{"paper_id":"62437855-80b3-42f9-8a1a-3051c034741f","body_text":"CA-125 concentration in serum and peritoneal fluid \nin patients with endometriosis – preliminary results\nMaria Szubert, Jacek Suzin, Tomasz Wierzbowski, Katarzyna Kowalczyk-Amico\nAbstract\nIntroduction: Cancer antigen 125 (CA-125), known as a biomarker for women\ngenital tract malignancies, could be also useful in detecting and monitoring\nendometriosis. The aim of this study was to evaluate CA-125 in serum and peri-\ntoneal fluid (PF) as an indicator of endometriosis.\nMaterial and methods: Fifty-six patients admitted to the First Department of Obstet-\nrics and Gynaecology for diagnostic or therapeutic laparoscopy conducted for infer-\ntility, pelvic pain, suspected endometriosis or ovarian cysts entered the study. Those\nwith laparoscopically confirmed endometriosis were assigned to group A, those\nwithout this condition to group B. Blood for CA-125 was taken prior to surgery, cen-\ntrifuged and assayed in accordance with the manufacturer's instructions (VIDAS\nCA-125 II). Peritoneal fluid and an endometrial biopsy were taken during laparoscopy.\nStatistical comparisons were performed using Statistica 7.1.\nResults: Group A consisted of 44 women with laparoscopically confirmed diag-\nnosis; 15 patients served as a control group. The mean value of CA-125 con-\ncentration in the endometriosis group was 33.98 U/ml, vs. 9.3 U/ml in the con-\ntrol group. The mean value of CA-125 in peritoneal fluid was 1241.88 U/ml in the\nnon-endometriosis group versus 2640.23 U/ml in the study group; both results\nwere statistically significant ( p < 0.05). There was a significant correlation\nbetween the stage of endometriosis and CA-125 plasma concentration (R = 0.5993,\np < 0.001). Cancer antigen 125 concentration in serum was a moderate predic-\ntor to distinguish between patients with and without endometriosis (AUC 0.794;\n95% CI 0.668-0.921; p = 0.001).\nConclusions: Cancer antigen 125 is a well-known biomarker for endometriosis\nand helpful in daily clinical practice when endometriosis is suspected. The cut-\noff value in serum suggesting endometriosis with 68% sensitivity is 11 U/ml.\nThis value is normal range for Ca-125 concentration.\nKey words: pelvic pain, infertility, laparoscopy, endometriosis.\nIntroduction \nCancer antigen 125 (CA-125) is a protein (cell surface antigen, member\nof the mucin family glycoproteins, encoded by the MUC 16 gene) known\nsince the early 1980s [1]. It serves as a biomarker of ovarian cancer, espe-\ncially for monitoring ovarian cancer therapy and early recurrences [2, 3].\nOther cancers characterized by CA-125 increase are those originating in\nthe endometrium, fallopian tubes, lungs, breast and gastrointestinal tract\n(i.e. pancreatic cancer). In normal women, plasma concentrations of \nCA-125 are increased slightly at ovulation and significantly during men-\nCorresponding author:\nMaria Szubert MD, PhD\nFirst Department \nof Gynaecology\nand Obstetrics\nClinic of Gynaecological \nSurgery and Oncology\nMedical University of Lodz\n37 Wileńska\n94-029 Lodz, Poland\nPhone: +48 42 680 47 22 \nFax: +48 42 636 64 37\nE-mail: igp@csk.umed.lodz.pl,\nmaja.szubert@interia.pl\nClinical research\nFirst Department of Gynaecology and Obstetrics, Clinic of Gynaecological Surgery \nand Oncology, Medical University of Lodz, Poland\nSubmitted: 24 October 2010\nAccepted: 19 April 2011\nArch Med Sci 2012; 8, 3: 504-508\nDOI: 10.5114/aoms.2012.29407 \nCopyright © 2012 Termedia & Banach\n\nArch Med Sci 3, June / 2012 505\nstruation. A marked increase is observed during\npregnancy [4]. Wilczak et al. described a patholog-\nical mass in the pelvis with highly elevated CA-125\nin serum, which suggested a malignant process of\nthe ovary. Laparotomy in this case revealed a large\ninflammatory tumour connecting the right adnexa\nand appendix vermiformis [5]. Cancer antigen 125\nis commonly elevated in endometriosis, especially\nin its moderate and severe degree. The disease is\nfound in women of reproductive age [6], mostly in\nthose with fertility problems. The prevalence of\nendometriosis is really unknown. It affects 15% to\n70% of women diagnosed with chronic pelvic pain\nsyndrome, endometrial ovarian cysts or infertility\n[7-9]. A recent study conducted by Barbosa et al.\nrevealed that 16.25% of fertile asymptomatic\npatients operated on for other reasons had mini-\nmal or mild endometriosis [10]. The degree of\nendometriosis can only be established by invasive\nprocedures: laparoscopy or open surgery. The \nCA-125 test has a specificity of 89% in detecting\nmoderate and severe endometriosis according to\na meta-analysis of 23 studies [9]. Its usefulness for\nmild endometriosis is limited by unsatisfactory sen-\nsitivity and specificity. Timing of blood collection for\nCA-125 in relation to the menstrual cycle signifi-\ncantly affects this test with higher values during\nand shortly after menstruation [11]. Abrao et al.\nfound that CA-125 was the marker presenting the\nhighest levels during the menstrual phase, between\nthe first and third day of the cycle compared to the\nluteal phase of the cycle [12]. \nNowadays the main goal is to find a group of \nbiomarkers for endometriosis. CA-125 plays an im -\nportant role in nearly all statistical analyses \nconducted on this topic recently. Mihalyi et al .\nrecently presented the results of a study on six \nbiomarkers. He proved that plasma levels of IL-6, \nIL-8 and CA-125 were increased in all women with\nendometriosis compared with the control group.\nMore important is the fact that the elevated con-\ncentration of mentioned cytokines and CA-125 was\nstatistically significant in minimal-mild endo metrio-\nsis compared with the control group [14]. \nOur research on serum and peritoneal fluid con-\ncentration of CA-125 is part of a major experiment\nwhose goal is to find a non-invasive procedure to\ndiagnose endometriosis. Additionally we are eval-\nuating vascular endothelial growth factor (VEGF),\nIL-1β and C-reactive protein (CRP) concentration in\npatients with endometriosis and after treatment of\nthis disease with danazol. The end of this research\nis planned for 2012.\nMaterial and methods\nThe study included 56 patients admitted to the\nFirst Department of Obstetrics and Gynaecology for\ndiagnostic or therapeutic laparoscopy. The patients\nunderwent laparoscopic surgery for infertility, pelvic\npain, suspected endometriosis or ovarian cysts.\nThose with laparoscopically confirmed endome trio-\nsis were assigned to group A, those without this\ncondition to group B (serving as a control group for\nthe statistical analysis). The control group consist-\ned of patients with tubal occlusion, benign ovarian\ncysts (except chocolate cysts) and polycystic ovary.\nWomen without endometriosis in whom no other\ncause of infertility was found during laparoscopy\nwere also assigned to group B. Women with any\nconditions known as influencing CA-125 concen-\ntration and with ovarian malignancy established by\nintraoperative histopathological examination as well\nas women in the luteal phase of the cycle were\nexcluded from the study. The study protocol was\napproved by the Ethics Committee of the Medical\nUniversity of Lodz. All subjects included in the study\nsigned informed consent. Thereafter all women\nwere asked to give blood and to complete the ques-\ntionnaire about regularity and complaints of men-\nstrual cycle. Blood samples were collected in ster-\nile tubes, immediately transported to the laboratory\nand centrifuged (3000/min). Serum CA-125 levels\nwere measured in accordance with the manufac-\nturer's instructions (VIDAS CA-125 II). During the\nprocedure peritoneal fluid and an endometrial biop-\nsy were taken. Peritoneal fluid was aspirated by\nlaparoscopic needle, immediately cooled and cen-\ntrifuged in the same conditions as blood samples,\ndiluted 11 times and assayed for CA-125 concen-\ntration. The dilution process was conducted using\nCA-125 diluent, a reagent included in the VIDAS kits.\nThe biopsy of the endometrium was histopatho-\nlogically examined to establish the phase of the\nmenstrual cycle. The presence and extent of\nendometriosis were carefully assessed, in accor-\ndance with the rASRM 1996 (the Revised American\nSociety for Reproductive Medicine) classification of\nendometriosis. In some cases a peritoneal biopsy\nor ovarian cyst excision was conducted. Specimens\nwere fixed in a 10% formalin solution and sent for\nhistopathological analysis. \nStatistical analysis\nStatistical comparisons were performed using\nStatistica 7.1 and the graphical analysis was con-\nducted using SPSS 12.0. The null hypothesis was\ntested with Shapiro-Wilk test. If the distribution of\ntested parameters was not normal non-parametric\nMann-Whitney U test was used for assessing\nwhether two independent samples of observations\nhad equally large values. For normal distribution\nStudent’s t-test was used to compare groups.\nKruskal-Wallis test and Spearman’s correlation –\nnon-parametric measurement tools of statistical\ndependence between two variables – were used to\nassess the relationship between measured vari-\nables. Regardless of the statistical test, only p val-\nues ≤ 0.05 were considered significant. \nCA-125 concentration in serum and peritoneal fluid in patients with endometriosis – preliminary results\n\n506 Arch Med Sci 3, June / 2012\nResults\nEndometriosis was confirmed laparoscopically\nin a group of 44 women; 15 women served as a con-\ntrol group. The differences in age and the men-\nEndometriosis (EEC) Number  of patients %\nControl group 15 27.78\nEEC I 10 18.52\nEEC II 17 31.48\nEEC III 11 20.37\nEEC IV 1 1.85\nTable I. Prevalence of endometriosis \n1.0\n0.8\n0.6\n0.4\n0.2\n0.0 Sensitivity\nFigure 1. ROC curve (marked thick line) and AUC (area\nunder curve) for CA-125 concentration in plasma\n1-Specificity\n0.0 0.2 0.4 0.6 0.8 1.0\n1.0\n0.8\n0.6\n0.4\n0.2\n0.0 Sensitivity\nFigure 2. ROC curve (marked thick line) and AUC\n(area under curve) for CA-125 concentration in peri-\ntoneal fluid\n1-Specificity\n0.0 0.2 0.4 0.6 0.8 1.0\nstruation length were not statistically significant\nbetween groups. Duration of the cycle was longer\nin the control group and was 37.3 days in compar-\nison to 30.2 days in the endometriosis group and\nthis difference was statistically significant. The \npercentage of smoking patients was higher in the\ncontrol group (46.7% for n = 7 vs. 19.6% n = 8 in\ngroup A) and this difference was also statistically\nsignificant (p = 0.0422). The mean value of CA-125\nconcentrations in the endometriosis group was\n33.98 U/ml, with the maximal value of 173.6 U/ml.\nThe mean level of that protein concentration in the\ncontrol group was 9.3 U/ml. The difference was sta-\ntistically significant ( p < 0.05). The same relation\nwas observed in CA-125 concentration in perito-\nneal fluid. The mean value of 1241.88 U/ml in the \nnon-endo metriosis group was compared with\n2640.23 U/ml in the study group and this difference\nwas also found to be statistically significant. \nThe rASRM (1996) staging was applied for all\npatients with endometriosis. There were 27 patients\nin initial stages (I and II) and 22 patients in advanced\nstages (III and IV). The distribution of each stage is\npresented in Table I. Endometriosis of second stage\nwas the most common diagnosis. The prevalence of\nendometriosis of stage I or II was 50%.\nA receiver operating characteristic (ROC) curve\nis a graphical plot of the sensitivity versus false pos-\nitive rate (two operating characteristics). In this case\nROC analysis is related directly to making the best\nprognosis of endometriosis. The area under the ROC\ncurve (AUC) estimates how good a predictor CA-125\ncould be for endometriosis (Figures I-II).\nCancer antigen 125 concentration in serum was\na moderate predictor to distinguish between\npatients with and without endometriosis (AUC\n0.794; 95% CI 0.668-0.921; p = 0.001). The statisti-\ncal power of CA-125 concentration in peritoneal flu-\nid to differentiate between the two study groups\nwas even worse than that of serum CA-125 (AUC\n0.691; 95% CI 0.53-0.852; p = 0.041). If the cut-off\nvalue for CA-125 in serum is 11 U/ml, the respective\nsensitivity of the test – which measures the pro-\nportion of actual positives which are correctly iden-\ntified as with endometriosis – is 68.29%. Specifici-\nty, which measures the proportion of negatives\nwhich are correctly identified (as without disease),\nis 66.67%. Positive predictive value (PPV) is 84.85%\n(the probability that a patient with positive test\nresults is correctly diagnosed) and negative pre-\ndictive value (NPV) (the probability that a patient\nwith negative test results really does not have the\ndisease) is 43.48%. For CA-125 tested in peritoneal\nfluid the cut-off point of 1295 U/ml was established\nfrom the ROC curve and the statistical analysis\nshowed results as listed in Table II. AUC for CA-125\nserum concentration in the control group and the\ngroup with the first stage of endometriosis I (EEC I)\nMaria Szubert, Jacek Suzin, Tomasz Wierzbowski, Katarzyna Kowalczyk-Amico\n\nArch Med Sci 3, June / 2012 507\ndid not differ statistically significantly. AUC for this\nmarker in the control and EEC II group was 0.798;\n95% CI 0.645-0.951; p = 0.004. With the cut-off val-\nue for CA-125 ≥ 9.7 U/ml the sensitivity of the test\nfor detecting EEC II was 82.35%. The statistical pow-\ner of serum CA-125 concentration was the best for\nadvanced stages of endometriosis (EEC III + EEC IV)\nin comparison to the control group – AUC 0.939;\n95% CI 0.849-1.029; p < 0.001. The sensitivity and\nspecificity for the cut-off point of CA-125 ≥ 14.7 U/ml\nestablished from the ROC curve are listed in Table\nIII. AUC for peritoneal fluid concentration of CA-125\n(for cut-off value of 1262.2 U/ml) in advanced\nstages of endometriosis is 0.654; 95% CI 0.418-\n0.890; p = 0.215.\nWe compared the frequency of occurrence of\nsymptoms typical for endometriosis such as pain\nbefore menstrual bleeding, dysmenorrhoea and\npain after intercourse. There was no significant dif-\nference in occurrence of listed symptoms between\npatients with and without endometriosis. \nThere is a significant correlation between the\nstage of endometriosis and CA-125 plasma con-\ncentration. The value of Spearman's rank correla-\ntion coefficient is R = 0.5993 (p < 0.001). It means\nthat this is a positive correlation; however, a per-\nfect Spearman correlation should be nearly +1 for\nvariables that are a perfect monotone function of\nthe other. There was no significant correlation\nbetween plasma and peritoneal concentration of\nCA-125. \nDiscussion\nOne percent of men and women have no reason\nfor higher concentration of CA-125. This protein can\nbe elevated in non-physiological conditions such as\nperitoneal infection, ascites, ovarian cysts, pancre-\natitis, heart or liver insufficiency and in patients\nafter surgery of the gastrointestinal tract. The \nrole of CA-125 in establishing the diagnosis of\nendometriosis is well known. However, the sensi-\ntivity of this biomarker alone is unsatisfactory. In\nour study the sensitivity of serum concentration of\nCA-125 in the diagnosis of disease was 68% reach-\ning up to 91.67% for the diagnosis of advanced\nstages of endometriosis. Bedaiwy and Falcone\nreviewed the Medline database for studies about\nCA-125 performance in testing endometriosis. His\nmeta-analysis showed that sensitivity of serum CA-\n125 varied in a wide range from 24% to 94%. The\nspecificity reached in our study was only about 67%,\nbut the cut-off point for CA-125 concentration was \n11 U/ml. Most studies included in the meta-analy-\nsis accepted the value of 35 U/ml as a cut-off point\nfor CA-125 serum concentration [15]. In one of the\nlargest studies on CA-125 the authors proved that\nin the diagnosis of endometriosis without\nendometriomas, combined use of two cut-off val-\nues for CA-125, 20 U/ml and 30 U/ml, provides\nimproved diagnostic performance [16]. There have\nbeen a lot of studies on the role of other biomark-\ners in endometriosis (e.g. TNF- α , IL-6, VEGF, CRP)\nconducted recently. The diagnostic accuracy of each\nmarker alone was either similar or worse than that\nof CA-125. The concentrations of various cytokines,\ngrowth and angiogenic factors, metalloproteinas-\nes, peptides, su bpopulations of leukocytes and\nexpression of various genes were examined in\nendo metriosis [17-19]. Nowadays researchers are\ntrying to develop a statistical model based on three\nor four serum biomarkers which could have enough\nstatistical power to diagnose endometriosis with-\nout the necessity of laparoscopy. Cancer antigen\n125 measurement is used in nearly all studies that\nraise this issue. \nCancer antigen 125 concentration is usually\nanalysed from a blood sample. It can also be meas-\nured in fluid from the chest or abdominal cavity.\nAssaying CA-125 in peritoneal fluid requires high\nsample dilutions or a modified immunoradiometric\nassay, and until now, its clinical value has been\nquestionable. All the tests currently in use are based\non the use of an antibody that is directed against\nthe CA-125 protein (monoclonal antibody tech-\nnique). Kraśnicki proved that the sensitivity of the\nperitoneal fluid CA-125 test for endometriosis was\nhigher than the respective serum test. He sug-\ngested that the measurement of CA-125 levels in\nperitoneal fluid could be useful in the detection of\nearly stage endometriosis, which seems to be\nmissed by the CA-125 serum test. In our opinion\nthis dependence is not so clear. However, this study\nis not readily comparable with ours because of the\ndifferent (luteal) menstrual cycle phase in which\nthe study was conducted [20]. There are a lot of\narguments that the CA-125 concentration is high-\ner in the first cycle phase [4]. In our study an\nSensitivity 60.53%\nSpecificity 61.54%\nPositive predictive value 82.14%\nNegative predictive value 34.78%\nTable II. Statistical data for CA-125 = 1295 U/ml in\nperitoneal fluid\nSensitivity 91.67%\nSpecificity 86.67%\nPositive predictive value 84.62%\nNegative predictive value 92.86%\nTable III. Statistical data for CA-125 ≥ 14.7 U/ml in\nserum to diagnose advanced stages of endometrio-\nsis (EEC III + EEC IV)\nCA-125 concentration in serum and peritoneal fluid in patients with endometriosis – preliminary results\n\n508 Arch Med Sci 3, June / 2012\nendometrial biopsy was taken from each patient to\neliminate the differences between CA-125 concen-\ntration in the follicular and luteal phase of the men-\nstrual cycle. Only women in the early follicular\nphase were included in the study.\nThe duration of the cycle in the control group in\nour study was probably influenced by other condi-\ntions such as polycystic ovary syndrome. Like oth-\ner researchers we found no relationship between\nseverity of symptoms which are believed to be typ-\nical for endometriosis and the extent of endo metri-\notic lesions at laparoscopy. There were no differ-\nences between minimal and severe disease. \nThere are publications reporting a positive cor-\nrelation between serum and peritoneal fluid values\nof CA-125 in women with and without endo metrio-\nsis [21] but we did not find such a correlation in our\nstudy. Cancer antigen 125 levels are much higher in\nperitoneal fluid but to compare results between\nstudies we should have information about the man-\nufacturer of the CA-125 test and the way of prepar-\ning the test (e.g. dilution used). \nIn conclusion, serum CA-125 measurement is an\ninexpensive test whose role is to improve diagnostic\naccuracy for endometriosis. In our study CA-125 con-\ncentration in serum was a moderate predictor to\ndistinguish between patients with and without this\ndisease. There is a need for further studies about\nits role in statistical analysis as a marker that\nenhances statistical power for the group of non-\ninvasive biomarkers for endometriosis. \nAcknowledgments\nThe study was part of research supported by\na grant: “The influence of danazol treatment on\nangiogenesis and inflammatory response in patients\nwith endometriosis” no. 2431/B/P01/2009/37 financ -\ned by the Polish Ministry of Science and Higher Edu-\ncation.\nReferences\n1. Yin BW, Dnistrian A, Lloyd KO. Ovarian cancer antigen \nCA-125 is encoded by the MUC16 mucin gene. Int J Can-\ncer 2002; 98: 737-40.\n2. Nowak-Markwitz E, Michalak M, Spaczyński M. Predic-\ntion value of serum Ca 125 level in benefit secondary\ncytoreduction in advanced ovarian cancer patients.\nWspółcz Onkol 2003; 7: 662-7.\n3. Berek JS, Taylor PT, Nicodemus CF . CA-125 velocity at\nrelapse is a highly significant predictor of survival post\nrelapse: results of a 5-year follow-up survey to a ran-\ndomized placebo-controlled study of maintenance ore-\ngovomab immunotherapy in advanced ovarian cancer. \nJ Immunother 2008; 31: 207-14.\n4. Muyldermans M, Cornillie FJ, Koninckx PR. CA-125 and\nendometriosis. Hum Reprod Update 1995; 1: 173-87.\n5. Wilczak M, Rzymski P, Stryjakowska K, Stanek R, Opala\nT. Highly elevated levels of the antigen Ca-125 associat-\ned with inflammatory abdominal masses. Arch Med Sci\n2007; 3: 278-80.\n6. Kennedy S, Bergqvist A, Charon C, et al. ESHRE guideline\nfor the diagnosis and treatment of endometriosis. Hum\nReprod 2005; 20: 2698-704.\n7. Mahmood TA, Templeton A. Prevalence and genesis of\nendometriosis. Hum Reprod 1991; 6: 544-9.\n8. Missmer SA, Hankinson SE, Spiegelman D, Barbieri RL,\nMarshall LM, Hunter DJ. Incidence of laparoscopically con-\nfirmed endometriosis by demographic, anthropometric,\nand lifestyle factors. Am J Epidemiol 2004; 160: 784-96. \n9. Child TJ, Tan SL. Endometriosis: aetiology, pathogenesis\nand treatment. Drugs 2001; 61: 1735-50.\n10. Barbosa CP, de Souza AM, Bianco B, Christofolini DM,\nMafra FA, de Lima GR. OC-125 immunostaining in\nendometriotic lesion samples. Arch Gynecol Obstet 2009;\n281: 43-7.\n11. Spaczynski RZ, Duleba AJ. Diagnosis of endometriosis.\nSemin Reprod Med 2003; 21: 193-208.\n12. Mol BW, Bayram N, Lijmer J G, et al. The performance of\nCA-125 measurement in the detection of endometriosis:\na meta-analysis. Fertil Steril 1998; 70: 1101-8.\n13. Abra~o MS, Podgaec S, Filho BM, Ramos LO, Pinotti JA, de\nOliveira RM. The use of biochemical markers in the diagno-\nsis of pelvic endometriosis. Hum Reprod 1997; 12: 2523-7.\n14. Mihalyi A, Gevaert O, Kyama CM, et al. Non-invasive diag-\nnosis of endometriosis based on a combined analysis of\nsix plasma biomarkers. Hum Reprod 2010; 25: 654-64.\n15. Bedaiwy MA, Falcone T. Laboratory testing for endome-\ntriosis. Clin Chim Acta 2004; 340: 41-56.\n16. Kitawaki J, Ishihara H, Koshiba H, et al. Usefulness and\nlimits of CA-125 in diagnosis of endometriosis without\nassociated ovarian endometriomas. Hum Reprod 2005;\n20: 1999-2003.\n17. Górski J, Szyłło K, Banasik M, Lewkowicz P, Tchórzewski\nH. CD4+, CD8+ and CD4+CD25+ T lymphocytes in periph-\neral blood and peritoneal fluid of women with endometrio-\nsis – preliminary report. Arch Med Sci 2007; 3: 37-42.\n18. Kajihara H, Yamada Y, Kanay ama S, et al. New insights \ninto the pathophysiology of endometriosis: from chronic\ninflammation to danger signal. Gynecol Endocrinol 2011;\n2: 73-9. \n19. Velasco I, Acién P, Campos A, Acién MI, Ruiz-Maciá E. Inter-\nleukin-6 and other soluble factors in peritoneal fluid and\nendometriomas and their relation to pain and aromatase\nexpression. J Reprod Immunol 2010; 84: 199-205.\n20. Kraśnicki D. Serum and peritoneal fluid CA-125 concen-\ntration in women with endometriosis. Ginekol Pol 2001;\n72: 1365-9.\n21. Amaral VF, Ferriani RA, Sá MF, et al. Positive correlation\nbetween serum and peritoneal fluid CA-125 levels in\nwomen with pelvic endometriosis. Sao Paulo Med J 2006;\n124: 223-7.\nMaria Szubert, Jacek Suzin, Tomasz Wierzbowski, Katarzyna Kowalczyk-Amico","source_license":"CC0","license_restricted":false}