endometriosis is a frequent gynecologic disease with a prevalence of about 10 - 15% of reproductive - aged women , and it represents the existence of endometrial tissue outside the uterine cavity . most common symptoms are represented by infertility , dysmenorrhea and most frequently by pelvic pain , and at the same time a reduced quality of life . the etiology and pathogenesis of the disease are not clearly understood , tough it is well known that susceptibility to endometriosis depends on complex interactions between genetic , immunologic , hormonal , and environmental factors . a general concept is that endometriosis is a local pelvic inflammatory process with altered function of immune - related cells , thus it was suggested that the serum of women with endometriosis contains an increased number of activated macrophages that secrete products as growth factors and cytokines . there is a body of evidence which suggests that the inflammation and immune responses play a pivotal role in the pathogenesis of endometriosis , especially a series of cytokines and angiogenic factors . the interleukin-1 ( il-1 ) system , which comprises 11 cytokines , is believed to play an important role in the pathophysiology of endometriosis , although this finding is controversial . both il-1 alpha and il-1beta ( il-1 ) are the most potent pro - inflammatory cytokines , and il-1 receptor antagonist is a naturally occurring anti - inflammatory cytokine . recent studies have shown decreased levels of il-1 receptor antagonist ( il-1ra ) in peritoneal fluid ( pf ) from patients with endometriosis and disease - related dysmenorrhea . at the same time , it was seen that the level of il-4 , a typical th2 cytokine , is increased in endometriotic tissues , with other authors observing that th2-type immune responses are activated in women with endometriosis . it is believed that il-4 stimulates the proliferation of endometriotic stromal cells ( escs ) , suggesting a key role in the development of endometriosis . being closely related to il-4 , il-13 has some biological activities in common with il-4 , although their target cells may display variable response , thus with a possible involvement in endometriosis . one of the most studied anti - inflammatory cytokines in endometriosis , il-10 , plays an important role in eliminating unwanted cells and cellular debris in a silent way . a very recent study has suggested that il-10 may suppress the immunity against endometrial implants , contributing to the development of endometriosis . the awareness that the immune cell activation is one of most important features of endometriosis and that anti - inflammatory cytokines could have a pivotal role in the development of it , has lead us to investigate the most important anti - inflammatory serum cytokines in the patients with endometriosis . the present study aimed to investigate the serum anti - inflammatory cytokine profile in the patients with endometriosis compared with the healthy controls and the possible role of these markers in the prediction of the disease . a case  control study was conducted between june 2013 and june 2014 in  dominic stanca  obstetrics and gynecology clinic , cluj - napoca , romania . the study included 160 patients admitted to the clinic , 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 severity of endometriosis was staged according to the revised american society for reproductive medicine ( rasrm ) classification ; almost all the patients included were staged as iii ( 30 - 37.50% ) or iv ( 47 - 58.75% ) according to rasrm staging criteria , and only 3 cases ( 3.75% ) were in stage ii . group ii ( control group)-80 healthy nonpregnant women aged between 18 and 40-year - old , without the clinical and paraclinical evidence of endometriosis . previous pelvic surgeries , history of cancer , suspected malignancy , adenomyosis or leiomyoma , presurgical suspicion of evidence of premature ovarian failure , or the use of ovarian suppressive drug such as oral contraceptives , gonadotropin - releasing hormone agonists , progestins , or danazol in the preceding 6 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 . university of medicine and pharmacy , cluj - napoca , romania , and signed informed consent was received from each woman before the sample collection . data was collected for each subject included in the study in a form containing general and anthropometric data ( weight , height ) , the heredo - collateral history , personal 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 meters ) . five ml of venous blood was collected from each patient before breakfast , which was centrifuged and the serum obtained was stored at 70c for future determinations . we used multiplex cytokine kits ( invitrogen human cytokine 30-plex panel , lhc6003 ) in order to measure serum levels of il-1ra , il-2 , il-2r , il-4 , il-10 , il-13 , and il-15 . dose measurements were performed with the use of a luminex 200 system ( luminex corporation , austin , tx , usa ) in accordance with the manufacturer 's specifications ( invitrogen corporation , carlsbad , ca , usa ) . the sensitivity of the test was specified by the manufacturer ( invitrogen corporation , carlsbad , ca , usa ) in the informative material included in the kits . the average sensitivity of the test for il-1ra was < 20 pg / ml with an inter - assay variation coefficient of 5.1% . for il-2 , the average sensitivity of the test was < 0.5 pg / ml with an inter - assay variation coefficient of 9.6% . the average sensitivity of the test for il-2r was < 10 pg / ml with an inter - assay variation coefficient of 4.2% . in the case of il-4 , the average sensitivity of the test was < 0.5 pg / ml with an inter - assay variation coefficient of 8.7% . the sensitivity of the test for il-10 was < 0.5 pg / ml , and the inter - assay variation coefficient of 9.8% . the test for il-13 revealed an average sensitivity of < 5 pg / ml , with inter - assay variation coefficient of 9.6% , and for il-15 , the average sensitivity of the test was < 15 pg / ml with an inter - assay variation coefficient of 7.6% . armonk , ny : ibm corp . and graphpad prism version 5.00 for windows , graphpad software , san diego , california usa . smirnov test with the lilliefors correction ( p > 0.05 was considered for a normal distribution ) . a case  control study was conducted between june 2013 and june 2014 in  dominic stanca  obstetrics and gynecology clinic , cluj - napoca , romania . the study included 160 patients admitted to the clinic , 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 severity of endometriosis was staged according to the revised american society for reproductive medicine ( rasrm ) classification ; almost all the patients included were staged as iii ( 30 - 37.50% ) or iv ( 47 - 58.75% ) according to rasrm staging criteria , and only 3 cases ( 3.75% ) were in stage ii . group ii ( control group)-80 healthy nonpregnant women aged between 18 and 40-year - old , without the clinical and paraclinical evidence of endometriosis . previous pelvic surgeries , history of cancer , suspected malignancy , adenomyosis or leiomyoma , presurgical suspicion of evidence of premature ovarian failure , or the use of ovarian suppressive drug such as oral contraceptives , gonadotropin - releasing hormone agonists , progestins , or danazol in the preceding 6 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 . university of medicine and pharmacy , cluj - napoca , romania , and signed informed consent was received from each woman before the sample collection . data was collected for each subject included in the study in a form containing general and anthropometric data ( weight , height ) , the heredo - collateral history , personal 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 meters ) . five ml of venous blood was collected from each patient before breakfast , which was centrifuged and the serum obtained was stored at 70c for future determinations . previous pelvic surgeries , history of cancer , suspected malignancy , adenomyosis or leiomyoma , presurgical suspicion of evidence of premature ovarian failure , or the use of ovarian suppressive drug such as oral contraceptives , gonadotropin - releasing hormone agonists , progestins , or danazol in the preceding 6 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 . university of medicine and pharmacy , cluj - napoca , romania , and signed informed consent was received from each woman before the sample collection . data was collected for each subject included in the study in a form containing general and anthropometric data ( weight , height ) , the heredo - collateral history , personal 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 meters ) . five ml of venous blood was collected from each patient before breakfast , which was centrifuged and the serum obtained was stored at 70c for future determinations . we used multiplex cytokine kits ( invitrogen human cytokine 30-plex panel , lhc6003 ) in order to measure serum levels of il-1ra , il-2 , il-2r , il-4 , il-10 , il-13 , and il-15 . dose measurements were performed with the use of a luminex 200 system ( luminex corporation , austin , tx , usa ) in accordance with the manufacturer 's specifications ( invitrogen corporation , carlsbad , ca , usa ) . the sensitivity of the test was specified by the manufacturer ( invitrogen corporation , carlsbad , ca , usa ) in the informative material included in the kits . the average sensitivity of the test for il-1ra was < 20 pg / ml with an inter - assay variation coefficient of 5.1% . for il-2 , the average sensitivity of the test was < 0.5 pg / ml with an inter - assay variation coefficient of 9.6% . the average sensitivity of the test for il-2r was < 10 pg / ml with an inter - assay variation coefficient of 4.2% . in the case of il-4 , the average sensitivity of the test was < 0.5 pg / ml with an inter - assay variation coefficient of 8.7% . the sensitivity of the test for il-10 was < 0.5 pg / ml , and the inter - assay variation coefficient of 9.8% . the test for il-13 revealed an average sensitivity of < 5 pg / ml , with inter - assay variation coefficient of 9.6% , and for il-15 , the average sensitivity of the test was < 15 pg / ml with an inter - assay variation coefficient of 7.6% . armonk , ny : ibm corp . and graphpad prism version 5.00 for windows , graphpad software , san diego , california usa . smirnov test with the lilliefors correction ( p > 0.05 was considered for a normal distribution ) . table 1 presents the biometry data of the patients considered for the study , showing the significant differences in age , weight , and bmi between groups . the biometry data of the patients considered for the study the descriptive statistics of the studied cytokines is presented in table 2 , showing a significantly higher mean serum level of il-1ra , il-4 , and il-10 in the endometriosis group ( mean 30.15 , 138.45 , and 1.48 , respectively , compared to 14.10 , 84.71 , and 0.68 , respectively ) , and no significant differences in the mean serum levels of il-2 , il-13 , and il-15 between the studied groups . after testing the distributions normality with the kolmogorov smirnov test with lilliefors correction , we have applied independent t - tests and a nonparametric test mann whitney u - test for independent samples , both test showing the same results as presented in table 2 . the detection rate for il-1ra , il-2 , il-4 , il-10 , il-13 , and il-15 , respectively , in the studied groups , was 78.75 , 90.00 , 95.00 , 83.75 , 67.50 , and 90.00% , respectively . comparative statistics of the studied cytokines in endometriosis and control groups assuming the differences observed for il-1ra , il-4 , and il-10 we have built the roc curves and we have calculated areas under the curve for the studied cytokines , showing an area of 0.957 ( 0.029 ) for il-1ra , 0.751 ( 0.070 ) for il-4 , and 0.777 ( 0.061 ) , respectively , for il-10 [ figures 13 ] . comparison between endometriosis and control groups interleukin-1 receptor antagonist values ( pg / ml ) in the left pane and the receiver operating characteristic curve for interleukin-1 receptor antagonist in the right pane comparison between endometriosis and control groups interleukin-4 values ( pg / ml ) in the left pane and the receiver operating characteristic curve for interleukin-4 in the right pane comparison between endometriosis and control groups interleukin-10 values ( pg / ml ) in the left pane and the receiver operating characteristic curve for interleukin-10 in the right pane endometriosis is an inflammatory disease , affecting women of reproductive age , and it is associated with various immune - inflammatory processes and cytokines activation . in the present study , we have found significantly higher serum level of il-1ra , il-4 , and il-10 , compared to healthy controls . we have also showed that there is no significant difference in il-2 , il-13 , and il-15 serum levels between women with endometriosis and women free of the disease . il-2r had a very low detection rate in the studied groups , so we were unable to draw any conclusions regarding its implication in the pathogenesis of endometriosis . cytokines are small proteins involved in intercellular communication , playing an important role in the link between immune and endometriosis tissues . a large number of studies attest a possible implication of pro - inflammatory and anti - inflammatory cytokines in the occurrence and progression of endometriosis . il-1 receptor antagonist is a naturally occurring anti - inflammatory cytokine , with both il-1 alpha and il-1 being the most potent pro - inflammatory cytokines . the effects of il-1 are inhibited by il-1 receptor type 2 ( il-1r2 ) , soluble forms of il-1r2 and il-1ra . elevated serum and pf levels of il-1 and il-1ra were observed in women with endometriosis in comparison with the healthy controls and also higher levels of il-1ra were observed in the early stages of the disease . on the other hand , a study that tried to determine the levels of il-1ra in pf and serum from the patients with endometriosis , showed no significant difference in serum il-1ra between the patients with and without endometriosis , and a lower il-1ra concentrations in pf from the patients with endometriosis than in the patients without endometriosis . at the same time , a study that aimed to investigate if il-1 and il-1ra gene polymorphism could be used as markers of susceptibility in endometriosis found no association of endometriosis with il-1-511 promoter , il-1 exon 5 , and il-1ra gene polymorphisms . a recent study , suggests a possible link between the endometrium , the tissue ectopic endometriosis and endometrioid ovarian cancer with the level expression of il-1 ligands system ( il-1alpha , il-1 , and il-1ra ) . il-4 and il-10 family are the main th2 cytokines , with an anti - inflammatory effect . il-4 is a cytokine with both stimulatory and inhibitory effects on the inflammatory system such as macrophage inhibition and t - cell activation . increased concentrations of il-4 , a typical th2 cytokine , were previously reported in endometriotic tissues , but its role in the pathogenesis of endometriosis is not clear . previous authors have reported the elevated levels of il-4 in endometriosis patients , both in plasma and peripheral blood mononuclear cell supernatants and at the same time , no difference in the concentrations of il-4 in pf between women with and without endometriosis . moreover , no difference was found between the il-4 concentrations in women with different stages of endometriosis . a very recent study investigating serum and pf immunological markers in adolescent girls with chronic pelvic pain , found that adolescents with endometriosis had significantly higher concentrations of serum and pf il-4 and lower pf il-2 compared with the controls , concluding that serum il-4 , peritoneal il-2 , and il-4 are a good method of discrimination between the subjects with endometriosis and controls . in the same line of ideas , a study that aimed to investigate a possible role of il-4 in the development of endometriosis suggested that proliferation of endometriotic stromal cells induced by locally produced il-4 is involved in the development of endometriosis . same authors have reported that il-4 induces eotaxin in ectopic endometrial stromal cells ( escs ) , which might promote angiogenesis and the subsequent development of endometriosis . the anti - inflammatory cytokine il-10 plays an important role in eliminating the unwanted cells and cellular debris in a silent way . il-10 is an acritical anti - inflammatory cytokine that is known to suppress th1-like immune responses and promote th2 responses . the levels of il-10 in pf are significantly increased in the patients with endometriosis compared with controls , and increased il-10 production may partially contribute to the disturbed immune regulation in the patients with endometriosis . a study focused on serum level of il-10 in the patients with endometriosis observed that the serum level of il-10 in the patients with endometriosis was significantly higher than that in healthy subjects or in control subjects with other gynecological disease and at the same time , il-10 administration promoted the growth of endometrial lesions in a murine model . thus , the authors suggested that il-10 may suppress the immunity against the endometrial implants , contributing to the development of endometriosis . also , other authors found decreased serum levels of il-19 and il-22 , but not il-10 , in women with ovarian endometriomas ( oe ) , involving il-10 family in the pathogenesis of endometriosis . moreover , it was shown that the antibody - mediated targeted delivery of il-10 inhibits endometriosis in a syngeneic mouse model . a higher concentration of il-10 was also observed in the patients with ovarian endometriosis when compared to those without this type of disease , as well as when compared to control group patients in a recent study . on the other hand , a recent meta - analysis , this meta - analysis on il-10 genetic polymorphisms associated with endometriosis , suggests that the il-10 -592 a / c polymorphisms conferred susceptibility to endometriosis . moreover , different studies investigating il-2 , il-4 , il-10 , and other inflammatory markers have found higher peritoneal concentrations of pro - inflammatory cytokines ( il-1 , tumor necrosis factor- , and il-6 ) , but also of il-10 and lower concentrations of interferon- , il-1ra , and il-15 in women with endometriosis . no statistical differences were found in il-2 , il-4 , il-12 , and il-13 concentrations . authors concluded that endometriosis development is accompanied by a shift toward th2 immune response at the systemic and local levels , supporting the hypothesis regarding the immune imbalance and autoimmune nature of endometriosis . the values obtained for the areas under the roc curves showed a good discriminative capacity for il-1ra , il-4 , and il-10 , especially il-1ra can be retained as a good test for correctly classify those with and without the disease . our results are in accordance with some of the mentioned studies , but at the same time , the current literature is characterized by contradictory results regarding the involvement of anti - inflammatory th2 cytokines in the pathogenesis of endometriosis . on the other hand , it is highly improbable that one single cytokine , either pro - inflammatory or anti - inflammatory , could be responsible for the occurrence and progression of endometriosis , and further studies focused especially on an association of inflammatory markers could bring some light in the entangled pathogenesis of this disease . one main limitation of our study could be the lack of differentiation between the patients with oe and the patients with deep infiltrating endometriosis ( die ) . oe and die are considered two distinct entities of endometriotic disease , and thus endometriosis can progresses to cystic ovarian disease and pelvic adhesions in some women , and to deeply infiltrating disease in other women , and sometimes to both stages of severe disease in the same woman . studies on pro - inflammatory cytokines showed differences between the patients with oe and die . in consequence , these differences could be present in the relationship with anti - inflammatory cytokines also , and so our results could be influenced by the lack of cleavage of the two forms of the disease in the investigation . almost all of the patients had advanced endometriosis , and in consequence a differentiation in serum levels of the studied markers between the patients with superficial endometriosis and oe or die was not possible . one study has shown that while multiplexed immunoassays have a sensitivity comparable to conventional elisa , it is possible that the robustness may vary among different multiplex bead arrays . due to the nonhomogenous data , the statistical analysis was conducted with the maximum parsimony assuming significance only when both parametric independent t - test for equality of means and the nonparametric mann we have evaluated the differences in main anti - inflammatory cytokines serum levels in the patients with endometriosis compared to the healthy controls using a multiplexed cytokine assay . our study has showed significantly higher serum levels of il-1ra , il-4 , and il-10 in women with endometriosis compared to controls . also , we showed that there is no difference in the serum levels of il-2r , il-13 , and il-15 between women with and without endometriosis . at the same time , our study showed that il-1ra , il-4 , and il-10 could be used as prognostic factors for endometriosis . further studies are needed to shed light on the possible involvement of anti - inflammatory cytokines in the pathogenesis of endometriosis . this paper was funded by  iuliu hatieganu  university of medicine and pharmacy internal grant number 1491/7/28.01.2014 . amm contributed in the conception of the work , study design , conducting the study , participated in analysis and interpretation of data , prepared the article draft , approval of the final version of the manuscript , and agreed for all aspects of the work . td contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . rc contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . rfmh contributed in conducting the study , carried out the analysis and interpretation of data , statistics , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . cb contributed in the acquisition of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . pmr contributed with assistance for the acquisition of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . md contributed in the conception and design of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
background : endometriosis is a frequent gynecologic disease with a severe impact on the quality of life in the affected women ; its pathogenesis is yet to be fully understood , with an altered immunity as a possible key factor . the present study aimed to investigate the serum anti - inflammatory cytokine profile in the patients with endometriosis compared with the healthy controls.materials and methods : 
 one hundred and sixty women were included , divided into two study groups ( group i  endometriosis ; group 2  healthy women ) . 
 we evaluated the serum levels of interleukin-1 receptor antagonist ( il-1ra ) , il-2 , il-2r , il-4 , il-10 , il-13 , and il-15 with the use of human multiplex cytokine panels . 
 statistical analyses ( normality distribution analysis , independent t - test , mann  whitney u - test ) were performed using ibm spss software ( version 22.0 ) and graphpad prism ( version 5.00 ) ; receiver operating characteristic curve were used to demonstrate the diagnostic performance of the studied markers.results:the mean serum level of il-1ra , il-4 , and il-10 were significantly higher in women with endometriosis compared to women free of disease from the control group ( 30.155 , 138.459 , and 1.489 , respectively , compared to 14.109 , 84.710 , and 0.688 , respectively ; p < 0.001 , p < 0.001 , and p = 0.002 , respectively . ) . 
 no significant differences in the mean serum levels of il-2 , il-13 , and il-15 were observed between the studied groups and il-2r had a very low detection rate.conclusion:endometriosis is associated with elevated levels of anti - inflammatory cytokines , il-1ra , il-4 , and il-10 , markers that have a potential role as a prognostic factor for endometriosis .