Role of Some Biomarkers in Chronic Pelvic Pain for Early Detection of Endometriosis in Infertile Women

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1 Med. J. Cairo Univ., Vol. 79, No. 2, September: 3-21, 11 Role of Some Biomarkers in Chronic Pelvic Pain for Early Detection of Endometriosis in Infertile Women ASHRAF A. FODA, M.D.* and IBRAHIM A. ABDEL AAl, M.D.** The Departments of Obstetrics & Gynecology* and Clinical Pathology**, Faculty of Medicine, Mansoura University, Egypt Abstract Objective: Diagnosis of endometriosis in infertile women with chronic pelvic pain depends mainly on laparoscopy, but it has many risks. The aim of this study was to evaluate the value of some biomarkers as a reliable non invasive method for the diagnosis of early stages of endometriosis in infertile women with chronic pelvic pain. Material and Methods: A Total of 95 women who underwent laparoscopy were divided into two groups: Control cases (3 cases) with no pathologic findings; and endometriosis patients (65 cases) [subdivided into stages 1-2 or minimalmild (MM) and stages 3-4 or moderate-severe (MS) cases]. Blood was drawn on cycle s days 6-1 and stored at 7 C for subsequent analysis of IL-6, CA-125, TNF- α, HsCRP and VEGF levels. Results: High levels of serum IL-6 and serum TNF- α were observed in MM endometriosis as compared to controls (p) and to MS endometriosis (p<.5). Levels of serum CA-125, Hs-CRP & VEGF were significantly elevated in MS endometriosis when compared with MM endometriosis and controls (p, p & p<.8 respectively). Conclusion: Serum IL-6 and TNF-α levels are reliable non-invasive biomarkers for diagnosis of early stages of endometriosis because they significantly increased in early cases than in late cases. CA125, Hs-CRP & VEGF are significantly increased in late cases, so they can not be used for early diagnosis. Key Words: Chronic pelvic pain Endometriosis Biomarkers IL-6 TNF- α Us-CRP CA-125 VEGF. Introduction CHRONIC pelvic pain is a common gynaecologic problem and it is defined as non-cyclic pain that persists for 6 or more months and leads to degrees of functional disability [1]. Endometriosis is a common cause of pelvic pain. Pain from endometriosis correlates with the depth of invasion and results from neuronal inva- Correspondence to: Dr. Ashraf Ahmed Foda, Lecturer of OB/ GYN, Faculty of Medicine, Mansoura University, Egypt. drashraf.foda@yahoo.com sion of endometriotic implants with sensory and sympathetic nerve supply [2,3]. Endometriosis is associated with infertility [4] and it has been reported that infertile women are 6-8 times more likely than fertile women to have the disease [5]. Endometriosis is generally associated with an immune-inflammatory process that takes place in the peritoneal cavity of patients [6]. The cytokines, interleukins and tumor necrosis factor- α (TNF-α ) have been implicated in the pathogenesis of endometriosis [7] as evidenced by elevated levels of several cytokines in the peritoneal fluid (PF) of women with endometriosis [8]. The serum measurement of IL-6 has been used in order to discriminate between women with and without the disease [8], and to identify stages of the disease [9]. Elevated serum CA-125 level is usually employed as a marker of recurrence following surgical intervention and in advanced endometriosis [1]. Tumor necrosis factor- α (TNF-α) may play a role in the progression of endometriosis and its associated infertility. It is produced in the PF of endometriosis patients by peripheral blood monocytes [11] and peritoneal macrophages [12]. C-reactive protein (CRP) has the ability to modulate the behavior of phagocytic cells in proinflammatory and anti-inflammatory ways. The association of CRP with IL-6 and TNF- α in endometriosis, indicates its potential use in the diagnosis of the disease [13]. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic factor. In endometriosis patients, VEGF is localized in the epithelium of endometriotic implants, particularly in hemorrhagic red implants [14,15]. 3

2 4 Role of Some Biomarkers in Chronic Pelvic Pain for Early Detection In recent years, many efforts have been made in trying to identify an easy way to diagnose endometriosis. Laparoscopic surgery is used for diagnosis and assessment but it has risks. Indeed, ultrasonography is solely effective in detecting ovarian endometriomas. Thus, a non-surgical diagnostic approach would be of great benefit to both physicians and women alike. Aim of the work: The main purpose of this study was to determine the clinical usefulness of IL-6, TNF-α, CA-125, high sensitive CRP (Hs- CRP) and VEGF levels in infertile women with pelvic pain as markers of the early stages of peritoneal endometriosis during which imaging is not effective. Patients and Methods This study was conducted at the Obstetrics & Gynecology department, Mansoura University Hospital. The study was approved by the ethics committee of Faculty of Medicine, Mansoura University. Informed written consent was taken from each patient prior to any procedure. This is a prospective study of 95 infertile women complaining of chronic pelvic pain undergoing laparoscopy between January 9 and May 1. The patients were divided into the following groups: Group-1 (endometriosis group): Consisted of 65 patients diagnosed to have endometriosis during laparoscopy. The severity of the disease was graded according to the revised four-stage scoring system of American Society of Reproductive Medicine (rasrm) [13]. Cases of this group were subdivided into two subgroups: Group-1A: Consisted of 37 cases with stages 1-2 or minimal-mild (MM) endometriosis. Group-1B: Consisted of 28 cases with stages 3-4 or moderate severe (MS) endometriosis. Group-2 (non-endometriosis group): Consisted of 3 cases with no detected pelvic pathology. Inclusion criteria were as follows: Reproductive age (between 18 and 35 years), regular menstrual cycles, and they don t smoke or drink alcohol. Exclusion criteria were: Patients above 35 years, any current infection (genital or systemic), and any medication within one month prior to laparoscopy, patients with minimal amount or bloody peritoneal fluid, or patients using intrauterine device. Study design: These patients were subjected to the following: Full history, general and abdominal examination, local pelvic examination and laparoscopic visualization of presence or absence of endometriosis. Sampling: Venous blood samples were obtained from the fasting patients in the follicular phase (days 5-1). Blood was withdrawn in the morning the day before laparoscopy to avoid the effect of stress on the biomarkers levels. Five ml of venous blood was withdrawn aseptically into dry plastic tubes. Then the collected blood samples were centrifuged at 3g for 15min, and the separated sera were stored at 7 C until taken for analysis [8]. Peritoneal fluid samples were aspirated from the peritoneal cavity during laparoscopy and from the Douglas pouch. The cellular constituents of the peritoneal fluid were removed by centrifugation at 3g for min, after which the supernatants was removed and stored in aliquots at 7 C until cytokines and other parameters will be assessed [8]. Methods: Blood and peritoneal fluid samples were taken for estimation of Interleukin-6, and TNF-α level by using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA, DRG, Germany). CA-125 and Hs-CRP were measured by automated electro-chemilumiescent immunoassay instrument (Elecsys 1, Roche, Germany). VEGF was determined by a competitive enzyme immunoassay technique using Accucyte human VEGF kit. Statistical analysis: The collected data will be tabulated, compared and proper statistical analysis will be performed. Mean and standard deviation were used to describe data. Student t-test was used to test for significance of difference in quantitative variables between groups. Pearson product moment correlation was used to test for linear relationship between quantitative variables. Kendall s nonparametric correlation was used to test for linear relationship between quantitative variables and disease stage (ordinal variable). Receiver Operating Characteristic (ROC) curve was used to evaluate the performance of the various values of biomarkers as diagnostic test for endometriosis. The area under the curve (AUC) is considered significantly different from the null hypothesis if >.5. The p-value was considered significant if less than.5. These tests were run on an IBM compatible personal computer using the Statistical Package for Social Scientists (SPSS) for windows (SPSS Inc., Chicago, IL, USA).

3 Ashraf A. Foda & Ibrahim A. Abdel Aal 5 Results Tables (1-5) show the serum levels and peritoneal fluid levels of IL-6. CA-125, TNF- α, Hs-CRP & VEGF respectively. Serum IL-6 and serum TNF- α levels were significantly higher in women with endometriosis when compared with the controls (p). Serum IL-6 and TNF- α were significantly elevated in women with MM endometriosis when compared with the control cases (p) and with MS endometriosis (p<.6 & p<.3 respectively). The serum levels of CA-125, Hs-CRP and VEGF were significantly elevated in women with endometriosis when compared with the control cases (p). The levels were significantly elevated in women with MS endometriosis when compared with MM (p<.1) & total endometriosis (p). PF levels of IL-6 & TNF- α were significantly elevated in MM than in MS endometriosis (p & p<.25 respectively). On the other hand, PF levels of CA-125 & VEGF were significantly elevated (p) in MS endometriosis than in MM endometriosis. Table (6) shows a significant positive correlation between the levels of serum and PF markers (p). Table (7) shows a significant negative correlation between the stage of the disease and serum levels of IL-6 (p) & TNF-α (p). The serum levels of CA-125, HsCRP & VEGF showed a significant positive correlation ( p<.5) with the stage of the disease. Table (8) shows a significant negative correlation between the stage of the disease and the PF levels of IL-6 (p), and a significant positive correlation (p) between the stage of the disease and the PF levels of CA-125 & VEGF. Table (9) shows the Receiver Operating Characteristic (ROC) curve analysis data. The ROC curve analysis of serum IL-6 levels revealed a high diagnostic value for serum IL-6 in respect to diagnosis endometriosis. The optimal serum IL-6 threshold found using the ROC curve was 12.2pg/ml, with a sensitivity of 95.58%, specificity of 83.33% and the diagnostic accuracy of 91.1%. ROC curve analysis revealed that the sensitivity, specificity, and diagnostic accuracy of serum CA- 125 in the diagnosis of endometriosis using 17.6 IU/ml as the threshold value were %, % & 73.33% respectively. The ROC curve analysis revealed that sensitivity, specificity and diagnostic accuracy for serum TNF-α in the diagnosis of endometriosis using a level > 12.45pg/ml as the cut-off value were 89.23%, 86.87% & 86.67% respectively. ROC curve analysis revealed that sensitivity, specificity and the diagnostic accuracy for serum Hs-CRP in the diagnosis of endometriosis using a level >438µg/ml as the cut-off value were 83.33%, 86.67% & 84.44% respectively. ROC curve analysis revealed that sensitivity and specificity for serum VEGF in the diagnosis of endometriosis using a level >236pg/ml as the cut-off value were 91.67% and 76.67%, respectively. The diagnostic accuracy was 86.68%. Table (1) represents the sensitivity & specificity on combining 2 or 3 biomarkers. It was found that improvement in sensitivity is associated with a marked reduction in specificity. So, the results of individual marker are of more value than combined results. Table (1): Serum and peritoneal fluid (PF) levels of IL-6 (pg/ml). Mean ± SD Range Mean ± SD Range p2: Control versus gr.1a p3: Control versus gr.1b 24.1 ± ± ± ± ± ± ± ± p<.5 is significant.

4 6 Role of Some Biomarkers in Chronic Pelvic Pain for Early Detection Table (2): Serum and peritoneal fluid (PF) levels of CA-125 (IU/ml). p2: Control versus gr. 1A p3: Control versus gr. 1B Mean ± SD Range Mean ± SD Range ± ± ± ± ± ± ± <.6 p<.5 is significant Table (3): Serum and peritoneal fluid (PF) levels of TNF- α (pg/ml). p2: Control versus gr. 1A p3: Control versus gr. 1B p<.5 is significant Mean ± SD Range Mean ± SD Range ± ± ± ± ± ± ± <.6 Table (4): Serum and peritoneal fluid (PF) levels of Hs-CRP (ng/ml). Mean ± SD Range Mean ± SD Range p2: Control versus gr. 1A p3: Control versus gr. 1B 71.28± ± ± ± ± ± ± ± >.615 (not sig.) p<.5 is significant Table (5): Serum and peritoneal fluid (PF) levels of VEGF (pg/ml). p2: Control versus gr. 1A p3: Control versus gr. 1B p<.5 is significant Mean ± SD 419.4± ± ± ±9.2 <.8 Range Mean ± SD Range.8 ± ± ± ±

5 Ashraf A. Foda & Ibrahim A. Abdel Aal 7 Table (6): Correlation between serum & peritoneal fluid levels of the biomarkers. IL-6 CA-125 TNF-α Hs-CRP VEGF r p (significant) (significant) (significant) (significant) (significant) Table (1): Sensitivity & specificity on combining two or more markers. Pair of markers IL6 + TNF-α IL6 + CRP TNF + CRP CA VEGF IL6 + TNF + CRP Specificity 7% 76% 73.3% 76.7% 63.3% Sensitivity % % % 93.8% % + r: Positive correlation coefficient (increase in serum levels with increase in PF levels). Table (7): Correlation of the serum biomarker levels with the stage of endometriosis. IL-6 CA-125 TNF-α Hs-CRP VEGF IL-6 CA-125 TNF-α Hs-CRP VEGF r r p (significant) (significant) (significant) <.4 (significant) <.43 (significant) r: Negative correlation coefficient (it decreases with increase in stage). + r: Positive correlation coefficient (it increases with increase in stage. Table (8): Correlation of the PF biomarker levels with the stage of endometriosis. p (significant) (significant) >.3 (not significant) >.239 (not significant) (significant) r: Negative correlation coefficient (it decreases with increase in stage). + r: Positive correlation coefficient (it increases with increase in stage. Table (9): ROC curve analysis of biomarkers serum levels. IL-6 CA-125 TNF-α Hs-CRP VEGF Level >12.2 >17.6 >12.45 >438 >236 Area under curve (AUC) Sensitivity (%) Specificity (%) Diagnostic accuracy (%) Positive predictive value % (PPV) Negative predictive value (NPV) SIL6 Specificity Curve (1): ROC curve of serum IL-6. SCA 125 Specificity Curve (2): ROC curve of Serum CA-125. STNFA Specificity Curve (3): ROC curve of serum TNF- α.

6 8 Role of Some Biomarkers in Chronic Pelvic Pain for Early Detection SCRP Specificity Curve (4): ROC curve of serum Hs-CRP SVEGF no value for serum IL-6 in the diagnosis of any stage of endometriosis []. The high levels of serum IL-6 in MM endometriosis can be explained by the finding that more inflammatory markers are secreted during the early stages rather than the late stages of a given disease, when most cytokines, chemokines and growth factors are secreted into peritoneal fluid and there was an inverse relationship between the total number of peritoneal macrophages and the extension of pelvic endometriosis when most cytokines are secreted into peritoneal fluid [21]. In the present study by using ROC curve analysis, the serum IL-6 optimal threshold value of >12.2pg/ml was found to be able to diagnose endometriosis with a sensitivity, specificity & diagnostic accuracy of 95.58%, 83.33% & 91.58% respectively. To diagnose MM endometriosis, Martinez et al. [9], established an optimal threshold value of 25.75pg/ml with a sensitivity of 75% and a specificity of 83%. On the other hands, Bedaiwy et al. [8], suggested a serum IL-6 discriminatory threshold of 2pg/ml with a sensitivity of 9% and a specificity of 67% for endometriosis patients, but this level is lower than the levels observed in the control cases in the present study. Specificity Curve (5): ROC curve of serum VEGF Discussion IL-6 is a pro-inflammatory multi-factorial cytokine secreted by the T-cells [8] and is involved in numerous immunological and proliferative processes [17]. IL-6 in the PF may play a role in the pathogenesis of endometriosis by initiating or sustaining inflammatory responses in the PF [17]. In the present study, high IL-6 levels were reported in the peritoneal fluid (PF) of patients with endometriosis. Our results are agreement with the finding of Cheong et al. [18]. In the present study, patients with MM endometriosis had significantly high levels of serum IL-6 than patients with MS endometriosis (p), and this finding is in agreement with previous findings [8,19], but diverge with the reports of other authors who reported high serum IL-6 levels in women with MS rather than in MM endometriosis and controls [11]. Some authors found These findings including ours make serum IL- 6 determination a promising tool for screening and early detection purposes and, combined with clinical data, will allow gynecologists to detect which women are at high risk of endometriosis. CA-125 is a classic marker of endometriosis. It was shown to have a greater diagnostic value in MS rather than in MM endometriosis indicating that CA-125 is usually employed as a marker of recurrence or of success in a surgical intervention [22,23]. In the present study, the serum CA-125 was significantly elevated (p<.1) in MS than in MM endometriosis, indicating that CA-125 can not be used as a measure for early diagnosis of peritoneal endometriosis. By using ROC analysis, the serum CA-125 optimal threshold value of >17.6IU/ml was found to be able to diagnose endometriosis with sensitivity, specificity, diagnostic accuracy and AUC of %, %, 73.3% &.851 respectively. Our results are in agreement with the previous findings [9,1,22,23]. The area under the ROC curve (AUC) of serum CA-125 for mild endometriosis was.788 as reported by Kitawaki et al. [23]. As a result, they established and 3IU/ml CA-125 as threshold

7 Ashraf A. Foda & Ibrahim A. Abdel Aal 9 values for screening of early stages of the disease. On the other hand, Martinez et al. [9] reported that CA-125 was better in the diagnosis of MS endometriosis with an AUC of.812. They concluded that CA-125 was not effective in its diagnosis of MM endometriosis (area under ROC curve, or AUC, <.5). In the present study, a marked elevation in TNF-α level was apparent in the PF of patients with endometriosis, with a higher value in MM than in MS endometriosis (p). Our results are in agreement with previous studies [11,18,19]. By using ROC curve analysis, the serum TNF- α optimal threshold value of >12.45pg/ml was found to be able to diagnose endometriosis with a sensitivity, specificity, diagnostic accuracy & AUC of 89.23%, 86.87%, 86.67% &.89 respectively. TNF-soluble high-affinity receptor complex (TNF-SHARC) was introduced as a therapeutic agent for treating endometriosis in human. TNF- SHARC inhibited TNF- α induced secretion of interleukins and granulocyte macrophage-colonystimulating factor in immortalized human endometriotic cells [24]. In the present study, there was a high serum Hs-CRP levels in the endometriosis group when compared with the non-endometriosis group (p). MS endometriosis had a significantly higher levels than MM endometriosis (p). This finding indicates that Hs-CRP can not be used for early diagnosis of endometriosis. These findings are in agreement with the findings of Lermann et al. [25]. There were contradictory results regarding VEGF serum levels in endometriosis. Matalliotakis et al. [26] reported a slight but significant increase of serum VEGF in diseased females. In contrast, Gagne et al. [27] found no significant difference in VEGF serum levels of patients with endometriosis compared with cases without the disease. In the present study, a marked elevation in VEGF level was apparent in the serum and PF of patients with endometriosis, with a higher values in MS than in MM endometriosis (p<.8). This is in agreement with the findings of Matalliotakis [26]. The inflammation associated with endometriosis, through increased levels of peritoneal fluid VEGF, may promote angiogenesis for progressive growth of endometriosis. In the present study, the high VEGF levels in PF are in agreement with the previous findings of Mahnke [28]. Thus, anti-angiogenic drugs are con- sidered potential therapeutic agents for treatment of endometriosis. Conclusion: The results of the present study indicate that serum IL-6 and TNF- α level can be used to discriminate between patients with or without endometriosis. Also, MM endometriosis patients display higher serum IL-6 and TNF- α level than MS endometriosis patients or the control cases. This throws light on markers of the early stages of the disease. Additionally, serum IL-6 and TNFα allow for discrimination between MM endometriosis and MS endometriosis. Measurements of CA-125, VEGF and Hs-CRP appears to be advantageous only for the diagnosis of severe endometriosis and positively correlate with the stage of the disease. Nevertheless, very low levels might serve as a marker for an absence of endometriosis. The cost of analysis of the markers per case was about 11 L.E. that is far more less than the costs of the hospital stay and diagnostic laparoscopy. Conflict of interest: To the best of our knowledge, we have no relevant financial relationships. Acknowledgement: To all the laboratory technicians in the Clinical Pathology Research team Unit, Clinical Pathology Department, Faculty of Medicine, Mansoura University, Egypt for their help and effort and also for all our patients for their cooperation. References 1- American College of Obstetricians and gynecologists: Chronic pelvic pain. Practice. Bulletin., No. 51, March GIUDICE I.C. and KAO I.C.: Endometriosis. Lancet, 364: 1789, BERKLEY K.J., RAPKIN A.J. and PAPKA R.E.: The pains of endometriosis. Science, 38: 1587, MITWALLY M.F. and CASPER R.F.: Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate. Fertil. Steril., 75: 35-39, OZKAN S., MURK W. and ARıCı A.: Endometriosis and infertility. Ann. N. Y. Acad. Sci., 1127: 92-, KOYAMA N., MATSUURA K. and OKAMURA H.: Cytokines in the peritoneal fluid of patients with endometriosis. Int. J. Gynecol. Obstet., 43: 45-5, KEENAN J.A., CHEN T.T., CHADWELL N.L., TORRY D. S. and CAUDLE M.R.: IL-1 beta, TNF-alpha, and IL-

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