Role of cystatin C levels as an inflammatory marker in predicting endometriosis

Endometriosis, which is a common chronic inflammatory disease associated with infertility and pelvic pain, is characterized by the presence of endometrium-like glands and tissues outside the uterus 1 . Currently, diagnosis relies on visualizing endometriotic lesions during surgery, as there is no reliable serum marker available 2 . Moreover, the origin of endometriosis is still largely unknown 1 . Therefore, medical history and biochemical markers were investigated together with ultrasonographic methods as an alternative to invasive methods in diagnosis 3 . Although the role of various gene expressions has been demonstrated in recent studies, serum biomarkers (e.g., TNF-α, IL-6, IL-1β, CA-125, and CA 19-9) remain uncertain as suitable candidates for non-invasive methods, even though they may be suitable candidates for non-invasive methods 4 . Furthermore, one study revealed an increased prevalence of endometriosis during the severe acute respiratory syndrome coronavirus 2 pandemic, raising more questions regarding the etiogenesis of endometriosis 5 . Cystatin C is a cysteine protease inhibitor produced by all nucleated cells. It is thought that cystatin C reduces endogenous cysteine protease and neutrophil migration activity in the inflammatory process 6 . Cystatin C is an important marker, especially in demonstrating kidney function and glomerular filtration rate 7 . However, recent studies have revealed the significance of cystatin C in various fields. In addition to studies suggesting that it is an early predictor of cardiovascular diseases 8 , some studies show that it can be a good biomarker for cerebrovascular diseases and peripheral vascular diseases 9 . Furthermore, studies have reported that cystatin C shows renal damage in patients with preeclampsia 10 . However, as cystatin C is considered to be a predictor of inflammation, it has been shown to be associated with malignancies. Cystatin C has been shown to be associated with various malignancies, in particular, urogenital malignancies 11 . However, no study in the literature investigates whether it is a suitable biomarker for endometriosis. In this respect, this study is significant in that it is the first of its kind and will make a valuable contribution to the literature. Hence, the aim of our study was to search for a non-invasive method that could assist in the diagnosis of endometriosis, the etiogenesis of which is still unclear, and to investigate the role of cystatin C in predicting endometriosis.

Our study included 90 women, 45 of whom were volunteer patients between the ages of 18 and 40 years who applied to the city hospital endometriosis polyclinic between January 2022 and June 2022, and the other 45 patients were in the control group who applied to the gynecology polyclinic. Our study was conducted in accordance with the Declaration of Helsinki and in compliance with the country's ethical standards. Ethics committee approval was obtained from the same hospital (21/1046). An informed consent form was signed by all patients. Endometriosis patients were selected using the revised American Fertility Society classification as patients who had undergone surgery for pelvic pain or infertility and whose pathology results were compatible with endometriosis. The control group was selected from healthy women volunteers between the ages of 18 and 40 years without infertility and no additional diseases. Patients who were pregnant and had gynecological comorbidities, active infections, kidney disease, cardiovascular disease, cerebrovascular disease, malignancy, and chronic autoimmune diseases were not included in the study. The demographic characteristics, obstetric histories, body mass index (BMI) values, ultrasonographic findings, physical examination findings, pathology results, and serum biochemical and hormonal parameters (hemoglobin (Hb), white blood cell (Wbc), neutrophil, lymphocyte, sodium, potassium, AST, ALT, urea, creatinine, hs-CRP, procalcitonin, CA-125, anti-Müllerian hormone (AMH), and cystatin C levels) of each patient were recorded. When calculating BMI, the patients’ height and weight were measured, and it was calculated using the formula: BMI=weight (kg)/height (m) 2 . All these parameters were compared between the endometriosis and control groups. Cystatin C levels were measured using a commercial ELISA kit (Elabscience, Elabscience Biotechnology Co. Ltd. Wuhan, P.R.C., Catalog No: E-EL-H3643, LOT: ER04688F5606). The measurement range is 0.31–20 ng/mL. Its sensitivity is 0.19 ng/mL, and the intra-assay and inter-assay %CV values are <10%. In the endometriosis group, serum cystatin C levels were taken preoperatively. Blood samples were collected in yellow-capped, vacuum-sealed, plastic gel tubes from both the endometriosis and control groups between 8:00 a.m. and 12:00 p.m. after 12 h of fasting. Statistical analyses were performed using SPSS version 22. The conformity of the variables to the normal distribution was examined using visual (histogram and probability graphs) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk tests). Descriptive analyses were performed using the mean and standard deviations for normally distributed variables. The means of parametric data determined by Levene's test, which showed normal distribution, were compared using Student's t-test. The Mann-Whitney U test was used to compare the parametric and ordinal data, which were determined not to be normally distributed. The presence of correlation between parametric data was tested using the Pearson test, and the presence of correlation between nonparametric and non-normal distributed data was tested using the Spearman test. Categorical data were compared using chi-square or Fisher's exact test (where values observed in cells did not meet the chi-square test assumptions) as appropriate. Cases with a p<0.05 were considered statistically significant. The role of cystatin C in predicting endometriosis was investigated using the ROC curve analysis method.

The comparison of demographic characteristics and biochemical and hormonal parameters between the endometriosis and control groups is shown in Table 1 . While there was no significant difference between the groups in terms of mean age and BMI, gravida and parity variables were found to be significantly lower in the endometriosis group. When serum cystatin C levels were compared, a statistically significant difference was found between the endometriosis and control cases (p<0.001). Moreover, the hs-CRP (p=0.002) and CA-125 (p<0.001) values of endometriosis patients were found to be significantly higher than those of the control subjects ( Table 1 ). N/L: neutrophil-lymphocyte ratio. According to the ROC curve analysis ( Figure 1 ), the cystatin C level was a discriminating parameter in patients with endometriosis. The area under the curve for cystatin C was 0.92 (0.86–0.98) at 95% confidence interval ( Figure 1 ). The threshold value for cystatin C was 5.14 ng/mL with a sensitivity of 86.7% and a specificity of 77.8%. In the linear regression analysis, it was observed that the probability of endometriosis increased 2.5 times when cystatin C levels exceeded the 5.14 ng/mL threshold (OR: 2.5; 95%CI 2.24–2.76) ( Table 2 ).

This study evaluated the possible association between serum cystatin C levels and endometriosis, and to the best of our knowledge, this is the first study to evaluate the relationship between endometriosis and the proinflammatory marker cystatin C. In our study, serum cystatin C levels were found to be statistically significantly higher in the endometriosis group when compared with the control group (p<0.001). Furthermore, the hs-CRP (p=0.002) and CA-125 (p<0.001) values of endometriosis patients were found to be significantly higher than those of the control subjects. According to the ROC curve analysis, cystatin C levels were a distinctive parameter in patients with endometriosis. The area under the curve for cystatin C was 0.92 (0.86–0.98) at 95%CI. The threshold value for cystatin C was found to be 5.14 ng/mL, with a sensitivity of 86.7% and a specificity of 77.8%. In the linear regression analysis, it was observed that the probability of endometriosis increased by 2.5 times when cystatin C levels increased above the 5.14 ng/mL threshold (OR: 2.5; 95%CI 2.24–2.76). As cystatin C is a protease inhibitor that plays a role in inflammatory processes, it has been investigated in many diseases associated with inflammation 7 – 11 , but research in the field of obstetrics and gynecology is limited. In one meta-analysis, it was demonstrated to be a promising biomarker in the detection of preeclampsia 12 . Additionally, another study reported that serum cystatin C levels in late pregnancy were associated with negative birth outcomes 13 . On the contrary, Zhang et al., suggested that serum cystatin C levels are significantly higher in patients with gestational diabetes mellitus (GDM) compared with the control group 14 . A study reported that increased serum cystatin C levels may be a risk factor for pregnant women with PCOS and GDM 15 . Studies on cystatin C in gynecology in the literature are mostly related to polycystic ovary syndrome (PCOS), and one study stated that cystatin C levels were significantly higher in women with PCOS compared with the control group and could be an important indicator for reducing cardiovascular risks 16 . Moreover, a study conducted in adolescents with PCOS suggested that the risk of PCOS increased 1.556 times when cystatin C increased by one unit and that there was a significant relationship between them 17 . Another study in patients with adolescent PCOS suggested that cystatin C may be a promising indicator in predicting future metabolic risks 18 . Various cytokines and markers have been shown in both the peritoneal cavity and serum in patients with endometriosis 4 , but the question of their role in the development of endometriosis and whether they are the cause or the result of endometriosis has not yet been clearly elucidated. In addition, although there is a difference between superficial and deep endometriosis, studies have shown that biomarkers contribute to the diagnosis of both superficial and deep pelvic endometriosis 4 . Although serum CA-125 is the most studied marker, studies have shown that its diagnostic performance is poor 19 . A meta-analysis conducted by Sokolov et al., stated that other markers such as CA 19-9 and CA 72-4 are more valuable in differentiating endometriosis from other pathologies and may help clarify the effect of circulating micro-RNA in the pathology of endometriosis 20 . Furthermore, a study investigating the role of autoantibodies and enzymes in the diagnosis of endometriosis suggested that autoantibodies against tropomyosin 3, α-enolase, and estradiol could be included in the panel of biomarkers for the non-invasive diagnosis of endometriosis 21 . Another study evaluated the hormonal etiologies of endometriosis and showed that, in rat models, gestrinone antagonizes the effects of estrogen on rat peritoneal endometrial implants when given combined estrogen therapy with gestrinone 22 . In their research, Soto et al., stated that numerous potential biomarkers for non-invasive tests for endometriosis, including glycoproteins, inflammatory cytokines, immune molecules, angiogenesis factors, hormones, microRNAs (miRNAs), proteomics, metabolomics, genomics, and microbiomes, have been investigated. However, they explained that the most promising and progressing areas for the non-invasive diagnosis of endometriosis are miRNAs, proteomics, metabolomics, genomics, and microbiome 23 . A study investigating the genetic origin of endometriosis compared the expression of stem cell-related genes in the endometrium, superficial endometriosis, and deep infiltrating endometriosis. It has been revealed that deep and superficial endometriosis tissues have similar stem cell-related genes; however, there are differences in gene expression between them 24 . Despite all these studies, a recent study stated that more confirmatory studies are required to fully establish these markers in the diagnosis, progression, and staging of endometrial lesions 25 . Many markers have been studied and continued to be investigated for the non-invasive diagnosis of endometriosis. The strengths of this study are as follows: this is the first study in the literature showing the relationship between endometriosis and cystatin C and diagnosis of endometriosis was supported by pathological examination in all patients. The limitations of our study are the small number of participants and its non-randomized design.

Cystatin C levels seem to be a promising non-invasive indicator in predicting endometriosis associated with inflammation.