Identification of Serum Biomarkers for Diagnosis of Endometriosis Using Multiplex Immunoassays

Endometriosis is a common gynecologic disorder characterized by chronic pelvic pain, dysmenorrhea, and infertility. Although this condition places significant financial burden on the healthcare system and negatively affects patient’s quality of life, the pathophysiology of the disease remains unclear, and noninvasive diagnostic methods are insufficient. The object of this study was to identify potential biomarkers for endometriosis from peripheral blood. We hypothesized that serum biomarkers modified in endometriosis patients would be detected by multiplex cytokine panel, and identification of a combination of these biomarkers would improve diagnostic power. A total of 141 women, aged 15–52 years with regular menstruation, participated in this study. Twenty-one serum cytokines were detected using the commercially available MILLIPLEX MAP Human Cytokine/Chemokine Kit Panel IV. Among these cytokines, breast- and kidney-expressed chemokine (BRAK)/chemokine (C-X-C motif) ligand 14 (CXCL14) was significantly decreased, and proliferation-inducing ligand (APRIL)/tumor necrosis factor ligand superfamily member 13 (TNFSF13) was significantly increased in endometriosis group. APRIL/TNFSF13 and BRAK/CXCL14 alone or in combination, however, failed to show adequate sensitivity or specificity for the diagnosis of endometriosis. Combination of APRIL/TNFSF13 and BRAK/CXCL14 with serum CA-125 levels yielded significantly higher sensitivity (71.2%) for detecting endometriosis without compromising specificity (80.8%) than CA-125 alone in a logistic regression model (P = 0.050). In conclusion, we identified a biomarker combination that detects endometriosis better than CA125 alone. Therefore, we conclude that multiplex cytokine panel is an efficient method for detecting endometriosis, and analysis of additional cytokine panels may lead to identification of a novel biomarker combination with superior diagnostic power. Similar content being viewed by others

Coutinho LM, Ferreira MC, Rocha ALL, Carneiro MM, Reis FM. New biomarkers in endometriosis. Adv Clin Chem. 2019;89:59–77. Ferrero S. Proteomics in the diagnosis of endometriosis: opportunities and challenges. Proteomics Clin Appl. 2018:e1800183. O DF, Flores I, Waelkens E, D’Hooghe T. noninvasive diagnosis of endometriosis: review of current peripheral blood and endometrial biomarkers. Best Pract Res Clin Obstet Gynaecol. 2018;50:72–83. Barcz E, Kaminski P, Marianowski L. Role of cytokines in pathogenesis of endometriosis. Med Sci Monit. 2000;6(5):1042–6. May KE, Conduit-Hulbert SA, Villar J, Kirtley S, Kennedy SH, Becker CM. Peripheral biomarkers of endometriosis: a systematic review. Hum Reprod Update. 2010;16(6):651–74. Huang RP, Huang R, Fan Y, Lin Y. Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system. Anal Biochem. 2001;294(1):55–62. De Andrade VT, Nacul AP, Dos Santos BR, Lecke SB, Spritzer PM, Morsch DM. Circulating and peritoneal fluid interleukin-6 levels and gene expression in pelvic endometriosis. Exp Ther Med. 2017;14(3):2317–22. Bedaiwy MA, Falcone T, Sharma RK, Goldberg JM, Attaran M, Nelson DR, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod. 2002;17(2):426–31. Malutan AM, Drugan T, Costin N, Ciortea R, Bucuri C, Rada MP, et al. Pro-inflammatory cytokines for evaluation of inflammatory status in endometriosis. Cent Eur J Immunol. 2015;40(1):96–102. Zhou WJ, Yang HL, Shao J, Mei J, Chang KK, Zhu R, et al. Anti-inflammatory cytokines in endometriosis. Cell Mol Life Sci. 2019;76(11):2111–32. Vodolazkaia A, El-Aalamat Y, Popovic D, et al. Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis. Hum Reprod. 2012;27(9):2698–711. Kocbek V, Vouk K, Bersinger NA, Mueller MD, Lanisnik RT. Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis. J Mol Diagn. 2015;17(3):325–34. O DF, El Aalamat Y, Waelkens E, De Moor B, D’Hooghe T, Fassbender A. multiplex immunoassays in endometriosis: an array of possibilities. Front Biosci (Landmark Ed). 2017;22:479–92. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology. 1982;143(1):29–36. Schisterman EF, Perkins NJ, Liu A, Bondell H. Optimal cut-point and its corresponding Youden index to discriminate individuals using pooled blood samples. Epidemiology. 2005;16(1):73–81. Kurth I, Willimann K, Schaerli P, Hunziker T, Clark-Lewis I, Moser B. Monocyte selectivity and tissue localization suggests a role for breast and kidney-expressed chemokine (BRAK) in macrophage development. J Exp Med. 2001;194(6):855–61. Frederick MJ, Henderson Y, Xu X, Deavers MT, Sahin AA, Wu H, et al. In vivo expression of the novel CXC chemokine BRAK in normal and cancerous human tissue. Am J Pathol. 2000;156(6):1937–50. Lendak DF, Mihajlovic DM, Novakov-Mikic AS, Mitic IM, Boban JM, Brkic SV. The role of TNF-alpha superfamily members in immunopathogenesis of sepsis. Cytokine. 2018;111:125–30. Vincent FB, Saulep-Easton D, Figgett WA, Fairfax KA, Mackay F. The BAFF/APRIL system: emerging functions beyond B cell biology and autoimmunity. Cytokine Growth Factor Rev. 2013;24(3):203–15. Chung HW, Jang S, Lim JB. Clinical implications and diagnostic usefulness of correlation between soluble major histocompatibility complex class I chain-related molecule a and protumorigenic cytokines in pancreatic ductal adenocarcinoma. Cancer. 2013;119(1):233–44. Fu Q, Zhu J, Van Eyk JE. Comparison of multiplex immunoassay platforms. Clin Chem. 2010;56(2):314–8. Krishhan VV, Khan IH, Luciw PA. Multiplexed microbead immunoassays by flow cytometry for molecular profiling: basic concepts and proteomics applications. Crit Rev Biotechnol. 2009;29(1):29–43. Hermann N, Dressen K, Schildberg FA, Jakobs C, Holdenrieder S. Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay. World J Methodol. 2014;4(4):219–31. Dressen K, Hermann N, Manekeller S, Walgenbach-Bruenagel G, Schildberg FA, Hettwer K, et al. Diagnostic performance of a novel multiplex immunoassay in colorectal cancer. Anticancer Res. 2017;37(5):2477–86. Hermann N, Dressen K, Schroeder L, Debald M, Schildberg FA, Walgenbach-Bruenagel G, et al. Diagnostic relevance of a novel multiplex immunoassay panel in breast cancer. Tumour Biol. 2017;39(6):1010428317711381. Cho S, Choi YS, Yim SY, Yang HI, Jeon YE, Lee KE, et al. Urinary vitamin D-binding protein is elevated in patients with endometriosis. Hum Reprod. 2012;27(2):515–22. Funding This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI18C2047). Author information Authors and Affiliations Contributions All authors (H.K., Y.S.C., J.S.K., S.K., Y.B.W., I.L., J.H.L., B.H.Y., S.K.S., J.H.P., S.C., J.H.S., J.B.L., and B.S.L.) were responsible for acquisition of data, analysis and interpretation of data, and final approval of the version to be published. Additionally, H.K., Y.S.C., S.C., and J.B.L. were responsible for substantial contributions to conception and design and drafting the manuscript. Especially, S.C. and J.B.L. were responsible for revising the manuscript critically for important intellectual content. Corresponding authors Ethics declarations Conflict of Interest The authors declare that they have no conflict of interest. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions About this article Cite this article Kim, H., Choi, Y.S., Kim, J.S. et al. Identification of Serum Biomarkers for Diagnosis of Endometriosis Using Multiplex Immunoassays. Reprod. Sci. 27, 1139–1147 (2020). https://doi.org/10.1007/s43032-019-00124-2 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-019-00124-2