Clinical immunologyPro-inflammatory cytokines for evaluation of inflammatory status in endometriosis

The aim of the study was to investigate the serum pro-inflammatory cytokine profile in patients with diagnosed endometriosis.

The study included 160 women, who were divided in two study groups (Group I – endometriosis; Group 2 – healthy). We evaluated the serum levels of interleukin (IL)-1 β, IL-5, IL-6, IL-7, and IL-12, and of tumour necrosis factor α (TNF-α) with the use of Human Multiplex Cytokine Panels.

The serum level of IL-1β, IL-6, and TNF-α is significantly higher in women with endome- triosis compared to women free of disease, from the control group (mean 10.777, 183.027, and 131.326, respectively, compared to 3.039, 70.043, and 75.285, respectively; p = 0.002, p < 0.001, and p = 0.015, respectively). No significant differences in the serum levels of IL-5 and IL-12 were observed between the studied groups, and IL-7 had a very low detection rate.

Women with endometriosis have elevated levels of key pro-inflammatory cytokines, i.e. IL-1β, IL-6, and TNF-α. At the same time, IL-1β and IL-6 could be used as predictors for endometriosis. Key words: inflammation, endometriosis, cytokine, interleukin. (Centr Eur J Immunol 2015; 40 (1): 96-102)

Endometriosis is a pelvic inflammatory condition de- fined as the presence of ectopic deposits of endometrial tissue outside of the uterine cavity. The disease manifests clinically through various forms of pelvic pain or subfer- tility. The presence of pelvic endometriosis is about 5-10% in the general population, but in women with pelvic pain, infertility, or both, the prevalence is 35-50% [1], with some authors reporting as high as 82% prevalence in these patients [2]. This variability could be due to the difficulty in non-surgical diagnosis. The gold standard in diagnos- ing endometriosis is diagnostic laparoscopy. On the other hand, it is an invasive procedure with potential hazards, which can include major vessel or bowel injury [3]. A sim- ple blood test for prediction and diagnosis of endometriosis would overcome these problems and have a major impact on women’s health. Immune system alterations are thought to be involved in the development of endometriosis, especially a dysfunc- tion in immune-related cells and macrophages within the peritoneum secreting a number of products, mainly cy- tokines and growth factors [4, 5]. At this level, there is an immune-inflammatory reaction that activates immune cells, together with endometriotic implants, producing high amounts of cytokines, growth factors, and angiogenic products [6]. However, systemic immune alterations have also been described in endometriosis, with activation of peripheral blood monocytes, which secrete high levels of cytokines [7]. Several cytokines, including interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), and tu- mour necrosis factor α (TNF-α), have been studied in the pathogenesis of endometriosis. Interleukin 6 is considered to play a potential role in the growth and/or maintenance of ectopic endometrial tissue. Interleukin 6 is a regulator of inflammation and immunity that modulates secretion of other cytokines, promotes T-cell activation and B-cell differentiation, and inhibits growth of various cell lines. The role of IL-6 in the pathogenesis of endometriosis has been extensively studied, but the levels of IL-6 detected both in the peritoneal fluid and in the serum of patients with endometriosis have been inconsistent [8, 9]. Tumor necrosis factor α is secreted from activated macrophages, and it has potent inflammatory, cytotoxic, and angiogenic effects. It is known to stimulate the expression of matrix metalloproteinases by endometriotic tissues, and matrix metalloproteinases actively participate in the invasion and matrix remodelling of endometriotic lesions [10]. More - over, etanercept, an anti-TNF therapy, was found to effec- Central European Journal of Immunology 2015; 40(1) Pro-inflammatory cytokines for evaluation of inflammatory status in endometriosis 97 tively reduce the development of endometriosis [11]. Some authors observed that increased production of these factors in the peritoneal fluid was associated with the elevation of similar factors in the peripheral blood of patients with endometriosis [12]. The present study aimed to investigate the serum pro-inflammatory cytokine profile in patients with endo- metriosis compared with healthy controls, and to evaluate the sensitivity and specificity of any single cytokine or a combination of cytokines in the prediction of the inflam- matory status in endometriosis.

Study population and design A case-control study was conducted between June 2013 and June 2014 in “Dominic Stanca” Obstetrics and Gynaecology Clinic, Cluj-Napoca, Romania. The study included 160 patients admitted to the clin- ic, who were divided into two groups as follows: Group I (endometriosis group) – 80 women with regular menses, and with no history of pelvic infections, autoimmune and neoplastic diseases, undergoing laparoscopy or laparotomy for suspected endometriosis. The evidence of endometriosis was verified by histopathological analysis. The severity of endometriosis was staged according to the revised American Society for Reproductive Medicine (rASRM) classification [13]. Group II (control group) – 80 healthy non-pregnant women aged between 18 and 40 years old, without clinical and paraclinical evidence of endometriosis. Exclusion criteria were as follows: previous pelvic surgeries, history of cancer, suspected malignancy, adeno- myosis or leiomyoma, pre-surgical suspicion of evidence of premature ovarian failure, or the use of ovarian suppres- sive drugs, such as oral contraceptives, GnRH agonists, progestins, or danazol in the preceding six months. None of the patients had taken anti-inflammatory medications or had been diagnosed with an inflammatory or infectious condition for ≥ 6 months before the study. The study protocol was approved by the Local Eth- ics Committee of “Iuliu Haţieganu” University of Med- icine and Pharmacy, Cluj-Napoca, Romania, and signed informed consent was received from each woman before sample collection. The study was conducted under the te- nets of the Helsinki Declaration. Data was collected for each subject included in the study in a form containing general and anthropometric data (weight, height), the heredo-collateral history, per- sonal pathological history, and data on the age and onset of symptoms. The body mass index (BMI) was calculated as the ratio between the weight (kg) and the squared height (in metres). A total of 5 ml of venous blood was collected from each patient before breakfast, which was centrifuged, and the serum obtained was stored at –70 oC for future de- terminations. Cytokine evaluation We used multiplex cytokine kits (Invitrogen Human Cytokine 30-Plex Panel, LHC6003) in order to measure serum levels of IL-1β, IL-5, IL-6, IL-7, IL-12, and TNF-α. Dose measurements were performed with the use of a Lu- minex 200 system (Luminex Corporation, Austin, TX, USA) in accordance with the manufacturer’s specifica- tions (Invitrogen Corporation, Carlsbad, CA, USA). The sensitivity of the test was specified by the manufacturer (Invitrogen Corporation, Carlsbad, CA, USA) in the infor- mative material included in the kits. The average sensitivity of the test for IL-1β was 5 pg/ ml, with an inter-assay variation coefficient of 4.8%. For IL-5, the average sensitivity of the test was 0.5 pg/ml with an inter-assay variation coefficient of 7.5%. The average sensitivity of the test for IL-6 was 0.5 pg/ml with an in- ter-assay variation coefficient of 7%. In the case of IL-7, the average sensitivity of the test was 10 pg/ml with an inter-assay variation coefficient of 9.8%. The sensitivity of the test for IL-12 was 1 pg/ml, and the inter-assay vari- ation coefficient of 8.1%. The test for TNF-α revealed an average sensitivity of 0.5 pg/ml, with inter-assay variation coefficient of 8.3%. Statistical analysis Statistical analyses were performed using Microsoft Excel and IBM SPSS software (version 22.0). Data is pre- sented as median and quartiles for the groups because the standard deviation was of importance. We performed an analysis for the significances of the observed differences with the median test for independent samples; also Pear- son’s χ 2 test with and without the continuity correction, and Fisher exact test were used as statistical tests. P val- ues less than 0.05 were regarded as significant. Because the study we had conducted was a case-control study, the predictive values (PPV – positive predictive value and NPV – negative predictive value) were calculated with the Bayesian approximation, assuming an endometriosis prevalence of 10%.

Tables 1 and 2 present the biometry data and inflam - matory markers considered for the study. Almost all of the cases from the endometriosis group were staged as III (30 – 37.50%) or IV (47 – 58.75%), and only 3 cases (3.75%) were in stage II of endometriosis, according to rASRM staging criteria. Table 3 presents the data obtained by inferential sta- tistic (the independent samples median-test probability), which show that mean serum levels of IL-1β , IL-6, and Central European Journal of Immunology 2015; 40(1) Andrei M. Malutan et al. 98 TNF-α were significantly higher in patients with endo- metriosis compared to healthy controls (mean 10.777, 183.027, and 131.326, respectively, compared to 3.039, 70.043, and 75.285, respectively; p = 0.002, p < 0.001, and p = 0.015, respectively). Figures 1-5 show the distribution and median of the studied markers’ serum levels between the groups with a significantly higher serum level of IL-1 β, IL-6 and TNF-α in the endometriosis group, and no significant dif- ferences in the serum levels of IL-5 and IL-12 between the studied groups. Interleukin 7 had a very low detection rate. Because the independent samples median test showed significant differences between the median serum levels of IL-1β, IL-6, and TNF-α in endometriosis and control groups, we have also evaluated the use of these markers as a predictive factor for endometriosis. Threshold values chosen for the three parameters (IL-1 β – 7 pg/ml, IL-6 – 125 pg/ml, and TNF-α – 100 pg/ml) have minimised overlapping confidence intervals from the observed distri- Table 1. Descriptive statistics of the endometriosis and control groups Variable Calculated parameters Endometriosis group Control group Average age (yr) mean ± SD 30.600 ±5.486 26.350 ±2.131 28.475 ±4.655 weight (kg) mean ± SD 62.050 ±9.067 56.925 ±8.094 59.488 ±8.920 height (cm) mean ± SD 164.725 ±5.114 167.225 ±6.773 165.975 ±6.094 BMI (kg/cm2) mean ± SD 22.912 ±3.520 20.307 ±2.126 21.609 ±3.173 Table 2. Descriptive statistic of the studied pro-inflammatory parameters Parameter IL-1β IL-5 IL-6 IL-12 TNF-α N valid 110 38 146 136 146 missing 50 122 4 24 14 median 4.170 0.035 69.052 2.080 88.552 minimum 0.0 0.0 11.3 0.2 0.5 maximum 53.2 0.5 542.3 16.1 584.5 percentiles 25 0.363 0.035 61.854 1.325 26.704 50 4.170 0.035 69.052 2.080 88.552 75 10.325 0.071 144.192 4.180 143.325 Table 3. Inferential statistic for the studied markers Independent samples median-test probability* IL-1β 0.010 IL-5 1 (using Fisher exact test) IL-6 < 0.001 IL-12 0.467 TNF-α 0.026 *p < 0.05 significant difference compared to control group Fig. 1. Distribution of interleukin 1β serum levels among groups 60 50 40 30 20 10 0 IL-1β serum level (pg/ml) Endometriosis Control 53.2 16.0 10.9 bution. We have divided the studied groups according to the three new tests as positives and negatives. Tables 4 and 5 show the results obtained for IL-1β and IL-6 as a predictor of endometriosis, with statistically sig- nificant results. Pearson’s χ2 test validated the observed differences, with a probability of 0.005 for IL-1β and < 0.001 for IL-6 (with the continuity correction proba- bility was < 0.001). Table 6 shows the results obtained for TNF-α as a predictive test for endometriosis, showing a lack of statistical significance. Pearson’s χ 2 test failed to validate the observed differences, with a probability of 0.074 (the probability with the continuity correction was also 0.072). Central European Journal of Immunology 2015; 40(1) Pro-inflammatory cytokines for evaluation of inflammatory status in endometriosis 99 Fig. 2. Distribution of interleukin 5 serum levels among groups Fig. 4. Distribution of interleukin 12 serum levels among groups Fig. 3. Distribution of interleukin 6 serum levels among groups Fig. 5. Distribution of tumor necrosis factor α serum levels among groups 0.30 0.25 0.20 0.15 0.10 0.05 0.00 20 15 10 5 0 600 500 400 300 200 100 0 600 500 400 300 200 100 0 IL-5 serum levels (pg/ml)IL-12 serum levels (pg/ml) IL-6 serum levels (pg/ml)TNF-α serum levels (pg/ml) Endometriosis Control Endometriosis Control Endometriosis Control Endometriosis Control 0.05 8.3 542.3 584.5 365.6 245.3 0.02 16.1 541.3523.2 283.3278.0262.1 150.2 149.0 91.9 84.8 Table 4. Contingency table for interleukin 1β as the gold- en standard Endometriosis group Control group Total Interleukin β positive 32 8 40 Interleukin 1β negative 24 46 70 Total 56 54 110 Sensitivity 0.571 Specificity 0.851 Positive predictive value* 0.300 Negative predictive value* 0.947 *Calculated with the Bayesian approximation for case-control studies, assuming a endometriosis prevalence of 10% Table 5. Contingency table for interleukin 6 as the golden standard Endometriosis group Control group Total Interleukin 6 positive 38 4 42 Interleukin 6 negative 38 76 114 Total 76 80 156 Sensitivity 0.500 Specificity 0.950 Positive predictive value* 0.526 Negative predictive value* 0.945 *Calculated with the Bayesian approximation for case-control studies, assuming a endometriosis prevalence of 10% Central European Journal of Immunology 2015; 40(1) Andrei M. Malutan et al. 100 Regarding staging of the endometriosis group, no sig- nificant differences were observed in the means or median values of IL-1β, IL-5, IL-6, IL-12, and TNF-α if the stage of endometriosis was used as a grouping parameter, be- tween patients with endometriosis stage III and stage IV (Table 7).

In the present study we found that in women with en- dometriosis there is a significantly higher serum level of IL-1β, IL-6, and TNF-α, compared to healthy controls. We also found that IL-5 and IL-12 did not differ significantly between women with endometriosis and women free of the disease. On the other hand, IL-7 had a very low detection rate in the studied groups, so we were unable to draw any

regarding its implication. At this moment, there are a large number of studies that support the pathophysiological implication of pro-in- flammatory and anti-inflammatory cytokines such as IL-2, IL-4, IL-6, IL-8, IL-10, interferon γ (IFN-γ), and TNF-α in endometriosis [14, 15]. Interleukin 6 is a cytokine derived from T cells, se- creted by macrophages, lymphocytes, fibroblasts, and endothelial cells. Its secretion is increased by peritoneal macrophages in the case of endometriosis [16]. Generally, IL-6 inhibits the growth of endometrial cells, the effect of which seems to be lost in endometriotic tissues [17]. Cytokines, and specifically IL-6, have been investigated regarding the pathogenesis of endometriosis, and also as a predictor of the disease [18, 19]. Elevated serum CA-125 and IL-6 are considered to be biological markers for dif- ferential diagnosis in endometriosis [20]. Previous studies found conflicting results regarding serum levels of IL-6 in endometriosis; however, most of them were in favour of a significant increase in IL-6 serum or peritoneal fluid (PF) levels in the case of endometriosis. Anterior reports [21, 22] showed significantly higher IL-6 levels in early stages of endometriosis as compared to the healthy group. Moreover, one study showed a correlation between IL-6 and pain, as well as a relation between cytokine expression and recurrences of endometriosis [23]. On the other hand, a recent study showed no statistically significant difference in serum IL-6 concentration between subjects with endo- metriosis and controls; consequently, the authors do not recommend measuring IL-6 in the serum as a predictor of endometriosis. The same authors also suggested that IL-6 provides the best discrimination between subjects with en- dometriosis and healthy controls, and the diagnostic value of this marker increased with the finding that serum IL-6 levels did not change significantly during any phase of the menstrual cycle in either group [3, 24]. Other similar stud- ies have shown a positive relationship between PF levels of IL-6 with severity of endometriosis [25, 26]. Tumor necrosis factor α is a pro-inflammatory cyto- kine produced mainly by activated macrophages. It pro- motes the production of other pro-inflammatory cytokines, such as IL-1, IL-6, and additional TNF-α . It is involved in the normal physiology of endometrial proliferation and shedding [27]. It was shown that peritoneal fluid TNF-α concentrations are elevated in women with endometriosis, and some studies showed that higher concentrations cor- relate with the stage of disease. One study observed that serum TNF- α levels and urinary sFlt-1 levels corrected for creatinine excretion were significantly increased in the endometriosis group [28]. Tumor necrosis factor α may play a central role in the local and systematic manifesta- tions of endometriosis, on the basis of evidence showing that it promotes the growth of endometriotic cells. More- over, some studies explored the association of TNF-α gene polymorphisms and endometriosis, and it seems that some polymorphisms are involved in the pathogenesis of endo- metriosis [29, 30]. On the other hand, blocking TNF-α ap- pears to inhibit the development of the disease in animal models [27]. Interleukin 1 is a cytokine that plays an important role in inflammation and immune response. The IL-1 family consists of IL-1α , IL-1β , and IL-1 receptor antagonist. Both IL-1α and IL-1β are the most potent pro-inflamma- tory cytokines, and IL-1 receptor antagonist is a naturally Table 6. Contingency table for tumor necrosis factor (TNF-α) as the golden standard Endometriosis group Control group Total TNF-α positive 38 20 58 TNF-α negative 34 54 88 Total 72 74 146 Sensitivity 0.527 Specificity 0.729 Positive predictive value* 0.178 Negative predictive value* 0.933 *Calculated with the Bayesian approximation for case-control studies, assuming a endometriosis prevalence of 10% Table 7. Endometriosis stages III and IV comparison Independent samples median-test probability* Interleukin 1β 1 (using Fisher exact test) Interleukin 5 1 (using Fisher exact test) Interleukin 6 0.892 Interleukin 12 0.839 TNF-α 0.591 *p < 0.05 significant difference compared to control group Central European Journal of Immunology 2015; 40(1) Pro-inflammatory cytokines for evaluation of inflammatory status in endometriosis 101 occurring anti-inflammatory cytokine [31, 32]. A series of studies showed that there are increased concentrations of IL-1β , IL-6, IL-10, and TNF-α , as well as decreased VEGF in the folicular fluid (FF) of endometriosis pa- tients [33, 34]. Moreover, Lambert et al. have shown both a significant increase in serum IL-1β and IL-1sRII lev- els in deep infiltrating endometriosis (DIE) compared to superficial endometriosis (SUP) and normal women, and suggested that a defect in the control of IL-1 can impact the pathophysiology of endometriosis [35]. On the other hand, Kalu et al. [36] found no significant difference in serum levels of platelet-derived growth factor (PDGF), IL- 6, Regulated on Activation, Normal T Cell Expressed and Secreted chemokine (RANTES), IL-1β, TNF-α, and sFas in patients with endometriosis, compared to controls. Applying IL-1β and IL-6 for the prediction of endome- triosis, we found that they can be successfully used, hav - ing a specificity of 0.85, respectively, and a specificity of 0.95. Because IL-1β is not very sensitive (0.57), it is rec- ommended for use in conjunction with other tests. Tumor necrosis factor α, used in the prediction of endometriosis, has limited value with a specificity of 0.72. Our results are in accordance with a recent study, which reported increased peritoneal concentrations of IL-6 and IL-10 in women with endometriosis. Analysis of the concentrations of these cytokines showed differ- ential diagnostic usefulness for endometriosis. Peritone- al concentrations of IL-6 offer relatively good diagnostic accuracy [37]. At the same time, a very recent study has shown that at the cut-off value of 3.00 pg/ml peritoneal TNF-α can be a reliable screening marker for the predic - tion of endometriosis in adolescents [38]. In contrast with our observations, Bedaiwy et al. [12] failed to show the significance of peritoneal IL-6 levels in diagnosing women with endometriosis. One of our main limitations is represented by the study population, which are only Caucasian women. Most of the studies present in the literature had mainly Asian origin subjects, this being considered a possible bias mark [39]. From this point of view, our population could be consid- ered also a strength, but a more heterogenic study group would probably give more accurate results. Another limita- tion could be the fact that most of the patients included in the study were stage III or IV (96.25%) and the lack of dif- ferentiation between patients with ovarian endometriomas (OE) and patients with DIE. As the study was conducted in a University Clinic, the patients addressing for treatment and included in the study had late stages of endometriosis, thus the low rate of early stages. Probably future studies could include patients presenting for unexplained infertili- ty undergoing exploratory laparoscopy, and thus including patients with early stages discovered incidentally. On the other hand, OE and DIE are considered two distinct enti- ties of endometriotic disease, and accordingly it is accept- ed that endometriosis progresses to cystic ovarian disease and pelvic adhesions in some women, to deeply infiltrating disease in other women, and sometimes to both stages of severe disease in the same woman [5]. A recent study has found a high positive correlation between serum levels of IL-6 and IL-8 in patients with OE, but not in the DIE and control groups [7]. One last limitation could be represented by the severity of endometriosis in the study population. Most of the recruited patients had advanced endometrio- sis, OE, or DIE, and consequently a differentiation in se- rum levels of the studied markers between patients with SUP and OE or DIE was not possible. We could also take in consideration the detection sensitivity of multiplexed immunoassays. It was shown that, while multiplexed im- munoassays have sensitivity comparable to conventional ELISA, it is possible that the robustness may vary among different multiplex bead arrays [40]. In conclusion, we examined the possible differences in pro-inflammatory cytokine serum levels from patients with endometriosis compared to healthy controls using a mul- tiplexed cytokine assay. We have shown that IL-1β, IL-6, and TNF-α serum levels are significantly higher in women with endometriosis compared to women free of disease, from the control group, and that there is no difference in the serum levels of IL-5 and IL-12 between these patients. More importantly, our study showed that IL-1β and IL-6 could be used as a non-surgical diagnostic test for endome- triosis. Further studies are necessary to clarify and confirm the role of pro-inflammatory cytokines in the pathogenesis of endometriosis, and moreover to find a suitable predic- tive model for early diagnosis. The authors declare no conflict of interest. This paper was published under the frame of the Eu- ropean Social Fund, Human Resources Development Operational Programme 2007-2013, project no. POS- DRU/159/1.5/S/138776.

1. Giudice LC, Kao LC (2004): Endometriosis. Lancet 364: 1789-1799. 2. Mounsey A, Wilgus A, Slawson DC (2006): Diagnosis and management of endometriosis. Am Fam Physician 74: 594- 600. 3. Othman Eel-D, Hornung D, Salem HT, et al. (2008): Serum cytokines as biomarkers for nonsurgical prediction of endo- metriosis. Eur J Obstet Gynecol Reprod Biol 137: 240-246. 4. Yagmur E, Bastu E, Karamustafaoglu-Balci B, et al. (2013): Non-invasive diagnosis of endometriosis based on a com- bined analysis of four plasma biomarkers. Centr Eur J Im- munol 38: 154-158. 5. Bulun SE (2009): Endometriosis. N Engl J Med 360: 268-279. 6. Kubatova A, Erdem A, Erdem M, et al. (2013): Serum cyto- kine and growth factor levels in patients with endometriosis. Centr Eur J Immunol 38: 500-504. 7. Carmona F, Chapron C, Martínez-Zamora MA, et al. (2012): Ovarian endometrioma but not deep infiltrating endometriosis Central European Journal of Immunology 2015; 40(1) Andrei M. Malutan et al. 102 is associated with increased serum levels of interleukin-8 and interleukin-6. J Reprod Immunol 95: 80-86. 8. Moini A, Malekzadeh F, Amirchaghmaghi E, et al. (2013): Risk factors associated with endometriosis among infertile Iranian women. Arch Med Sci 9: 506-514. 9. Agic A, Xu H, Finas D, et al. (2006): Is endometriosis associ- ated with systemic subclinical inflammation? Gynecol Obstet Invest 62: 139-147. 10. K lllc SH, Evsen S, Tasdemir N, et al. (2007): Follicular fluid vascular endothelial growth factor and tumour necrosis fac- tor a concentration in patients with endometriosis undergoing ICSI. Reprod Biomed Online 15: 316-320. 11. Zulfikaroglu E, K lllc S, Islimye M, et al. (2011): Efficacy of anti-tumor necrosis factor therapy on endometriosis in an experimental rat model. Arch Gynecol Obstet 283: 799-804. 12. Bedaiwy MA, Falcone T, Sharma RK, et al. (2002): Predic - tion of endometriosis with serum and peritoneal fluid mark- ers: a prospective controlled trial. Hum Reprod 17: 426-431. 13. Revised American Society for Reproductive Medicine clas- sification of endometriosis: 1996 (1997): Fertil Steril 67: 817-821. 14. Wu MY, Ho HN (2003): The role of cytokines in endometri- osis. Am J Reprod Immunol 49: 285-296. 15. OuYang Z, Hirota Y, Osuga Y, et al. (2008): Interleukin-4 stimulates proliferation of endometriotic stromal cells. Am J Pathol 173: 463-469. 16. Berkkanoglu M, Arici A (2003): Immunology and endome- triosis. Am J Reprod Immunol 50: 48-59. 17. Dmowski WP, Braun DP (2004): Immunology of endome- triosis. Best Pract Res Clin Obstet Gynaecol 18: 245-263. 18. Bedaiwy MA, Falcone T (2004): Laboratory testing for endo- metriosis. Clin Chim Acta 340: 41-56. 19. Ohata Y, Harada T, Miyakoda H, et al. (2008): Serum inter- leukin-8 levels are elevated in patients with ovarian endome- trioma. Fertil Steril 90: 994-999. 20. Martínez S, Garrido N, Coperias JL, et al. (2007): Serum in- terleukin-6 levels are elevated in women with minimal-mild endometriosis. Hum Reprod 22: 836-842. 21. Mier-Cabrera J, Jiménez-Zamudio L, García-Latorre E, et al. (2011): Quantitative and qualitative peritoneal immune pro- files, T-cell apoptosis and oxidative stress-associated char- acteristics in women with minimal and mild endometriosis. BJOG 118: 6-16. 22. Khan KN, Masuzaki H, Fujishita A, et al. (2004): Higher activity by opaque endometriotic lesions than nonopaque le- sions. Acta Obstet Gynecol Scand 83: 375-382. 23. Velasco I, Acién P, Campos A, et al. (2010): Interleukin-6 and other soluble factors in peritoneal fluid and endometri- omas and their relation to pain and aromatase expression. J Reprod Immunol 84: 199-205. 24. Somigliana E, Vigano P, Tirelli AS, et al. (2004): Use of the concomitant serum dosage of CA 125, CA 19-9 and interleu- kin-6 to detect the presence of endometriosis. Results from a series of reproductive age women undergoing laparoscopic surgery for benign gynaecological conditions. Hum Reprod 19: 1871-1876. 25. Milewski Ł, Barcz E, Dziunycz P, et al. (2008): Association of leptin with inflammatory cytokines and lymphocyte sub- populations in peritoneal fluid of patients with endometriosis. J Reprod Immunol 79: 111-117. 26. Milewski Ł, Dziunycz P, Barcz E, et al. (2001): Increased levels of human neutrophil peptides 1, 2, and 3 in peritoneal fluid of patients with endometriosis: association with neutro- phils, T cells and IL-8. J Reprod Immunol 91: 64-70. 27. Lu D, Song H, Shi G (2013): Anti-TNF-α treatment for pel- vic pain associated with endometriosis. Cochrane Database Syst Rev 3:CD008088. 28. Cho SH, Oh YJ, Nam A, et al. (2007): Evaluation of serum and urinary angiogenic factors in patients with endometriosis. Am J Reprod Immunol 58: 497-504. 29. Lee GH, Choi YM, Kim SH, et al. (2008): Association of tumor necrosis factor-a gene polymorphisms with advanced stage endometriosis. Human Reproduction 23: 977-981. 30. Teramoto M, Kitawaki J, Koshiba H, et al. (2004): Genet- ic contribution of tumor necrosis factor (TNF)-alpha gene promoter (-1031,-863 and -857) and TNF receptor 2 gene polymorphisms in endometriosis susceptibility. Am J Reprod Immunol 51: 352-357. 31. Rogus J, Beck JD, Offenbacher S, et al. (2008): IL1B gene promoter haplotype pairs predict clinical levels of interleu- kin-1beta and C-reactive protein. Hum Genet 123: 387-398. 32. Singh H, Sachan R, Goel H, Mittal B (2008): Genetic vari- ants of interleukin-1RN and interleukin-1b genes and risk of cervical cancer. BJOG 115: 633-638. 33. Wunder DM, Mueller MD, Birkhauser MH, Bersinger NA (2006): Increased ENA-78 in the follicular fluid of patients with endometriosis. Acta Obstet Gynecol Scand 85: 336-342. 34. Gupta S, Goldberg JM, Aziz N, et al. (2008): Pathogenic mechanisms in endometriosis-associated infertility. Fertil Steril 90: 247-257. 35. Lambert S, Santulli P, Chouzenoux S, et al. (2014): Endo- metriosis: Increasing concentrations of serum interleukin-1β and interleukin-1sRII is associated with the deep form of this pathology. J Gynecol Obstet Biol Reprod (Paris) 43: 735-743. 36. Kalu E, Sumar N, Giannopoulos T, et al. (2007): Cytokine profiles in serum and peritoneal fluid from infertile women with and without endometriosis. J Obstet Gynaecol Res 33: 490-495. 37. Wickiewicz D, Chrobak A, Gmyrek GB, et al. (2013): Di- agnostic accuracy of interleukin-6 levels in peritoneal fluid for detection of endometriosis. Arch Gynecol Obstet 288: 805-814. 38. Drosdzol-Cop A, Skrzypulec-Plinta V (2012): Selected cyto- kines and glycodelin A levels in serum and peritoneal fluid in girls with endometriosis. J Obstet Gynaecol Res 38: 1245- 1253. 39. Clayton DG, Walker NM, Smyth DJ, et al. (2005): Popu- lation structure, differential bias and genomic control in a large-scale, case-control association study. Nat Genet 37: 1243-1246. 40. Elshal MF, McCoy JP (2006): Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods 38: 317-323.