{"paper_id":"d7fb9314-803e-4302-b15f-9d60f6f5fba0","body_text":"Gene Cell Tissue. 2022 April; 9(2):e118656.\nPublished online 2021 October 15.\ndoi: 10.5812/gct.118656.\nResearch Article\nEvaluation of Plasma MMP-9 and VEGF-A mRNAs as Non-invasive\nDiagnostic Biomarkers in Women with Endometriosis\nSepideh Abdollahi 1, Pantea Izadi 1, *, Shahla Noori Ardebili 2, Samaneh Chegeni 3 and Mir Saead\nYekaninejad 4\n1Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran\n2Department of Gynecology and Laparoscopy , Atieh Hospital, Tehran, Iran\n3Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran\n4Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran\n*Corresponding author: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Email: p-izadi@sina.tums.ac.ir\nReceived 2021 August 13; Revised 2021 September 06; Accepted 2021 September 15.\nAbstract\nBackground: Endometriosis is one of the common gynecological diseases and can lead to pelvic pain, dysmenorrhea, dyspareunia,\nand infertility in women. Thus, accurate and early diagnosis is a pivotal issue and an essential need for managing this disorder. At\nthe present, the gold standard diagnostic method for endometriosis is laparoscopic surgery that is an invasive method and can lead\nto delay in diagnosis. Thus, there is an immediate necessity to search for non-invasive diagnostic biomarkers, such as blood-based\nones.\nObjectives: Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A) have essential roles in the patho-\ngenesis of endometriosis. Therefore, in this study , we evaluated the plasma mRNA levels of MMP-9 and VEGF-A, as potential non-\ninvasive diagnostic biomarkers for endometriosis.\nMethods: This study included 48 women (24 cases and 24 controls) who underwent laparoscopy for suspected endometriosis. Pre-\noperative plasma samples were collected, and after RNA extraction, the levels of MMP-9 and VEGF-A mRNAs were determined by\nreverse transcription-quantitative polymerase chain reaction (RT-qPCR).\nResults: Plasma MMP-9 mRNA level was statistically higher in endometriosis patients compared with the control group (P value =\n0.01). However, plasma VEGF-A mRNA level did not show a significant difference between the two groups (P value =0.5).\nConclusions: It seems that the plasma level of MMP-9 mRNA in endometriosis patients is significantly higher than in non-\nendometriosis women. This finding can provide new insights regarding this mRNA’s applicability as a non-invasive diagnostic\nbiomarker for discovering new cases of endometriosis (newly diagnosed). According to our results, despite the suggested role of\nVEGF-A in endometriosis pathogenesis, it seems that the plasma level of VEGF-A mRNA does not have the potential to be used as a\nnon-invasive diagnostic biomarker.\nKeywords: Endometriosis, Matrix Metalloproteinase 9, Diagnosis, Biomarkers, VEGF-A, Plasma\n1. Background\nEndometriosis is a debilitating and chronic disease\namong women. In this disorder, the endometrial tissue\ngrows in ectopic locations. It is diagnosed in approxi-\nmately 10-15% of reproductive-aged women and 35-50% of\ninfertile women. In addition, it is observed in 50-70% of\nwomen with pelvic pain (1). The common signs of en-\ndometriosis comprise pelvic pain, dysmenorrhea, dyspare-\nunia, and infertility . Thus, it can influence the quality of life\nand impose health-related costs (2).\nThe gold standard diagnostic method for endometrio-\nsis is laparoscopic surgery and biopsy (3). Laparoscopic\nsurgery is an invasive and costly procedure. In addition,\nit harbors the risks associated with general anesthesia and\npost-procedure adhesions. Thus, a non-invasive diagnostic\ntest for endometriosis can lead to timely diagnosis and bet-\nter disease management (4). Blood biomarkers have been\nconsidered as non-invasive diagnostic parameters for this\ndisease. Blood-based diagnostic markers for endometrio-\nsis can lead to cost-effective, less invasive, and easily accept-\nable procedures for patients and provide quick results (5).\nDespite many efforts, no non-invasive biomarker has been\napproved for the diagnosis of endometriosis yet.\nExtracellular circulating RNAs in biological fluids such\nas plasma, urine, and saliva are becoming optimistic non-\ninvasive and cost-effective diagnostic tools (6). A previous\nCopyright © 2021, Gene, Cell and Tissue. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International\nLicense (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is\nproperly cited.\n\nAbdollahi S et al.\nstudy showed the capacity of some miRNA panels as blood-\nbased non-invasive biomarkers for endometriosis (7), and\nsome studies have suggested the usefulness of plasma mR-\nNAs as biomarkers for different cancers (8-11). Accordingly ,\ncirculating mRNAs in serum may be helpful non-invasive\ntools for the diagnosis of endometriosis.\nAccording to previous investigations, matrix\nmetalloproteinase-9 (MMP-9) and vascular endothelial\ngrowth factor-A (VEGF-A) make an essential contribution\nto the pathogenesis of endometriosis (12, 13). Also, the\nmRNAs of these genes are overexpressed in endometrial\nlesions in women with endometriosis (14, 15). The MMP-9\ngene encodes a matrix metalloproteinase involved in\nextracellular matrix (ECM) degradation (16). This feature is\nvital for the invasion of the endometrial tissue to ectopic\nlocations (17). The VEGF-A gene encodes a factor essential\nfor angiogenesis; a process that is necessary for the sur-\nvival and propagation of ectopic endometrial lesions (18).\nSome studies have detected the mRNAs of these genes\nin the peripheral blood of endometriosis patients (19,\n20). However, the diagnostic potential of these mRNAs\ncirculating in the plasma of endometriosis patients is\nuncertain.\n2. Objectives\nThe aim of this study was to quantify the mRNA levels\nof MMP-9 and VEGF-A in endometriosis patients’ plasma\nsamples by RT-qPCR.\n3. Methods\n3.1. Patients and Sample Collection\nThe study population consisted of 48 women with\nsuspected endometriosis referred to a gynecologist\nwith complications such as dysmenorrhea or pelvic\npain. All the patients were scheduled to undergo la-\nparoscopy and signed written informed consent before\nsurgery . This study was approved by the Research Ethi-\ncal Committee of Tehran University of Medical Sciences\n(IR.TUMS.MEDICINE.REC.1398.579). Before surgery , 5 mL of\nblood was obtained from each participant and transferred\ninto a tube containing EDTA as an anticoagulant. Then\nblood samples, without delay , were transferred to the lab-\noratory for subsequent processing and plasma separation.\nAccording to the gold standard for endometriosis diagno-\nsis (direct visualization of lesions in laparoscopic surgery\nand confirmation by pathological assessment of the tis-\nsue), the patients were categorized into two groups: 24\npatients with a confirmed diagnosis of endometriosis (as\nthe case group) and 24 patients without endometriosis (as\nthe control group). Endometriosis stage was defined based\non the guidelines of the American Society of Reproductive\nMedicine (ASRM).\nThe characteristics of study participants, including\nthe menstrual cycle phase and medical background, were\nrecorded in a clinical questionnaire based on their medi-\ncal records. Moreover, none of the patients received pre-\noperative treatments, such as hormone therapy , which was\nregarded as one of important inclusion criteria due to the\npossible effects of drugs on gene expression. In addition,\nall the participants were in reproductive age and referred\nto a gynecologist with endometriosis symptoms. Patients\nwith a previous diagnosis of diseases such as cancer, au-\ntoimmune diseases, and endocrine disturbances (polycys-\ntic ovarian syndrome, etc.) were excluded from this study .\n3.2. Plasma Separation and RNA Extraction\nBy centrifugation (at 5000 rpm and then 1500 rpm for\n15 min at 4 ºC), plasma samples were initially separated\nfrom whole blood. Thermo Scientific Nanodrop 2000 was\nused to measure plasma quality at 414 nm for detecting\nhemolysis in samples. Then the plasma samples that had\nno hemolysis were stored at -80 °C. The TRIzol reagent (Ri-\nboEx LS, GeneAll, Korea) was used to isolate total RNA from\n400 uL plasma. Afterward, the RNA samples were sub-\njected to standard ethanol precipitation, and the pellet was\nresuspended in 10 µL of RNAase-free water. Finally , the\nquality and quantity of the extracted RNAs were appraised\nby a spectrophotometer.\n3.3. Reverse Transcription-quantitative Polymerase Chain Reac-\ntion\nThe extracted RNA samples were converted to cDNA us-\ning a Reverse Transcription kit (Geneall, Korea) according\nto the manufacturer’s protocol. The mRNA levels of MMP-\n9 and VEGF-A were measured by SYBR Green RealQ Plus 2x\nMaster Mix Green (Ampliqon, Denmark) using Roche Light-\nCycler® 96 System. Normalization was carried out employ-\ning β-Actin as a reference gene. The reactions included an\ninitial step at 95°C for 30 sec, followed by 40 cycles of am-\nplification; 95 °C for 10 s (denaturation), 60 °C for 15 s (an-\nnealing), and 72 °C for 30 s (extension). The qRT-PCR reac-\ntion mixture consisted of 5 µL SYBR Green master mix, 1µL\nof each of forward and reverse primers (primer concentra-\ntion: 5 pmol), 1 µL of target cDNA, and 2 µL sterile water\nin a total volume of 10 µL. Each reaction was performed\nin duplicate. The specificity of RT-qPCR amplification was\nconfirmed by melting curve analysis and observing a sin-\ngle peak. The 2 -∆∆Cq method was used for calculating the\nrelative expression of each target mRNA. The sequences of\nthe primers used in this study have been represented in Ta-\nble 1.\n2 Gene Cell Tissue. 2022; 9(2):e118656.\n\nAbdollahi S et al.\nTable 1. The Sequences of the Primers Used for Gene Expression Analysis\nPrimers Sequences\nMMP-9\nForward 5’- CAGGCAGCTGGCAGAGGAAT-3’\nReverse 5’- TTCGACTCTCCACGCATCTC-3’\nVEGF-A\nForward 5’- CTCCACCATGCCAAGTGGT-3’\nReverse 5’- TCTCGATTGGATGGCAGTAGC-3’\nβ-Actin\nForward 5’- CCCAGCACAATGAAGATCAAGATCAT-3’\nReverse 5’- ATCTGCTGGAAGGTGGACAGCGA-3’\nAbbreviations: MMP-9, matrix metalloproteinase-9; VEGF-A, vascular endothe-\nlial growth factor-A.\n3.4. Statistical Analysis\nThe Kolmogorov-Smirnov test showed that the data\nhad a non-normal distribution (P < 0.05). Thus, a non-\nparametric test, the Mann-Whitney U test, was used to com-\npare the studied variables between the groups. All statisti-\ncal tests were performed in IBM SPSS Statistics 22 (SPSS Inc.,\nChicago, IL, USA), and P < 0.05 denoted a statistically sig-\nnificant difference between the two groups.\n4. Results\n4.1. Participants’ Descriptive Parameters\nThe participants’ clinicopathological information has\nbeen briefed in Table 2. A total of 24 endometriosis patients\nwith stage III (n = 10) and IV (n = 14) were enrolled in this\nstudy (mean age: 33.5 ± 5.1 years). The control group in-\ncluded 21 women with benign gynecological diseases and\nthree healthy women (mean age: 37.5 ± 9.1 years). A def-\ninite diagnosis in all the patients was made by histolog-\nical analysis. The menstrual cycle of all the participants\nwas determined; there was no significant difference in\nthe menstrual phase between patients with endometriosis\nand control subjects (P value > 0.05).\n4.2. MMP-9 and VEGF-A mRNAs’ Plasma Levels\nThe relative levels of the mRNAs of MMP-9 and VEGF-\nA were measured in plasma samples using RT-qPCR (Fig-\nure 1). The plasma mRNA level of MMP-9 was significantly\nhigher in endometriosis patients compared with the con-\ntrol group (P value = 0.01). In contrast, no statistically sig-\nnificant difference was observed between the two groups\nregarding VEGF-A mRNA plasma level (P value = 0.5).\n20 \n15 \n10 \n5\n0\nmRNA Fold Change \nN-EMS \nEMS \nMMP-9 VEGF-A \nGroups\nNS \nFigure 1. The mRNA fold-change of MMP-9 and VEGF-A in the plasma of endometrio-\nsis (EMS) patients compared with non-endometriosis (N-EMS) counterparts. (*: P\nvalue < 0.05, NS: non-significant).\n4.3. Association of mRNA Levels of MMP-9 and VEGF-A with Men-\nstrual Cycle Phase and Disease Stage\nThere was no significant association between the\nmRNA level of MMP-9 and the menstrual cycle (P value >\n0.05). However, there was a significant positive association\nbetween VEGF-A mRNA level and the proliferative phase\nof the menstrual cycle in the study participants (P value\n< 0.05). Moreover, the expression levels of both target\nmRNAs in this study were independent of disease stage (P\nvalue > 0.05).\n5. Discussion\nEndometriosis is a common and chronic gynecologi-\ncal disease that affects women’s reproductive health and\nquality of life. Endometriosis can have complications such\nas pelvic pain, dysmenorrhea, dyspareunia, and infertil-\nity (21), and its diagnosis requires a surgical intervention\nthat is accompanied by risks and complications for women\n(22). The lack of non-invasive diagnostic tests for en-\ndometriosis leads to a considerable delay in disease detec-\ntion (3). So, developing non-invasive diagnostic biomark-\ners for endometriosis is an urgent need. In this context,\nblood-based biomarkers can be helpful. In recent years,\nmost efforts for finding non-invasive diagnostic biomark-\ners for endometriosis have been directed towards blood\nmiRNAs. Also, it seems that circulating mRNAs can be use-\nful as potential biomarkers (5, 7). Recently , it has been\nindicated that diffusion RNAs are protected by lipopro-\ntein complexes or phospholipids. Thus, these molecules\nGene Cell Tissue. 2022; 9(2):e118656. 3\n\nAbdollahi S et al.\nTable 2. Participants’ Characteristics in This Study .\nCharacteristics Endometriosis Women (n = 24) Non-endometriosis Women (n = 24)\nAge (y , mean± SD) 33.5 ± 5.1 37.5 ± 9.1\nCycle phase, No. (%)\nFollicular 20 (83) 19 (79)\nLuteal 4 (17) 5 (21)\nARSM stage, No. (%)\nIII 10 (42) NA\nIV 14 (58) NA\nOther diagnoses, No. (%)\nHealthy NA 3 (13)\nOvarian cysts NA 8 (33)\nMyoma NA 11 (46)\nOther NA 2 (8)\nAbbreviations: ARSM, American Society of Reproductive Medicine; NA, Not Available.\nare stable in circulation despite the high amounts of the\nRNases present in blood (23). It has been shown that\nvarious kinds of RNAs, including mRNAs and non-coding\nRNAs, can be extracted and detected in body fluids such as\nplasma and serum (24, 25). Indeed, several investigations\nhave shown that cell-free mRNAs in plasma can be promis-\ning non-invasive diagnostic tools for cancer (26, 27). Hence,\nin this study , the plasma levels of MMP-9 and VEGF-A mR-\nNAs, as the genes relevant to endometriosis pathogenesis,\nwere evaluated by RT-qPCR.\nIn this study , for the first time, we measured plasma\nMMP-9 mRNA level via qRT-PCR in women with en-\ndometriosis. Our results showed a significant increase\nin serum MMP-9 mRNA in endometriosis compared with\nnon-endometriosis women. Moreover, this study showed\nthat the expression of MMP-9 was independent of the\nmenstrual phase. Interestingly , endometriosis, as a be-\nnign disease, shares a common feature with malignancies,\nand that is its invasive behavior (28). It has been shown that\nMMP-9, as a matrix metalloproteinase, is an essential fac-\ntor for the degradation of the extracellular matrix (ECM)\nand the basement membrane and a crucial factor for cells’\ninvasive behaviors (29). Several studies have reported\nthat MMP-9 can induce invasiveness in the endometrial\ntissue, leading to the degradation of ECM, invading other\nlocations, and creating ectopic endometriosis lesions (30,\n31). There are pieces of evidence suggesting that the MMP-9\ngene is significantly overexpressed in the endometriosis\ntissue, which can be expected according to its role in\nendometriosis pathogenesis (13, 27). The elevation of\nMMP-9 mRNA in the plasma of endometriosis patients,\nobserved in this study , was consistent with the results\nof previous investigations on the expression MMP-9 in\nthe endometriosis tissue, suggesting its potential as a\nnon-invasive diagnostic biomarker for endometriosis.\nThis study showed no significant difference in plasma\nVEGF-A mRNA level between endometriosis and non-\nendometriosis women. Moreover, we showed that VEGF-\nA mRNA level positively correlated with the proliferative\nphase of the participants’ menstrual cycles. This molecule\nis an essential factor for physiological and pathological an-\ngiogenesis and induces vascular endothelial cells’ prolifer-\nation and migration (18). Angiogenesis plays a significant\nrole in endometriosis progression because, similar to tu-\nmors’ behaviors and their metastatic spread, endometrio-\nsis lesions require neovascularization to survive in ectopic\nlocations. Indeed, the establishment, proliferation, and\nsurvival of endometriosis lesions depend on an adequate\nblood supply (12). Thus, regarding the role of VEGF-A in en-\ndometriosis pathogenesis, the overexpression of its gene\nin the endometriosis tissue can be expected, as shown in\na previous study (12). According to our results, however,\nthe plasma level of VEGF-A did not show a significant dif-\nference between endometriosis patients and controls. It\nseems that the plasma level of VEGF-A mRNA, in contrast\nto its expected role in endometriosis pathogenesis, cannot\nbe a useful blood-based biomarker for discriminating en-\ndometriosis in women. Furthermore, our finding regard-\ning the comparable levels of VEGF-A mRNA in plasma sam-\nples from endometriosis and non-endometriosis women\nwas inconsistent with that of a previous study , indicating\na significantly higher level of VEGF-A mRNA in endometrio-\nsis patients (32). This discrepancy may be due to the limited\nnumber of control subjects in the mentioned study (i.e.,\n4 Gene Cell Tissue. 2022; 9(2):e118656.\n\nAbdollahi S et al.\nonly 10 women, mostly infertile) and different samples in\nthe two studies (serum vs. plasma).\nWe must address some limitations of the present study .\nOur sample size was relatively small, and the patients\nmainly presented stage III and IV endometriosis. Hence,\nlarge-scale studies should be performed to clarify the ap-\nplicability of MMP-9 and VEGF-A mRNAs as non-invasive di-\nagnostic biomarkers for endometriosis.\n5.1. Conclusion\nIn the present study , the levels of MMP-9 and VEGF-\nA mRNAs, as non-invasive diagnostic biomarkers, were\nevaluated by RT-qPCR in the plasma samples of women\nendometriosis. A significant difference was detected in\nplasma MMP-9, but not VEGF-A, mRNA level between en-\ndometriosis and non-endometriosis women. In conclu-\nsion, it can be suggested that MMP-9 mRNA has the po-\ntential for being used as a diagnostic biomarker for en-\ndometriosis, and it can be considered for further investi-\ngations along with other mRNAs. However, VEGF-A seemed\nto be inapplicable as a non-invasive diagnostic biomarker\ndespite its role in endometriosis pathogenesis.\nFootnotes\nAuthors’ Contribution: Concept and design: S. A. and P.\nI.; Collecting and interpreting clinical data and revising the\nmanuscript: S. A., P. I., S. CH., and SH. N.; Statistical analysis:\nS. Y., S. A., and P. I.; Drafting the manuscript: S. A. and P. I. All\nauthors read and approved the final manuscript.\nConflict of Interests: No author has any potential conflict\nof interest.\nEthical Approval: This study was approved by the Re-\nsearch Ethical Committee of Tehran University of Medical\nSciences (IR.TUMS.MEDICINE.REC.1398.579).\nFunding/Support: This study was a part of a M.Sc. the-\nsis supported by Tehran University of Medical Sciences\n(Tehran, Iran; grant No: 43115).\nInformed Consent: All patients signed written informed\nconsent before surgery .\nReferences\n1. Hirsch M, Dhillon-Smith R, Cutner AS, Yap M, Creighton SM. The\nPrevalence of Endometriosis in Adolescents with Pelvic Pain: A\nSystematic Review . J Pediatr Adolesc Gynecol . 2020; 33(6):623–30. doi:\n10.1016/j.jpag.2020.07.011. [PubMed: 32736134].\n2. DiVasta AD, Vitonis AF, Laufer MR, Missmer SA. Spectrum of symp-\ntoms in women diagnosed with endometriosis during adolescence\nvs adulthood. Am J Obstet Gynecol . 2018; 218(3):324 e1–324 e11. doi:\n10.1016/j.ajog.2017.12.007. [PubMed: 29247637].\n3. Saunders PTK, Horne AW. Endometriosis: Etiology , pathobiol-\nogy , and therapeutic prospects. Cell. 2021; 184(11):2807–24. doi:\n10.1016/j.cell.2021.04.041. [PubMed: 34048704].\n4. Nisenblat V, Bossuyt PM, Farquhar C, Johnson N, Hull ML.\nImaging modalities for the non-invasive diagnosis of en-\ndometriosis. Cochrane Database Syst Rev . 2016; 2. CD009591. doi:\n10.1002/14651858.CD009591.pub2. [PubMed: 26919512]. [PubMed\nCentral: PMC7100540].\n5. Nisenblat V, Bossuyt PM, Shaikh R, Farquhar C, Jordan V, Schef-\nfers CS, et al. Blood biomarkers for the non-invasive diagnosis of\nendometriosis. Cochrane Database Syst Rev . 2016;(5). CD012179. doi:\n10.1002/14651858.CD012179. [PubMed: 27132058]. [PubMed Central:\nPMC7076288].\n6. Tzimagiorgis G, Michailidou EZ, Kritis A, Markopoulos AK,\nKouidou S. Recovering circulating extracellular or cell-free\nRNA from bodily fluids. Cancer Epidemiol . 2011; 35(6):580–9. doi:\n10.1016/j.canep.2011.02.016. [PubMed: 21514265].\n7. Zafari N, Tarafdari AM, Izadi P, Noruzinia M, Yekaninejad MS, Bahramy\nA, et al. A Panel of Plasma miRNAs 199b-3p, 224-5p and Let-7d-\n3p as Non-Invasive Diagnostic Biomarkers for Endometriosis. Re-\nprod Sci. 2021;28(4):991–9. doi: 10.1007/s43032-020-00415-z. [PubMed:\n33398851].\n8. Ng EK, Tsui NB, Lam NY, Chiu RW, Yu SC, Wong SC, et al. Presence of fil-\nterable and nonfilterable mRNA in the plasma of cancer patients and\nhealthy individuals. Clin Chem. 2002;48(8):1212–7. [PubMed: 12142376].\n9. Sueoka E, Sueoka N, Iwanaga K, Sato A, Suga K, Hayashi S, et al. De-\ntection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung\ncancer patients by quantitative real-time polymerase chain reaction.\nLung Cancer . 2005; 48(1):77–83. doi: 10.1016/j.lungcan.2004.10.007.\n[PubMed: 15777973].\n10. Wang H, Zhang X, Wang L, Zheng G, Du L, Yang Y, et al. Investiga-\ntion of cell free BIRC5 mRNA as a serum diagnostic and prognostic\nbiomarker for colorectal cancer. J Surg Oncol . 2014;109(6):574–9. doi:\n10.1002/jso.23526. [PubMed: 24338523].\n11. Dalle Carbonare L, Frigo A, Francia G, Davi MV, Donatelli L, Stranieri\nC, et al. Runx2 mRNA expression in the tissue, serum, and circu-\nlating non-hematopoietic cells of patients with thyroid cancer. J\nClin Endocrinol Metab . 2012; 97(7):E1249–56. doi: 10.1210/jc.2011-2624.\n[PubMed: 22511796].\n12. Cardoso JV, Abrao MS, Vianna-Jorge R, Ferrari R, Berardo PT, Machado\nDE, et al. Combined effect of vascular endothelial growth fac-\ntor and its receptor polymorphisms in endometriosis: a case-\ncontrol study . Eur J Obstet Gynecol Reprod Biol . 2017; 209:25–33. doi:\n10.1016/j.ejogrb.2016.10.046. [PubMed: 27836223].\n13. Yang M, Jiang C, Chen H, Nian Y, Bai Z, Ha C. The involvement of\nosteopontin and matrix metalloproteinase- 9 in the migration of\nendometrial epithelial cells in patients with endometriosis. Reprod\nBiol Endocrinol . 2015; 13:95. doi: 10.1186/s12958-015-0090-4. [PubMed:\n26289107]. [PubMed Central: PMC4545920].\n14. Danastas K, Miller EJ, Hey-Cunningham AJ, Murphy CR, Lindsay LA. Ex-\npression of vascular endothelial growth factor A isoforms is dysregu-\nlated in women with endometriosis. Reprod Fertil Dev. 2018;30(4):651–\n7. doi: 10.1071/RD17184. [PubMed: 29017687].\n15. Zhang L, Xiong W, Xiong Y, Liu H, Li N, Du Y, et al. Intracellular\nWnt/Beta-Catenin Signaling Underlying 17beta-Estradiol-Induced Ma-\ntrix Metalloproteinase 9 Expression in Human Endometriosis. Biol\nReprod. 2016; 94(3):70. doi: 10.1095/biolreprod.115.135574. [PubMed:\n26888969].\n16. Mott JD, Werb Z. Regulation of matrix biology by matrix met-\nalloproteinases. Curr Opin Cell Biol . 2004; 16(5):558–64. doi:\n10.1016/j.ceb.2004.07.010. [PubMed: 15363807]. [PubMed Central:\nPMC2775446].\n17. Weigel MT, Kramer J, Schem C, Wenners A, Alkatout I, Jonat W,\net al. Differential expression of MMP-2, MMP-9 and PCNA in en-\ndometriosis and endometrial carcinoma. Eur J Obstet Gynecol Re-\nprod Biol. 2012;160(1):74–8. doi: 10.1016/j.ejogrb.2011.09.040. [PubMed:\n22056701].\nGene Cell Tissue. 2022; 9(2):e118656. 5\n\nAbdollahi S et al.\n18. May K, Becker CM. Endometriosis and angiogenesis. Minerva Ginecol.\n2008;60(3):245–54. [PubMed: 18547987].\n19. Mabrouk M, Elmakky A, Caramelli E, Farina A, Mignemi G, Ven-\nturoli S, et al. Performance of peripheral (serum and molecular)\nblood markers for diagnosis of endometriosis. Arch Gynecol Ob-\nstet. 2012; 285(5):1307–12. doi: 10.1007/s00404-011-2122-4. [PubMed:\n22065163].\n20. De Sanctis P, Elmakky A, Farina A, Caramelli E, Seracchioli R, Mabrouk\nM, et al. Matrix metalloproteinase-3 mRNA: a promising peripheral\nblood marker for diagnosis of endometriosis. Gynecol Obstet Invest .\n2011;71(2):118–23. doi: 10.1159/000320752. [PubMed: 21150162].\n21. Dun EC, Kho KA, Morozov VV, Kearney S, Zurawin JL, Nezhat\nCH. Endometriosis in adolescents. JSLS. 2015; 19(2). doi:\n10.4293/JSLS.2015.00019. [PubMed: 26005317]. [PubMed Central:\nPMC4432718].\n22. Lier MCI, Vlek SL, Ankersmit M, van de Ven PM, Dekker J, Bleeker\nMCG, et al. Comparison of enhanced laparoscopic imaging tech-\nniques in endometriosis surgery: a diagnostic accuracy study . Surg\nEndosc. 2020;34(1):96–104. doi: 10.1007/s00464-019-06736-8. [PubMed:\n31028547]. [PubMed Central: PMC6946762].\n23. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-\nAgadjanyan EL, et al. Circulating microRNAs as stable blood-\nbased markers for cancer detection. Proc Natl Acad Sci U S A .\n2008;105(30):10513–8. doi: 10.1073/pnas.0804549105. [PubMed:\n18663219]. [PubMed Central: PMC2492472].\n24. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as\nbiomarkers in cancer patients. Nat Rev Cancer . 2011;11(6):426–37. doi:\n10.1038/nrc3066. [PubMed: 21562580].\n25. Tsui NB, Ng EK, Lo YM. Stability of endogenous and added RNA in\nblood specimens, serum, and plasma.Clin Chem. 2002;48(10):1647–53.\n[PubMed: 12324479].\n26. March-Villalba JA, Martinez-Jabaloyas JM, Herrero MJ, Santamaria J,\nAlino SF, Dasi F. Cell-free circulating plasma hTERT mRNA is a use-\nful marker for prostate cancer diagnosis and is associated with poor\nprognosis tumor characteristics. PLoS One . 2012; 7(8). e43470. doi:\n10.1371/journal.pone.0043470. [PubMed: 22916267]. [PubMed Central:\nPMC3423343].\n27. Cao W, Zhou D, Tang W, An H, Zhang Y. Discovery of plasma\nmessenger RNA as novel biomarker for gastric cancer identified\nthrough bioinformatics analysis and clinical validation. PeerJ. 2019;7.\ne7025. doi: 10.7717/peerj.7025. [PubMed: 31249732]. [PubMed Central:\nPMC6587939].\n28. Zeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an inva-\nsive, N-cadherin-expressing epithelial cell type in endometriosis us-\ning a new cell culture model. Am J Pathol . 2001; 159(5):1839–52. doi:\n10.1016/S0002-9440(10)63030-1. [PubMed: 11696444]. [PubMed Cen-\ntral: PMC1867070].\n29. Chung HW, Wen Y, Chun SH, Nezhat C, Woo BH, Lake Polan M. Ma-\ntrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-\n3 mRNA expression in ectopic and eutopic endometrium in women\nwith endometriosis: a rationale for endometriotic invasiveness.Fertil\nSteril. 2001;75(1):152–9. doi: 10.1016/s0015-0282(00)01670-8. [PubMed:\n11163831].\n30. Shaco-Levy R, Sharabi S, Benharroch D, Piura B, Sion-Vardy N. Ma-\ntrix metalloproteinases 2 and 9, E-cadherin, and beta-catenin ex-\npression in endometriosis, low-grade endometrial carcinoma and\nnon-neoplastic eutopic endometrium. Eur J Obstet Gynecol Reprod\nBiol. 2008; 139(2):226–32. doi: 10.1016/j.ejogrb.2008.01.004. [PubMed:\n18295959].\n31. Mousazadeh S, Khaksar F. Gene Expression Levels of Gelatinases in En-\ndometriosis. Stud Med Sci. 2018;28(12):795–804.\n32. Acimovic M, Vidakovic S, Milic N, Jeremic K, Markovic M, Milosevic-\nDjeric A, et al. Survivin and VEGF as Novel Biomarkers in Diagnosis of\nEndometriosis. J Med Biochem. 2016;35(1):63–8. doi: 10.1515/jomb-2015-\n0005. [PubMed: 28356866]. [PubMed Central: PMC5346803].\n6 Gene Cell Tissue. 2022; 9(2):e118656.","source_license":"CC0","license_restricted":false}