{"paper_id":"32cb2927-1fdd-4bea-b5b7-7d997cc1a7bf","body_text":"CORRESPONDENCE Open Access\nBiomarker ResearchRashes Gertel et al. Biomarker Research           (2026) 14:85 \nhttps://doi.org/10.1186/s40364-026-00976-2\nEGF as a non-invasive biomarker \nof endometriosis: a case control study\nEmma R. Rashes Gertel1, Cassandra C. Daisy1, Katherine Kaplan1, Steven J. Staffa2,3, David Zurakowski2,3,4,  \nAllison F. Vitonis4,7,8, Kathryn L. Terry4,7,8, Amy L. Shafrir6,7,9,10, Simon T. Dillon4,5, Stacey A. Missmer4,7,9,11,12,  \nMichael S. Rogers1,2,4,6,7, Towia A. Libermann4,5 and Marsha A. Moses1,2,4,6,13*\n  * C o r r e s p o n d e n c e :  \nMarsha A. Moses\nmarsha.moses@childrens.harvard.edu\n1Vascular Biology Program, Boston Children’s Hospital, Boston, MA, USA\n2Department of Surgery, Boston Children’s Hospital, Boston, MA, USA\n3Department of Anesthesiology, Boston Children’s Hospital, Boston, MA, \nUSA\n4Harvard Medical School, Boston, MA, USA\n5Genomics, Proteomics, Bioinformatics and Systems Biology Center, Beth \nIsrael Deaconess Medical Center, Boston, MA, USA\n6Department of Surgery, Harvard Medical School, Boston, MA, USA\n7Boston Center for Endometriosis, Boston Children’s Hospital and Brigham \nand Women’s Hospital, Boston, MA, USA\n8Department of Obstetrics, Gynecology and Reproductive Biology, \nBrigham and Women’s Hospital, Boston, MA, USA\n9Division of Adolescent and Young Adult Medicine, Department of \nMedicine, Boston Children’s Hospital, Boston, MA, USA\n10Department of Nutrition and Public Health, School of Nursing and \nHealth Sciences, Merrimack College, North Andover, MA, USA\n11Department of Epidemiology, Harvard T.H. Chan School of Public \nHealth, Boston, MA, USA\n12Department of Obstetrics and Gynecology, University of Michigan, Ann \nArbor, MI, USA\n13Vascular Biology Program and Department of Surgery, Boston Children’s \nHospital Boston and Harvard Medical School, Karp Family Research \nLaboratories, 300 Longwood Avenue, Boston, MA 02115, USA\n© The Author(s) 2026. Open Access  This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 \nInternational License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you \ngive appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the \nlicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or \nexceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit  h t t  p : / /  c r e  a t  i \nv e c o m m o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 /     .    \nAbstract\nEndometriosis is a chronic inflammatory condition that affects an estimated 1 in 10 women but is often mis- and \nunderdiagnosed due to its non-specific symptoms and the lack of a non-invasive diagnostic test. This study aimed \nto identify and validate a potential non-invasive biomarker for endometriosis. This study applied quantitative \nproteomics discovery approaches to identify and validate non-invasive biomarkers of endometriosis with a long-\nterm goal of leveraging them for purposes of diagnosis and therapeutic monitoring. Isobaric tags for relative and \nabsolute quantification (iTRAQ) combined with mass spectrometry were used to identify and quantitate proteins \nand peptides in urine samples from participants with surgically-confirmed endometriosis ( n = 73) and 1:1 age-\nmatched participants never diagnosed with endometriosis ( n = 73). Among those aged 25–47 at urine collection, \nEpidermal Growth Factor (EGF) (validated using monospecific enzyme-linked immunosorbent assays (ELISA)) was \npresent at significantly lower levels in the urine of participants with surgically-confirmed endometriosis compared \nto controls ( P = 0.02) and had an excellent negative predictive value (NPV) of 94.3% with a cutoff of > 800 pg/ug \nas determined by Bayes’ formula. Urinary EGF levels were also significantly lower in the urine of participants aged \n25–47 with endometriosis who experience acyclic pelvic pain compared to samples from age-matched controls \nwho did not report acyclic pelvic pain ( P = 0.007) such that the presence of acyclic pelvic pain increased the NPV of \nEGF to 96.8% as determined by Bayes’ formula. These data demonstrate that urinary EGF has potential as a novel, \nnon-invasive, accurate and objective biomarker for endometriosis diagnosis and prognosis of this disease.\nKeywords iTRAQ, Biomarker, Non-invasive, Endometriosis, Epidermal Growth Factor, Urine\n\nPage 2 of 5\nRashes Gertel et al. Biomarker Research           (2026) 14:85 \n  To the Editor,\nEndometriosis is a chronic inflammatory estrogen-\ndependent condition marked by growth of endometrial-\nlike tissue outside the uterus that affects approximately \n10% of women [ 1]. Advances in imaging have improved \ndiagnosis of certain subphenotypes of endometriosis, but \nsuperficial peritoneal lesions with a revised American \nSociety for Reproductive Medicine (rASRM) stage of I or \nII and patients with negative imaging findings or failure \nwith empirical treatment can only be definitively diag -\nnosed via surgical visualization [ 2]. While imaging can \nplay an important role in diagnosis, imaging alone cannot \nrule out disease [3, 4]. \nThe lack of biomarkers of endometriosis leaves lapa -\nroscopy or laparotomy as the only option for objective \ndiagnosis, contributing to the average 7-year delay from \nsymptom onset to confirmed presence of endometrio -\nsis, leading to an average age of diagnosis in the fourth \ndecade despite most patients experiencing symptoms \nbefore age 25 [ 5– 7]. The identification of biomarkers to \ndetect endometriosis could improve this timeline [ 8]. It \nwould also provide insight into the pathophysiology of \nthis disease, which could contribute to the development \nof personalized and more effective therapies.\nUrine is an ideal medium for non-invasive diagnostic \nand prognostic testing, rich with proteins and peptides \nthat can serve as biomarkers. With a person’s average \nurine output of approximately 1.5 L per day, urine collec-\ntion is simple, economical and non-invasive [ 9]. We set \nout to discover potential urinary biomarkers that could \ndifferentiate between those with and without endome -\ntriosis. We used an unbiased and quantitative proteomics \ndiscovery approach, using urine samples from an existing \ncohort sampled from participants with surgically-con -\nfirmed endometriosis (Supplemental Table 1).\niTRAQ was used in the discovery phase to identify and \nquantitate the urinary proteome of women with endome-\ntriosis and controls. We used an 8-plex iTRAQ protocol \nas previously described [ 9]. Employing a conservative \n2.0-fold cutoff for the differential expression of urinary \nproteins from participants with endometriosis compared \nto samples from sex- and age-matched controls, we iden -\ntified 26 proteins that were downregulated and 30 pro -\nteins that were upregulated in the urine of participants \nwith endometriosis (Fig. 1). Total protein quantitation \nwas determined using the Bradford Assay as previously \nreported [10, 11]. \n \nFig. 1 Four proteins identified via iTRAQ were selected for validation based on physiological relevance and availability of reliable ELISA kits. In participants \n25–47 years old, normalized EGF levels (pg/ug) were found to be significantly lower in the urine of endometriosis participants compared to controls \n(control n = 24; endometriosis n = 22; P = 0.02, Supplemental Table 2). Predictive accuracy of urinary EGF for diagnosis of endometriosis in this group was \n0.701 (Fig. 2A). The other three analyzed proteins (RRBP1, MAFA and HPX) showed no ability to discriminate between participants with endometriosis and \ncontrols (data not shown)\n \n\nPage 3 of 5\nRashes Gertel et al. Biomarker Research           (2026) 14:85 \nEGF ELISA results showed no significant difference in \nthe concentration of EGF in urine between women with \nand without endometriosis when considering the whole \ncohort (Supplemental Table 2). A commonly reported \nsymptom of endometriosis is acyclic pelvic pain [ 12]. \nOf those participants with endometriosis who reported \nthe presence of acyclic pelvic pain, normalized urinary \nEGF levels were significantly lower than in the urine of \ncontrols who did not report the presence of acyclic pel -\nvic pain (Supplemental Table 3). The accuracy of EGF in \npredicting the presence of endometriosis in women with \nacyclic pelvic pain is 0.767 (Fig. 2B).\nBayes’ formula was used with a population estimate of \nendometriosis prevalence of 10%, the prevalence of endo-\nmetriosis in the general population, to determine PPV \nand NPV (Supplemental Table 4) [ 1]. A cutoff of < 800 \npg/ug resulted in a negative predictive value of 94.3%, \nsensitivity of 64% and specificity of 67% in the urine of \nendometriosis participants vs. controls aged 25–47 years. \nIn those same samples from endometriosis participants \nwho also experienced acyclic pelvic pain compared to \ncontrol samples, the same cutoff of < 800 pg/ug gave an \neven higher NPV of 96.8%, sensitivity of 80% and speci -\nficity of 68%. These NPVs suggest urinary EGF has prom-\nise as a clinical tool to rule out endometriosis in those \nwith high levels, especially in the presence of acyclic pel -\nvic pain.\nIn addition to a binary cutoff value, we determined the \nprobability of endometriosis based on quartile ranges of \nurinary EGF levels (Fig. 2C and D). We found that women \n25–47 years of age with low urinary EGF levels had \napproximately double the odds of endometriosis com -\npared to those with higher urinary EGF levels (Fig.  2C). \nFurthermore, when the presence of acyclic pelvic pain is \nconsidered, the risk increases approximately three-fold \nfor those women with low urinary EGF levels (Fig.  2D). \nUrine from participants with much higher urinary EGF \nlevels (> 1200 pg/ug) had a 0–1% predicted probability of \ndisease, further supporting the clinical potential of uri -\nnary EGF testing as a tool to rule out endometriosis.\nFig. 2 ( A) ROC curve for urinary EGF (pg/ug) in participants aged 25-47 with endometriosis compared to age-matched controls ( p-value=0.01). (B) ROC \ncurve for urinary EGF (pg/ug) in participants ages 25-47 with endometriosis and acyclic pelvic pain compared to age-matched controls without acyclic \npelvic pain (P<0.001). (C) Quartiles of urinary EGF levels (pg/ug) with associated predicted probability of endometriosis in women 25-47 years of age and \n(D) in those same women who also have acyclic pelvic pain. Predicted probability was calculated for each quartile using Bayes’ formula assuming a 10% \nprevalence of endometriosis\n \n\nPage 4 of 5\nRashes Gertel et al. Biomarker Research           (2026) 14:85 \nControls were females without a known diagnosis of endometriosis. They \nwere recruited through local clinics, local advertisements, online postings \nand/or word of mouth and were identified to ensure sampling from the \ncommunities served by these two hospitals and thus the underlying \npopulation. All participants could have been receiving standard of care for \nany medical conditions including pelvic pain and are thus representative of \nthe underlying general population. Samples were collected following the \nWorld Endometriosis Research Foundation (WERF) Endometriosis Phenome \nand Biobanking Harmonization Project (EPHect) protocol. Written informed \nconsent was obtained, with parental consent and participant assent for girls \n<18 years of age at enrollment. All procedures involving human participants \nwere performed in accordance with the ethical standards of the U.S. Common \nRule, the principles of the Belmont Report and the ethical tenets outlined in \nthe Declaration of Helsinki.\nConsent for publication\nNot applicable.\nCompeting interest\nThe authors declare no competing interests.\nReceived: 4 March 2026 / Accepted: 23 July 2026\nReferences\n1. Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med. \n2020;382(13):1244–1256. https:/ /doi.or g/10.10 56/N EJMra1810764. PMID: \n32212520.\n2. Becker CM, Bokor A, Heikinheimo O, Horne A, Jansen F, Kiesel L, King K, \nKvaskoff M, Nap A, Petersen K, Saridogan E, Tomassetti C, van Hanegem N, \nVulliemoz N, Vermeulen N. ESHRE Endometriosis Guideline Group. ESHRE \nguideline: endometriosis. Hum Reprod Open. 2022;2022(2):hoac009.  h t t  p s : /  / \nd o  i .  o r g / 1 0 . 1 0 9 3 / h r o p e n / h o a c 0 0 9     . PMID: 35350465; PMCID: PMC8951218.\n3. Avery JC, Knox S, Deslandes A, Leonardi M, Lo G, Wang H, Zhang Y, \nHoldsworth-Carson SJ, Thi Nguyen TT, Condous GS, Carneiro G, Hull ML, \nImagendo Study Group. Noninvasive diagnostic imaging for endometriosis \npart 2: a systematic review of recent developments in magnetic reso-\nnance imaging, nuclear medicine and computed tomography. Fertil Steril. \n2024;121(2):189–211. Epub 2023 Dec 16. PMID: 38110143.\n4. Chen-Dixon K, Uzuner C, Mak J, Condous G. Effectiveness of ultrasound for \nendometriosis diagnosis. Curr Opin Obstet Gynecol. 2022;34(5):324–331. \nhttps:/ /doi.or g/10.10 97/G CO.0000000000000812. PMID: 36036477.\n5. Nnoaham KE, Hummelshoj L, Webster P , d’Hooghe T, de Cicco Nardone F, de \nCicco Nardone C, Jenkinson C, Kennedy SH, Zondervan KT, World Endome-\ntriosis Research Foundation Global Study of Women’s Health consortium. \nImpact of endometriosis on quality of life and work productivity: a multi-\ncenter study across ten countries. Fertil Steril. 2011;96(2):366–e3738. Epub \n2011 Jun 30. PMID: 21718982; PMCID: PMC3679489.\n6. Nnoaham KE, Hummelshoj L, Kennedy SH, Jenkinson C, Zondervan KT, World \nEndometriosis Research Foundation Women’s Health Symptom Survey \nConsortium. Developing symptom-based predictive models of endometrio-\nsis as a clinical screening tool: results from a multicenter study. Fertil Steril. \n2012;98(3):692–e7015. https:/ /doi.or g/10.10 16/j .fertnstert.2012.04.022 . Epub \n2012 May 30. PMID: 22657249; PMCID: PMC3679490.\n7. Koninckx PR, Ussia A, Adamyan L, Tahlak M, Keckstein J, Wattiez A, Martin DC. \nThe epidemiology of endometriosis is poorly known as the pathophysiology \nand diagnosis are unclear. Best Pract Res Clin Obstet Gynaecol. 2021;71:14–\n26. Epub 2020 Sep 1. PMID: 32978068.\n8. Vigano’ P , Vercellini P , Somigliana E, Chapron C, Petraglia F, Griffith LG, et al. \nI’m looking through you: What consumers and manufacturers need to know \nabout non-invasive diagnostic tests for endometriosis. J Endometr Uterine \nDisorders. 2023;2:100031.\n9. Jedinak A, Loughlin KR, Moses MA. Approaches to the discovery of non-inva-\nsive urinary biomarkers of prostate cancer. Oncotarget. 2018;9(65):32534–50. \nhttps:/ /doi.or g/10.18 632/ oncotarget.25946 . PMID: 30197761; PMCID: \nPMC6126692.\n10. Roy R, Zurakowski D, Wischhusen J, Frauenhoffer C, Hooshmand S, Kulke M, \nMoses MA. Urinary TIMP-1 and MMP-2 levels detect the presence of pancre-\natic malignancies. Br J Cancer. 2014;111(9):1772–9.  h t t  p s : /  / d o  i .  o r g / 1 0 . 1 0 3 8 / b j \nc . 2 0 1 4 . 4 6 2     . Epub 2014 Aug 19. PMID: 25137018; PMCID: PMC4453724.\nEGF shows promise as a potential novel, non-invasive \nbiomarker for endometriosis that could enable early and \nfrequent testing for this disease.\nAbbreviations\niTRAQ  Isobaric tags for relative and absolute quantification \nEGF  Epidermal Growth Factor \nELISA  Enzyme-linked immunosorbent assays\nNPV  Negative predictive value \nrASRM  Revised American Society for Reproductive Medicine\nNSAIDs  Nonsteroidal anti-inflammatory drugs\nBCH  Boston Children’s Hospital\nBWH  Brigham and Women’s Hospital\nWERF  World Endometriosis Research Foundation\nEPHect  Endometriosis Phenome and Biobanking Harmonization Project\nRRBP1  Ribosome-binding protein 1\nMAFA  Transcription factor MafA\nHPX  Hemopexin\nIQR  Interquartile range\nROC  Receiver operating characteristic\nAUC  Area under the curve\nPPV  Positive predictive value\nSupplementary Information\nThe online version contains supplementary material available at  h t t  p s : /  / d o  i .  o r \ng / 1 0 . 1 1 8 6 / s 4 0 3 6 4 - 0 2 6 - 0 0 9 7 6 - 2     .  \nSupplementary Material 1\nAuthor contributions\nERRG and CCD contributed to the design of the study, data curation, \nanalysis and were major contributors in writing and revising the manuscript. \nKK contributed to the design of the study, data curation and analysis. \nSJS contributed to analysis and writing and revising the manuscript. DZ \ncontributed to analysis, supervision and writing and revising the manuscript. \nAFV and ALS contributed resources and participated in manuscript review and \nediting. KLT contributed to manuscript review and editing. STD contributed to \nanalysis. SAM contributed resources and participated in writing and revising \nthe manuscript. MSR contributed to funding acquisition and manuscript \nreview and editing. TAL contributed to analysis and manuscript review and \nediting. MAM contributed to the design of the study, data curation, analysis, \nfunding acquisition, supervision and was a major contributor in writing and \nrevising the manuscript.\nFunding\nThe authors gratefully acknowledge the support of the Nile Albright Research \nFoundation (MAM), the Thea and James M. Stoneman Charitable Foundation \n(MAM) and the Marriott Daughters Foundation (MAM, MR, SAM, KLT, ALS, AFV). \nFinancial support for establishment of and data and urine collection within \nthe A2A cohort was provided by the J. Willard and Alice S. Marriott Foundation \n(SAM, KLT, ALS, AFV). None of these funders had a role in study design, data \ncollection and analysis, decision to publish, or preparation of the manuscript.\nData availability\nThe datasets used and/or analysed during the current study are available from \nthe corresponding author on reasonable request.\nDeclarations\nEthics and consent to participate\nThe study was approved by the Boston Children’s Hospital (BCH) Institutional \nReview Board (Assurance Identification #: FWA00002071) on behalf of \nboth BCH and Brigham and Women’s Hospital (BWH). Samples of urine \nwere collected and processed as part of the Women's Health Study: From \nAdolescence to Adulthood (A2A) cohort. This includes patients with a surgical \ndiagnosis from BCH or BWH or patients receiving follow-up treatments at \nBCH or BWH who received a prior documented surgical diagnosis elsewhere. \n\nPage 5 of 5\nRashes Gertel et al. Biomarker Research           (2026) 14:85 \n11. Jedinak A, Curatolo A, Zurakowski D, Dillon S, Bhasin MK, Libermann TA, Roy \nR, Sachdev M, Loughlin KR, Moses MA. Novel non-invasive biomarkers that \ndistinguish between benign prostate hyperplasia and prostate cancer. BMC \nCancer. 2015;15:259. https:   //d oi. or g/10 .11 86/ s12 885- 015-1284-z . PMID: \n25884438; PMCID: PMC4433087.\n12. Tsamantioti ES, Mahdy H. Endometriosis. In: StatPearls. Treasure Island (FL): \nStatPearls Publishing. 2026 Jan–23. PMID: 33620854.\nPublisher’s note\nSpringer Nature remains neutral with regard to jurisdictional claims in \npublished maps and institutional affiliations.","source_license":"public-domain-us","license_restricted":false}