{"paper_id":"898a0e6a-dc9c-4717-afcc-96892f77c771","body_text":"Central European Journal of Immunology 2013; 38(2) 154 \nExperimental immunology \nCorrespondence: Ercan Bastu, M.D., Istanbul University School of Medicine, Department of Obstetrics and Gynecology,Division of Infertility, \nCapa 34093, Istanbul, Turkey, phone +90 532 413 4195, e-mail: ercan.bastu@istanbul.edu.tr \nNon-invasive diagnosis of endometriosis based \non a combined analysis of four plasma\nbiomarkers \nERBIL YAGMUR 1, ERCAN BASTU 2, BURCIN KARAMUSTAF AOGLU-BALCI 2,\nSULEYMAN ENGIN AKHAN 2, F ARUK BUYRU 2\n1Bahceci IVF Center, Fulya, Istanbul, Turkey \n2Department of Obstetrics and Gynecology, Istanbul University School of Medicine, Istanbul, Turkey \nAbstract \nPurpose: The objective of the current study was to evaluate whether the analysis of different proin -\nflammatory and angiogenesis-regulating cytokines in a well-defined patient population can be accurate \nfor the diagnosis of endometriosis at different stages. \nMaterial and methods: For this prospective study, plasma samples were collected from women \nundergoing laparoscopic surgery for subfertility. The stage of endometriosis was confirmed during \nlaparoscopy. \nResults: Plasma levels of interleukin 6 (IL-6), carbohydrate antigen (CA)-125 were significantly \nhigher in women with endometriosis compared with controls (46.70 ±27.08 vs. 27.08 ±16.29, p < 0.05; \n49.39 ±28.32 vs. 9.78 ±3.87, respectively). In women with minimal-mild endometriosis, only the plas -\nma level of IL-6 was increased (56.45 ±23.21 vs. 27.08 ±16.29, p < 0.05). In women with moderate- \nsevere endometriosis, plasma levels of IL-6 and CA-125 were significantly increased, and those of Epo \nand tumor necrosis factor α (TNF- α) were significantly decreased (44.53 ±31.07 vs. 27.08 ±16.29,\np < 0.05; 57.84 ±61.44 vs. 9.78 ±3.87, p < 0.05; 26.60 ±10.19 vs. 30.32 ±7.94, p < 0.05; 58.61 ±7.93 \nvs. 65.40 ±9.86, p < 0.05, respectively). The area under curve (AUC) for Epo, TNF- α, IL-6 and CA-125 \nwere 0.280, 0.322, 0.729 and 0.864, respectively. \nConclusions: The results of our study show that progression of endometriosis is associated with the \nelevated level of serum IL-6. Clearly, larger prospective studies are required to determine the diagnos -\ntic potential of measuring circulating inflammatory cytokine levels like IL-6 in endometriosis. \nKey words: endometriosis, IL-6, CA-125, Epo, TNF- α.\n(Centr Eur J Immunol 2013; 38 (2): 154-158) \nIntroduction \nThis perplexing disease affects 6-10% of women in the \nreproductive age (ages of 12-80 years) [1]. Laparoscopy is \naccepted as the “gold” standard for the diagnosis of \nendometriosis. The extent of endometriosis is generally \nstaged by the American Society for Reproductive Medicine \n(ASRM) scoring system, which categorizes minimal and \nmild endometriosis as stage I and stage II, and moderate \nand severe endometriosis as stage III and stage IV [2]. \nA non-invasive diagnostic test for endometriosis might \nabolish the need for surgery. However, such a noninvasive \ndiagnostic test is currently unavailable [3]. In the current \npractice, unexpected preoperative findings potentially can \nlead to under-treatment or unnecessary surgery. Accurate \nnon-invasive diagnostic techniques are clearly necessary to \nmanage women with endometriosis more effectively. In \naddition, a survey [4], which was taken by 7025 women \nwith endometriosis, revealed that 65% of the women with \nendometriosis were initially diagnosed with another con -\ndition, which led to an average delay of 8 years between \nthe start of symptoms and the actual diagnosis of \nendometriosis [5, 6]. \nDOI: 10.5114/ceji.2013.35204 \n\nCentral European Journal of Immunology 2013; 38(2) 155 \nThe immune deviations in endometriosis, including \nincreased local production of some cytokines as well as ele -\nvated autoantibody production of local and systemic immu -\nnity,suggest that endometriosismay be an autoimmunedis -\norder [7-11]. \nThe objectiv e of the current study was to evaluate \nwhether the analysis of different proinfl ammatory and \nangiogenesis-regulating cytokines in a well-defined patient \npopulation can be accurate for the diagnosis of endometrio -\nsis at different stages. \nMaterial and methods \nFor this prospective study, plasma samples were col -\nlected from women undergoing the laparoscopic surgery \ndue to infertility at the gynecology clinic of the Department \nof Obstetrics and Gynecology, Istanbul University School \nof Medicin e (Istanbul, Turkey). The study had received \napproval from the Ethics Committee of the Istanbul Uni -\nversity School of Medicine and written consent from the \nparticipants before its initiation. Blood (4 ml) was drawn \non the day of the surgery, centrifuged at 1000 rpm for 15 \nmin, labeled and stored at –80°C till analysis. The stage of \nendometriosis was confirmed during laparoscopy accord -\ning to the revised American Fertility Society classification \n[12]. For each patient, relevant clinical information (age, \nstage of endometriosis, current medication and, number and \ntype of previous operations) was recorded. \nThe first comparison was of controls (22 patients in \nwhich endometriosis was excluded laparoscopically by an \nexperienc ed gynecologic surgeon) versus all stages of \nendometriosis (33 patients). Endometriosis patients were \nthen divided into three groups according to the stage of \nendometriosis: controls (33 patients; no endometriosis), \nminimal-mild endometriosis (16 patients; stages I-II) and \nmoderate-severe endometriosis (17 patients; stages III-IV). \nExclusioncriteria were as follows: women (1) who were \non hormonal medication; (2) who underwent an operation \nwithin 6 months; (3) who had other pelvic inflammatory \ndisease. \nSelection and measurement of biomarkers \nAfter a widespread literature search, four plasma bio -\nmarkers were selected due to reported significant differ -\nences in the plasma concentrations between women with \nand without endometriosis.Erythropoietin(Epo), interleukin \n(IL)-6, tumor necrosis factor α (TNF- α) and carbohydrate \nantigen (CA)-125, are suggested to play a role in the devel -\nopment of endometriosis due to either their angiogenic \npotential or as autocrine/paracrine factors or by encourag -\ning vascularization or survival and proliferation of ectopic \nendometrial cells [13-17]. \nPlasma concentrations of Epo, IL-6 and TNF- α were \ndetermined by using commercially available ELISA kits \n(R&D Systems Inc., Minneapolis, MN, USA) according to \nthe manufacturer’s instructions. Plasma concentrations of \nthe CA-125 level were measured using Microparticle \nEnzyme Immunoassay (MEIA) Abbott AxSYM instrument \n(Abbott Diagnostics, Abbott Park, IL, USA). Inter-assay \ncoefficient of variation for Epo, IL-6, TNF- α and CA-125 \nwas < 10, 6.4, 3.5 and < 10%, respectively.Intra-assay coef -\nficient for Epo, IL-6, TNF- α and CA-125 was 5.9, 4.2, 1.8 \nand < 10%, respectively. \nStatistical analysis \nData were expressed as mean ±SD. Statistical analysis \nwas performed using the Student’s t-test as appropriate, \nwhereas means were compared among groups by a one-way \nanalysis of variance (ANOVA). When homogeneity and \nnormality of the samples were not appropriate, non-para -\nmetric statistical methods (Kruskal-Wallisand Mann-Whit -\nney tests) were applied for comparison. If a significant over -\nall difference was found in a one-way ANOVA or \na Kruskal-Wallis test, post hoc Tukey or multiple compar -\nison test were performed to identify any significant differ -\nences among individual groups. Receiver operating char -\nacteristi c (ROC) curves were generated and confidence \nintervals for areas under ROC curves were calculated. Sta -\ntistical significancewas calculated using the Statistical Pack -\nage for Social Sciences (SPSS Inc., Chicago, IL, USA) and \nwas considered to be P < 0.05. \nResults \nControls and endometriosis patients were matched for \nage (controls: 26.86 ±9.13, endometriosis patients: 31.24 \n±7.24, p > 0.05). The plasma levels of IL-6, CA-125 were \nsignificantly higher, whereas the plasma levels of Epo and \nTNF- α were non-significantly decreased, in women with \nendometriosis compared with controls (IL-6: 46.70 ±27.08 \nvs. 27.08 ±16.29, p < 0.05; CA-125: 49.39 ±28.32 vs. 9.78 \n±3.87, p < 0.0001; Epo: 26.26 ±9.31 vs. 30.32 ±7.94,\np > 0.05; TNF- α: 60.22 ±9.11 vs. 65.40 ±9.86, p > 0.05, \nrespectively) (Table 1). \nTable1. Comparisonof serum erythropoietin(Epo), interleukin \n(IL)-6, tumor necrosis factor α (TNF- α) and carbohydrate anti -\ngen (CA)-125values in women with and without endometriosis \nEndometriosis Controls p\npatients (n = 22) \n(n = 33) \nIL-6, mIU/ml 46.70 ±27.08 27.08 ±16.29 0.016 \nCA-125, U/ml 49.39 ±28.32 9.78 ±3.87 0.0001 \nTNF- α, pg/ml 26.26 ±9.31 65.40 ±9.86 0.051 \nEpo, mIU/ml 60.22 ±9.11 30.32 ±7.94 0.099 \nNote: Student’s t-test was used to evaluate statistically significant differences \nbetween the values. \nNon-invasive diagnosis of endometriosis based on a combined analysis of four plasma biomarkers \n\nCentral European Journal of Immunology 2013; 38(2) 156 \nIn women with minimal-mild endometriosis, the only \nsignificant difference was the increased plasma level of\nIL-6 in comparison to the controls (IL-6: 56.45 ±23.21 vs. \n27.08 ±16.29, p < 0.05). In women with moderate-severe \nendometriosis, plasma levels of IL-6 and CA-125 were sig -\nnificantly increased, and those of Epo and TNF- α were sig -\nnificantly decreased, when compared with controls (IL-6: \n44.53 ±31.07 vs. 27.08 ±16.29, p < 0.05; CA-125: 57.84 \n±61.44 vs. 9.78 ±3.87, p < 0.05; Epo: 26.60 ±10.19 vs. \n30.32 ±7.94, p < 0.05; TNF- α: 58.61 ±7.93 vs. 65.40 ±9.86, \np < 0.05, respectively) (Table 2). \nReceiver operating characteris tic curve analysis was \nused to examine the diagnostic test performance of Epo, \nTNF- α, IL-6 and CA-125. The area under curve (AUC) for \nEpo, TNF- α, IL-6 and CA-125 were 0.280, 0.322, 0.729 \nand 0.864, respectively (Fig. 1). \nDiscussion \nThe results of this study show that minimal-mild \nendometriosis patients displayed higher serum IL-6 levels, \nwhile moderate -severe endometriosis patients displayed \nhigher serum Epo, TNF- α, IL-6 and CA-125 levels than \nother patients. \nIncreased serum IL-6 levels observed in patients with \nminimal-mild endometriosis in our study are in line with \nfindings of some reports [18-20] but depart from others\nthat found no value for serum IL-6 in the diagnos is\nof endometriosis at any stage [21, 22] and from yet others \nthat reported slightly elevated serum IL-6 levels in controls \n[23-25]. Additionally, it is worthwhile to note that serum \nIL-6 was the only marker that was constantly elevated in \nall our comparison groups (endometriosis group, minimal- \nmild endometriosis group and moderate-severeendometrio -\nsis group). \nInflammatory markers such as TNF- α and Epo were \nslightly higher in the controls than in the endometrios is \ngroup. This observation may be explained by the possibil -\nity that controls with adhesions, etc. had increased plasma \nconcentra tions of inflammatory cytokines. Comparable \nTNF- α [20, 23-25] levels were previously reported in \nwomen with and without endometriosis. However, other \ninvestigators reported elevated levels of TNF- α and Epo in \nendometriosis patients compared with controls [13, 18, 26]. \nThese discrepancies may be explained by differences in the \nstudy design (i.e. different inclusion criteria and different \nTable2. Comparison of serum erythropoietin (Epo), interleukin (IL)-6, tumor necrosis factor α (TNF- α) and carbohydrate anti -\ngen (CA)-125 values in women with and without endometriosis in relation to the stage of the disease \nStage I-II endometriosis Stage III-IV endometriosis Controls (n = 22) p\npatients ( n = 16) patients ( n = 17) \nMean ±SD Median Min-Max Mean ±SD Median Min-Max Mean ±SD Median Min-Max \nIL-6, mIU/ml 56.45 ±23.21 29.2 18.9-205.2 44.53 ±31.07 b 33.9 18.7-150.0 27.08 ±16.29 20.5 18.7-92.0 0.014 \nCA-125, U/ml 11.39 ±4.81 19.0 6.12-19.9 57.84 ±61.44 a,b 39.8 8.7-309.3 9.78 ±3.87 15.9 3.56-18.47 0.000 \nTNF- α, pg/ml 67.48 ±11.29 a 35.4 54.9-79.0 58.61 ±7.93 b 21.6 48.0-75.3 65.40 ±9.86 33.9 49.8-80.2 0.013 \nEpo, mIU/ml 24.75 ±3.39 25.8 19.1-28.0 26.60 ±10.19 b 22.6 18.0-60.0 30.32 ±7.94 35.3 21.1-46.5 0.021 \nap < 0.05: stage I-II vs. stage III-IV \nbp < 0.05: stage III-IV vs. controls \nNote: Kruskal-Wallis test was used to calculate the overall p-values; Mann-Whitney test was used to perform the pairwise comparisons. \n1.0 \n0.8 \n0.6 \n0.4 \n0.2 \n0.0 \n0.0 0.2 0.4 0.6 0.8 1.0 \nspecificity \nsensitivity \nEpo \nIL-6 \nTNF- α\nCA-125 \nreference line \nFig. 1. Receiver operating characteristic (ROC) curve for the \nidentification of endometriosis for serum erythropoietin (Epo), \ninterleukin (IL)-6, tumor necrosis factor α (TNF- α) and car -\nbohydrate antigen (CA)-125 \nErbil Yagmur et al. \n\nCentral European Journal of Immunology 2013; 38(2) 157 \nmenstrual cycle phases) as well as genetic diversity. It is \nimportant to note that although CA-125 is the most exten -\nsively investigated and used biomarker of endometriosis \n[27], it is well established that CA-125 levels lack diag -\nnostic power as a single biomarker of endometriosis [28]. \nRegarding the role of cytokines in the pathogenesis of \nendometriosis, our findings are consistent with previous \nreports [8-10, 29, 30], which state that endometriosis is asso -\nciated with the elevatedlevel of serum IL-6 and the elevated \nlevel of this cytokine correlates with a more advanced stage \nof the disease. However, we were unable to reveal any sta -\ntistically significant differences in the case of the other test -\ned cytokines (Epo and TNF- α), which is also consistent \nwith some studies [29, 30]. \nConclusions \nEstablishing a non-invasive diagnostic test for \nendometriosis can have a radical impact on the patients’ \nquality of life and the accuracy of the treatment by poten -\ntially reducing the number of unnecessary laparoscopies. \nThe results of our study show that progress ion of \nendometriosis is associated with the elevated level of serum \nIL-6. Undoubtedly, larger well-designed prospective stud -\nies are urgently needed to determine the diagnostic poten -\ntial of cytokines like IL-6 in endometriosis. \nWe certify that there is no conflict of interest with any \nfinancial organization regarding the material discussed in \nthis manuscript. \nReferences \n1. Giudice LC, Kao LC (2004): Endometr iosis. Lancet 364: \n1789-1799. \n2. 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Fertil Steril 92: \n1844-1849. \nErbil Yagmur et al.","source_license":"CC0","license_restricted":false}