRNAs in the serum, plasma17and ectopic / \neutopic tissues of endometriosis patients18.\nBased on current knowledge, a consensus biomarker for the diagnosis of endometriosis has not yet been \nidentified. However, studies have demonstrated that the number of EVs elevates in the early stages of the \ndisease19. The spread and stability of EVs, make them an ideal biomarker14.\nConsequently, we have chosen to measure the expression levels of candidate miRNAs found in plasma-\nderived EVs. This decision is based on the belief that these miRNAs could serve as non-invasive diagnostic \nbiomarkers for endometriosis, representing a promising avenue for exploration.\nResults\nResults of effect sized analysis\nCohen’s d values for the significantly dysregulated miRNAs ranged from 0.61 to 0.92 indicating moderate to \nlarge effect sizes. Despite the limited sample size, these effect sizes suggest meaningful biological differences \nbetween the endometriosis and control groups (Supplementary Table S1).\nSelection of MiRNAs and prediction of targets\nUsing bioinformatics tools, we evaluated potential targets and signaling pathways of microRNAs in the 60 \nselected articles. The analysis of the results ultimately yielded a list of 15 miRNAs (Table 1).\nMiRTarBase database, enrichment, and pathway analysis\nmiR-148a, miR-451a, miR-154, miR-23b, and miR-100 were selected as candidate microRNAs for further \ninvestigation due to their potential involvement in the development of endometriosis. The selection criteria were \nbased on the target genes regulated by these microRNAs and their established significance in the pathogenesis \nof endometriosis.\nmiR-451a\nNo pathogenic single nucleotide polymorphisms (SNPs)of miR-451a were identified. The five signaling pathways \nin which miR-451a received the highest scores were analyzed using Enrichr software (Supplementary Table S2).\nmiR-148a\nThe miR-148a molecule is an intergenic microRNA. The signaling pathways associated with this miRNA suggest \na potential link to cancer. To date, no pathogenic SNPs have been reported for this miRNA (Supplementary \nTable S3).\nmiR-23b\nmiR-23b is located in intron 3 of the AOPEP (aminopeptidase O) gene on chromosome 9q, and no pathogenic \nSNPs associated with this miRNA have been reported (Supplementary Table S4).\nmiR-100\nmiR-100 is a secretory microRNA that is studied in various fluids, tissues, and EVs27,33. It is an intergenic RNA \nlocated on chromosome 11q (Supplementary Table S5).\nmiR-154\nmiR-154 is an intergenic microRNA, and no SNPs have been reported for it. Bioinformatics analysis indicates \nthat miR-154 is involved in the Hippo signaling pathway (Supplementary Table S6).\nScientific Reports |        (2025) 15:24970 2| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nFinal list of MiRNAs selection\nThe dysregulation of miR-451a in plasma, serum, and endometriotic tissue has been investigated. miR-451a \nplays a role in regulating the expression of the MIF cytokine. miR-23b and miR-23a are downregulated in \nboth ectopic and eutopic endometrium when compared to normal endometrium 18. Microarray analysis \nhas demonstrated that miR-100-5p is expressed differentially in ectopic tissue compared to eutopic tissue 27. \nFurthermore, bioinformatics analysis has identified COX-2 as the target of miR-100.\nBioinformatics analyses have demonstrated that miR-154 plays a role in the Hippo signaling pathway. \nIn addition, miR-154, in conjunction with other microRNAs such as miR-196b-5p, may serve as a potential \ndiagnostic biomarker for endometriosis30. Research indicates that miR-148a regulates the expression of DNMT1 \nand HLA-G mRNA34 (Table 2). In the first step, studies conducted without any time limitations up to April 2023 \nwere reviewed. Subsequently, five miRNAs were selected for the laboratory phase using bioinformatics analysis.\nSTRING model\nProtein-protein interaction networks were analyzed using the STRING database (Fig. 1).\nThe expression level of Circulating and EV- associated candidate MiRNAs\nAfter successfully passing the normality test, the data were analyzed using a parametric test. The data were \nnormalized with miR-16. Our findings indicated that miR-451, miR-23b, miR-148a, and miR-100 were \nsignificantly downregulated in the endometriosis group compared to the control group. Surprisingly, miR-154 \nwas not detected in either group (Fig. 2).\nmiRNA name Reasons for selection of miRNAs\nmiR-451a Targeting of MIF\nmiR-23b-3p Targeting of SF-1, STAR\nAnd Cyp19A1\nmiR-100-5p Targeting of COX2\nmiR-154-5p Involved in the Hippo signaling pathway\nmiR-148a-3p Targeting of HLA-G, DNMT1\nTable 2. Candidate MiRNAs based on their targets. In the first step, the studies conducted in 2007–2023 were \nreviewed, then using bioinformatics analysis 5 miRNAs were selected for the laboratory phase.\n \nmiRNA name Up/Down regulation (controversy) Reasons for selection Some of references\nmiR-451a Up (5)*\nDown (2) Targeting of MIF 16,19\nmiR-196a-5p Up (1) Targeting of PGR 20\nmiR-503-5p Up (1)\nDown (1)\nTargeting of SMAD2\nLocation of host gene\n21\nmiR-29c-3p Up (4)\nDown (1)\nTargeting of C-JUN and MMP2\nDetection by NGS study and highlight it\n22\nmiR-196b-5p Up (1)\nDown (4)\nTargeting of TGFBR2\nExonic miR\nInvolved in estrogen signaling\n23\nmiR-200a-3p Up (1)\nDown (5) Targeting of TGFB and SMAD 24\nmiR-200b-3p Down (5) Targeting of TGFB 24\nmiR-23a Down (1) Targeting of SF-1 and STAR\nAnd Cyp19A1\n18\nmiR-23b Down (2) Targeting of SF-1 and STAR\nAnd Cyp19A1\n18,25\nmiR-126-3p Up (2)\nDown (4) Targeting of CXCL12 26\nmiR-100-5p Up (6) Targeting of COX2 27,28\nmiR-199a Up (2)\nDown (3) Targeting of TGFB2 and TGFR1 27,29\nmiR-154-5p Down (1) Involved in hippo signaling pathway 30\nmiR-214-3p Down (1) Use of it in fibrosis therapy of endometriosis 31\nmiR-148a Up (3)\nDown (2) Targeting of Hela-G 32\nTable 1. The list of candidate mirnas. *These numbers refer to count of articles that evaluation intended \nmiRNAs.\n \nScientific Reports |        (2025) 15:24970 3| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nqRT-PCR efficiency results\nAmplification efficiency determined via Lin Reg PCR indicated a range between 1.91 and 1.92 for all candidate \nmiRAN, except for miR-154, which indicate an efficiency of 1.0. This result supports the specificity and reliability \nof the qRT-PCR reactions, while indicating a lack of detectable expression for miR-154 in both case and control \ngroup (Supplementary Table S7).\nDiscussion\nThe aim of this study was to investigate whether the expression levels of plasma derived circulating and EV-\nassociated candidate miRNAs changes and the end can serve as a potential biomarker for the disease. EV-miRNAs \nhave higher stability, specificity and sensitivity compared other type biomarkers such as circulating cell- free \nDNA, making them potentially more effective for early disease detection and monitoring. The packaging of EVs \nis purposeful and they are tissue specific allowing for targeted diagnosis and monitoring. In additional they can \nmore accurate and reliable than traditional biomarker like proteins35–37.\nFig. 1. STRING model. The protein- protein interaction acquired via SRING database.\n \nScientific Reports |        (2025) 15:24970 4| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nIn the first step, we identified candidate miRNAs based on previous literature. Subsequently, through \nbioinformatics analysis, we performed a final selection of miRNAs. The expression levels of these miRNAs \nhad been evaluated in serum, plasma, and tissue in previous studies 17,18,30. The expression levels of individual \nmicroRNAs exhibit considerable variability across studies, attributable to multiple confounding factors, \nincluding: sample size, sample type (serum, plasma, tissue, or extracellular vesicles), menstrual cycle phase \nof subjects, experimental techniques used and sampling conditions. To minimize inter-individual variability, \nwe collected all samples in the morning and restricted sampling to early proliferative phase (menstrual cycle \ndays 3–5). Notably, the expression profile of EV-associated microRNAs diverges from cellular or tissue-derived \npatterns. This discrepancy arises from two biologically significant mechanisms: active sorting and cellular \nclearance.\nFig. 2. Relative expression of miRNAs in plasma. Plasma EVs derived candidate miRNAs were evaluated \nwith the RT-PCR from women with endometriosis (n = 13) and control group (n = 11). Data represent the \nmean ± SEM, and the miRNAs expression is normalized to the miR-16 expression. *P < 0.05.\n \nScientific Reports |        (2025) 15:24970 5| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nmiRNAs can regulate the expression levels of messenger RNAs (mRNAs) and play an essential role in both \nnormal and abnormal biological processes. In addition to cellular miRNAs, they can secrete from different \ncell types via EVs 38. EVs transport and also transduce miRNAs into target cells, even distant via the blood \ncirculation. EVs- derived miRNA changes the expression levels and functions of recipient cells 39. Therefore, \ninvestigating plasma-derived miRNAs will aid for endometriosis progression, therapy, and the identification of \nnovel diagnostic biomarker. We chose candidate miRNAs based on their targets and signaling pathway, which \nplay an important role in endometriosis.\nThe macrophage migration inhibitory factor (MIF) is one of the targets of miR-451a. Endometriotic cells \nsecrete the MIF cytokine and has mitogenic properties that promote the growth of these cells. MIF can stimulate \nthe secretion of factors associated with cell proliferation and angiogenesis. Elevated levels of MIF have been \nshown in endometriotic tissues and peritoneal fluid of patients with endometriosis 40. The expression level of \nmiR-451a is inversely related to the extent of lesions 17. Also, the miR-451a serves as a reproducible diagnostic \nserum biomarker1. Our findings indicate that miR-451a is downregulated in EV-derived plasma, which may \ninfluence the progression of the disease.\nThe steroidogenic factor-1 (SF-1) regulates through miR-23a and miR-23b. SF-1 and other estrogenic \nenzymes regulate estrogen formation that has a significant role in endometriosis. An increase in the expression \nlevel of SF-1 is associated with a reduction in the expression levels of miR-23a and miR-23b in endometrial \ntissue. The upregulation of SF-1 leads to an increase in the expression of steroidogenic acute regulatory protein \n(stAR) and cytochrome P450 aromatase (CYP19A1), both of which are essential components of the estrogen \nsynthesis pathway18. Also, our study indicates that the expression level of miR-23b reduce in EV-plasma derived \ncan lead to the dysregulation of target genes and contribute to the progression of endometriosis.\nBioinformatics analysis has demonstrated that COX-2 is a validated target of miR-100. COX-2 is expressed \nduring proliferation, differentiation, and inflammation, which are prominent indexes of endometriosis. COX-2 \nhas a significant role in pain associated with endometriosis41. Despite the downregulation of miR-100 in ectopic \nversus eutopic endometrium38,22 our study indicates that the expression level of EV-derived plasma miR-100 \ndecreases in the endometriosis group.\nThe expression level of miR-154 is not affected by the menstrual cycle. It can be considered, either alone \nor in combination with hsa-miR-196b-5p, hsa-miR-378a-3p, and hsa-miR-33a-5p, as a non-invasive potential \ndiagnostic biomarker30. In addition, miR-154 is involved in the Hippo signaling pathway which has crucial \nroles in both normal and abnormal functions of endometrial cells in endometriosis. This pathway regulates cell \nproliferation, apoptosis and epithelial mesenchymal transition (EMT) process. Therefore, dysregulation of Hippo \nsignaling pathway can develop endometriosis42. We could not detect miR-154 in the EVs derived from plasma \nof our participants, including both the case and control groups. We performed amplification efficiency analysis \nwith LinReg PCR software. While all other miRNAs showed amplification above 1.91, miR-154 exhibited an \nefficiency of 1.0 consistence with the true absence of miR- 154. This finding indicates that the lack of detectable \nexpression of miR-154 in circulating and EV- associated RNA derived from plasma of case and control groups \nin this study is probably due to inter- individual or population specific variability, differences in RNA isolation \nmethods or sample processing and technical limitation.\nHypoxia is a hallmark of endometriosis that leads to altered methylation of the promoters of several genes \ninvolved in the development of endometriosis. The miR-148a/AUF complex suppresses the function of DNMT1 \nmRNA, resulting in hypomethylation 34. HLA-G can protect the fetus from maternal immune surveillance \nand rejection. Finding has shown that serum levels of HLA-G are higher in women with endometriosis \ncompared to a control group, that indicatea the runaway power of ectopic endometrial tissue of the immune \nsystem34. Additionally, miR-148a is an element of the G protein-coupled estrogen receptor (GPER)/miR-148a/\nHLA-G signaling pathway in ovarian endometriosis. The expression level of miR-148a is reduced in both the \nendometrium and endometriosis-associated ovarian cancer32. Furthermore, miR-148a can affect the expression \nof BCL-2 and the activity of caspase-3/9, making it a potential candidate for treatment of endometriosis32.\nWe showed that the expression levels of miR-451a, miR-23b, miR-148 and miR-100 significantly decrease \nin plasma derived EV of endometriosis patients. We propose that plasma-derived EV-miRNAs could serve as a \npotential diagnostic biomarker for endometriosis. However, further studies with a larger case and control groups \nare suggested to validate these findings.\nMaterials and methods\nEligibility criteria and search strategy\nAt the first, we reviewed all published studies in the PubMed database, without any time limitations, up to April \n2023. The search terms applied were (“MicroRNAs“[Mesh]) OR (“microRNA “[Title/Abstract]) OR (“miR“[Title/\nAbstract]) AND (“endometriosis“[Title]) OR (“endometriosis“[Title/Abstract]) OR (“Endometriosis“[Mesh]). \nWe assessed studies involving patients with endometriosis that examined miRNAs in relation to their role in \ndisease development or their potential as diagnostic biomarkers. The study population consisted of women with \nendometriosis at any stage, diagnosed through laparoscopy and/or Doppler sonography (Fig. 3).\nStudy selection\nAll obtained references were independently screened on the keywords in title/abstract or Mesh terms. Five \nmiRNAs were selected for our next in vitro phase using bioinformatics analysis. Finally, the expression level \nof candidate miRNAs were evaluated in the plasma derived EV in the case (n = 13) and control group (n = 11).\nBioinformatics analysis\nBy utilizing the miRTarBase database ( https://mirtarbase.cuhk.edu.cn), the validated targets of miRNAs were \nacquired. Followed by the identification of associated miRNA pathway using the lists of uploaded genes in the \nScientific Reports |        (2025) 15:24970 6| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nFig. 3. Flow diagram of inclusion and exclusion steps and search strategy. This flow diagram illustrates the \nsystematic process of applying inclusion and exclusion criteria during the research, as well as the search \nstrategy used to identify relevant studies.\n \nScientific Reports |        (2025) 15:24970 7| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nEnrichr software ( https://maayanlab.cloud/Enrichr/). Ultimately, five miRNAs were selected for the invitro \nphase.\nCriteria for MiRNAs selection\nTwo criteria for selection of miRANs is considered. First, the role of miRNA in progrresion of endometriosis \nor intredused it as a potentially diagnostic biomarker based on previous litreture. Second, specific targets or \npathway miRNAs in roled in endometriosis based on bioinformatics analysis.\nSTRING model\nThe validated targets of candidate miRNAs were acquired from the miRTarBase database based on reports \nwith more consensus assay including q-PCR, micro array and NGS. After removing repetitive targets among \nfive miRNAs the overlapping targets between miRTarBase and TargetScan ( https://www.targetscan.org) were \nidentified. Then protein- protein interaction achieved by the STRING (https://string-db.org/) database.\nPatients and control groups\nTwenty-four women aged 20–37 years old with normal body mass index (BMI) were enrolled in this study \nbetween June and august 2019 at the Royan Institute (Tehran, Iran). The study was approved by the Research \nEthics Committee of the Royan Institute (Code No.: IR.ACECR.ROYAN.REC.1398.144), and written informed \nconsent was obtained from all participants.\nThe presence of the disease was confirmed either by the laparoscopy and histopathological examinations \n(n = five) or doppler sonography( n n = eight). All cases who selected by laparoscopy suffered of an ovarian \nendometrioma at stages III–IV according to the Revised American Fertility Society Classification System \n(ARMS).\nThe control group was composed of 11 normal ovulatory women without endometriosis who were infertile \ndue to male factors. The absence of the endometrioma cysts was confirmed with the doppler sonography. \nWomen with a history of an irregular menstruation, cancers, inflammatory and autoimmune disease, fibroma, \nand adenoma were excluded from the study.\nAt the sampling time, all women were on days two-five of the menstrual cycle phase. No subjects had taken \nany hormonal treatments at least three months ago. All of the samples were collected in the morning.\nEffect sized analysis\nTo show that sample size cannot affect the validity of the results, Cohen’s d was calculated to estimate effect size, \nusing conventional thresholds (α = 0.05, two tail).\nPlasma isolation\nPeripheral blood samples (6 ml) were collected into a tube containing citrate as the anticoagulant agent. Whole \nblood was centrifuged at 2500  g at room temperature for 15  min. The plasma was transferred into a new \nmicrotube, and then centrifuge procedure was repeated. Platelet-free plasma (PFP) was isolated by a high-speed \ncentrifugation at 13,000 g for 5 min. Finally, the PFPs were aliquoted and stored at −80 °C until use.\nRNA extraction of plasma and EV isolation\nTotal RNA including circulating and EV- associated RNA was extracted from 2  ml of plasma. we used the \nPlasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Cat. No. 51000, Norgen Biotek). Briefly \n2 ml of plasma was mixed with lysis buffer A to disrupt EV membranes. And release their RNA contents. Ethanol \nwas then added, and the lysate was passed through spin column to bind RNA. After several washing steps, RNA \nwas eluted in 50 ml of elution solution A and sorted at −80 °C until further analysis. This kit designed to extract \ntotal RNA from plasma/ serum including both circulating and EV-associated RNA. The kit lyses extracellular \nvesicles during the extraction process, allowing access to vesicle-contained RNA without requiring physical EV \nisolation (e.g., ultracentrifugation or NTA).\nQuantitative reverse transcription PCR assay\nImmediately after extracting RNA, cDNA was synthesized using reverse transcription (RT) enzyme (Takara, \nUSA) following the manufacturer’s protocol. Stem-loop primers were used for more specifics. Primers were \ndesigned based on miRNA sequences obtained from the miRbase ( https://mirbase.org/) database as follows \n(Supplementary Table S8). The prepared master mix contained 10ng RNA, 4 ml enzyme buffer, 1 ml RT enzyme, \nand 1 ml stem loop master primer. The expression level of the candidate miRNAs was measured by the RT-PCR \nSYBR Green. Then, the expression levels of miRNAs were normalized with the expression level of hsa-miR-16.\nAssessment of PCR efficiency\nLinReg PCR software was used to calculate the efficiency of each qRT-PCR reaction. This software analysis row \nfluorescence data, adjust the base line automatically, and identifies the exponential phase (Window of linearity) \nfor each sample. Then it uses linear regression to estimate the slope of that phase, which is used to determine the \nreaction efficiency for each sample.\nStatistical analysis\nThe expression level of plasma derived EVs candidate miRNAs were assessed between the normal and \nendometriosis group. All data was analyzed for normal distribution. After successfully passing the normality \nparametric test, the SPSS software was used. A P value < 0.05 was considered as statistically significant.\nScientific Reports |        (2025) 15:24970 8| https://doi.org/10.1038/s41598-025-09660-1\nwww.nature.com/scientificreports/\n\nData availability\nThe archived datasets analyzed in support of the conclusions of this article will be made available upon request \nby the first or corresponding authors.\nReceived: 13 April 2025; Accepted: 30 June 2025\nReferences\n 1. Moustafa, S., Burn, M., Mamillapalli, R., Nematian, S. & Flores, V . Accurate diagnosis of endometriosis using serum MicroRNAs. \nAm. J. 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The inactivation of Hippo signaling pathway promotes the development of adenomyosis by regulating EMT, \nproliferation, and apoptosis of cells. Reprod. Sci. 30, 2715–2727 (2023).\nAcknowledgements\nThis study was financially sponsored by the genetic department of Royan Institute Tehran, Iran. The authors \ndedicate this article to the memory of Dr. Saeid Kazemi Ashtiani, the late founder of Royan Institute.\nAuthor contributions\nSAS, PA, FSh, AM, MT, MSh, Contributed to conception and design; SAS, Contributed to all experimental work, \ndata and statistical analysis, and interpretation of data; PA, MT, Were responsible for overall supervision; MH, \nAM Cooperated in the sampling section; MH, ZCh, Was responsible for patients assessment and diagnosis, \nalso for patient recruitment to study; SAS Collected the samples; AGh, Calculated the number of samples; ZCh \nCooperated with some experimental tests; FSh helped in design of E.V , approval tests and supervision of man-\nuscript in EV isolation part; AGh analyzed the row data; MSh, AGh, RA helped with interpretation of results; \nSAS, ZCh Drafted the initial manuscript and wrote the manuscript; PA, MT, AM Contributed to revise and edit \nthe manuscript; All authors read and approved the final manuscript.\nFunding\nNo funds, grants, or other support was received except full financial support from the genetic department of \nRoyan Institute Tehran, Iran. Institutional Review Board Statement: The study was conducted in accordance \nwith the Declaration of Helsinki, and approved by the Research Ethics Committee of the Royan Institute (Code \nNo.: IR.ACECR.ROYAN.REC.1398.144).\nDeclarations\nCompeting interests\nThe authors declare no competing interests.\nInformed consent\nInformed consent was obtained from all subjects involved in the study.\nAdditional information\nSupplementary Information The online version contains supplementary material available at  h t t p s : / / d o i . o r g / 1 \n0 . 1 0 3 8 / s 4 1 5 9 8 - 0 2 5 - 0 9 6 6 0 - 1     .  \nCorrespondence and requests for materials should be addressed to M.T. or P .A.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access  This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives \n4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in \nany medium or format, as long as you give appropriate credit to the original author(s) and the source, provide \na link to the Creative Commons licence, and indicate if you modified the licensed material. 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