May serum advanced oxidation protein products levels be diagnostic markers in endometrioma?

This single-center, prospective case-control study was conducted between 2019 and 2021 at a university hospital and included women aged 25 to 42 years who underwent laparoscopic surgery for benign adnexal indications. Eligible participants were women of reproductive age undergoing laparoscopy who were diagnosed with ovarian endometrioma based on intraoperative visualization and histopathological confirmation, or with a simple ovarian cyst confirmed as benign serous cystadenoma. In addition, patients who underwent laparoscopic tubal ligation with normal pelvic anatomy and no evidence of endometriosis were included as controls. Patients were excluded if they were pregnant, had a history of previous pelvic surgery or pelvic inflammatory disease, had a history or suspicion of malignancy (including ovarian cancer), or suffered from chronic rheumatologic or systemic diseases, particularly hepatic disorders. Active smokers and those who did not provide written informed consent were also excluded. These inclusion and exclusion criteria ensured that all participants were comparable in age and reproductive status and that the study population was free from confounding systemic inflammatory or malignant conditions. The study protocol and data collection procedures were approved by the local institutional review board (Ankara Bilkent City Hospital No. 2 Clinical Research Ethics Committee; Decision No: E2-21–90). Written informed consent was obtained from all participants prior to enrollment. All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All data were fully anonymized before analysis. Peripheral venous blood samples were collected preoperatively from all participants. Samples were centrifuged at 2,000 rpm for 10 min, and the serum was stored at − 20 °C until analysis. Advanced oxidation protein product (AOPP) levels were determined spectrophotometrically using Witko’s method. Briefly, 200 µL of serum was diluted 1:5 in PBS, followed by the addition of 10 µL of 1.16 M potassium iodide and 20 µL of acetic acid. The absorbance was read immediately at 340 nm against a blank. Chloramine T was used as a calibrator, and results were expressed as µmol/L chloramine T equivalents. A post hoc power analysis was conducted using G*Power version 3.1 to verify the adequacy of the study sample. Based on the primary comparison between the endometrioma ( n  = 51) and combined comparator groups ( n  = 82), the effect size (Cohen’s d = 0.73) was derived from AOPP values, with standard deviations estimated from interquartile ranges using the formula SD ≈ IQR/1.349. Using a two-tailed test at α = 0.05, the achieved power was 0.95, indicating that the sample size was sufficient to detect meaningful differences. Continuous variables were expressed as mean ± standard deviation (SD). The normality of data distribution was assessed using the Shapiro–Wilk test. Associations between categorical variables were evaluated using the Chi-square test, while continuous variables were compared using Student’s t-test. Differences between the three groups were analyzed using one-way ANOVA followed by Bonferroni post hoc tests. Logistic regression with backward stepwise elimination was used to identify independent risk variables and calculate odds ratios (ORs). A p-value < 0.05 was considered statistically significant. All analyses were performed using SPSS version 25.0 (IBM Corp., Chicago, IL, USA).

Comparison of demographic and clinical characteristics among the endometrioma group ( n  = 51), the simple cyst group ( n  = 40), and the control group ( n  = 42) revealed no statistically significant differences in terms of age, body mass index (BMI), or age at menarche. However, the endometrioma group had a lower gravidity than the other two groups. The mean volume of the adnexal mass was significantly higher in the endometrioma group compared to the simple cyst group; adnexal masses were not observed in the control group. Serum levels of CA 125 and CA 19 − 9 were also significantly elevated in the endometrioma group. Furthermore, the levels of both calprotectin and advanced oxidation protein products (AOPPs) were markedly increased in patients with endometrioma (165.07 ± 33.25 µmol/L and 10.42 ± 3.62 µmol/L, respectively), compared to the corresponding values in the simple cyst and control groups (Table  1 ). Table 1 Comparison of study groups by demographics and biomarkers Variables Endometrioma ( n  = 51) Simple cyst ( n  = 40) Control ( n  = 42) p Post hoc Comprasions p value Endo-Cyst Endo-Ctrl Cyst- Ctrl Age 37.00 ± 7.97 37.56 ± 10.50 37.18 ± 4.30 0.95a BMI 27.32 ± 5.76 26.13 ± 3.76 27.13 ± 4.32 0.50a Menarche age 12.89 ± 1.18 13.28 ± 1.31 12.81 ± 0.82 0.17a Gravidy 2(3) 2(3) 4(1) < 0.001b 0.288 < 0.001 0.008 Mass (cm) 7.45 ± 2.53 6.09 ± 1.44 - 0.556 Ca 125 66.50(111.7) 16.9(20.7) 20.3 (7.7) < 0.001b < 0.001 < 0.001 1 Ca 19 − 9 18.2(26.2) 6.8(10.5) 5.7(3.6) < 0.001b 0.025 < 0.001 0.377 Calprotectin 52.75(49.92) 42.79(27.53) 36.44(33.9) 0.003 0.021 0.006 1 AOOP 8.77(11.38) 5.05(4.56) 4.16(3.83) < 0.001 0.008 < 0.001 0.820 AOOP Advanced oxidation protein products, BMI Body mass index, IQR Interquartile range, kg kilogram, m2 square meter, n number, SD Standard deviation. Data are expressed as mean±SD, median and interquartile range (IQR), or number (percentage) where appropriate. Endo: Endometrioma group; Cyst: Simple cyst group; Ctrl: Control group.A p value of <0.05 indicates a significant difference. Statistically significant p-values are in bold and asterisk Comparison of study groups by demographics and biomarkers AOOP Advanced oxidation protein products, BMI Body mass index, IQR Interquartile range, kg kilogram, m2 square meter, n number, SD Standard deviation. Data are expressed as mean±SD, median and interquartile range (IQR), or number (percentage) where appropriate. Endo: Endometrioma group; Cyst: Simple cyst group; Ctrl: Control group.A p value of <0.05 indicates a significant difference. Statistically significant p-values are in bold and asterisk Initially, the study population consisted of three groups: endometrioma, simple cyst, and control. However, because there were no statistically significant differences between the simple cyst and control groups in terms of serum CA125, CA19-9, calprotectin, and AOPP levels, these two groups were combined. This merged group was used as a single comparator in the binary logistic regression analysis. Consequently, binary logistic regression was performed to identify factors associated with the presence of endometrioma, using the combined simple cyst and control group as the reference. In univariate logistic regression analysis, chronic pelvic pain (OR: 0.126, 95% CI: 0.047–0.338, p  < 0.001) was significantly associated with lower odds of endometrioma. This association remained statistically significant in the multivariate model (aOR: 0.160, 95% CI: 0.043–0.595, p  = 0.006). Additionally, higher serum levels of CA125 (OR: 1.017, 95% CI: 1.006–1.027, p  = 0.002), calprotectin (OR: 1.005, 95% CI: 1.001–1.008, p  = 0.005), and AOPP (OR: 1.223, 95% CI: 1.112–1.346, p  < 0.001) were positively associated with the presence of endometrioma. Bilaterality also showed a statistically significant association (OR: 0.231, 95% CI: 0.065–0.827, p  = 0.024), while infertility demonstrated borderline significance (OR: 0.125, 95% CI: 0.013–1.161, p  = 0.067). In the multivariate logistic regression model, calprotectin (aOR: 1.004, 95% CI: 1.000–1.008, p  = 0.037), and AOPP (aOR: 1.238, 95% CI: 1.095–1.399, p  = 0.001) remained statistically significant independent predictors of endometrioma. Other variables, including age, BMI, smoking status, bilaterality, and CA19-9, did not retain significance in the adjusted model (Table  2 ). Table 2 Binary logistic regression analysis of predictors for endometrioma group membership p OR 95% CI Lower- Upper p aOR 95% CI Lower- Upper Age (years) 0.807 0.994 0.947–1.043 BMI 0.426 1.034 0.952–1.124 Smoking 0.589 0.777 0.310–1.944 Chronic pelvic pain < 0.001 0.126 0.047–0.338 0.006 0.160 0.043–0.595 Infertility 0.067 0.125 0.013–1.161 Bilaterality 0.024 0.231 0.065–0.827 0.717 1.416 0.216–9.301 CA125 (U/mL) 0.002 1.017 1.006–1.027 0.024 1.012 1.002–1.023 CA19-9 (U/mL) 0.217 1.003 0.998–1.009 Calprotectin (ng/mL) 0.005 1.005 1.001–1.008 0.037 1.004 1.000–1.008.000.008 AOPP (µmol/L) < 0.001 1.223 1.112–1.346 0.001 1.238 1.095–1.399 AOOP  Advanced oxidation protein products, BMI  Body mass index. A p value of < 0.05 indicates a significant difference. Statistically significant p -values are in bold and asterisk Binary logistic regression analysis of predictors for endometrioma group membership AOOP  Advanced oxidation protein products, BMI  Body mass index. A p value of < 0.05 indicates a significant difference. Statistically significant p -values are in bold and asterisk Receiver operating characteristic (ROC) curve analyses were performed for CA125, CA19-9, calprotectin, and AOPP. The area under the curve (AUC) was calculated to be 0.864 for CA125, 0.721 for CA19-9, 0.693 for calprotectin, and 0.732 for AOPP. The optimal cut-off values were identified as 34.7 for CA125, 16.6 for CA19-9, 44.02 for calprotectin, and 6.79 for AOPP. At these thresholds, the sensitivity and specificity were 79.5% and 88.7% for CA125; 59.0% and 90.1% for CA19-9; 77.5% and 60.3% for calprotectin; and 69.2% and 74.6% for AOPP, respectively. The corresponding Youden index values were 0.682 for CA125, 0.491 for CA19-9, 0.375 for calprotectin, and 0.439 for AOPP (Fig.  1 ; Table  3 ). Fig. 1 Comparison of ROC Curves for CA125, CA19-9, Calprotectin, and AOPP Comparison of ROC Curves for CA125, CA19-9, Calprotectin, and AOPP Table 3 Summary of ROC analysis parameters for CA125, CA19-9, Calprotectin, and AOPP Marker AUC Cut-off Sensitivity (%) Specificity (%) Youden Index LR⁺ LR⁻ CA125 0.864 34.7 79.5 88.7 0.682 7.04 0.23 CA19-9 0.721 16.6 59.0 90.1 0.491 5.96 0.46 Calprotectin 0.693 44.02 77.5 60.3 0.375 1.95 0.37 AOPP 0.732 6.79 69.2 74.6 0.439 2.72 0.41 AOOP  Advanced oxidation protein products, LR⁺  Positive likelihood ratio, LR  Negative likelihood ratio Summary of ROC analysis parameters for CA125, CA19-9, Calprotectin, and AOPP AOOP  Advanced oxidation protein products, LR⁺  Positive likelihood ratio, LR  Negative likelihood ratio

The demographic and clinical characteristics of the study groups were largely comparable in terms of age, BMI, and age at menarche. However, gravidity was significantly lower, and adnexal mass volume significantly higher, in the endometrioma group compared to the simple cyst and control groups. Moreover, serum levels of CA125, CA19-9, calprotectin, and AOPP were markedly elevated in the endometrioma group. Based on the absence of statistically significant differences between the simple cyst and control groups for these biomarkers, these two groups (simple cyst + control) were merged into a single comparator group (n:82) for the logistic regression analysis. The multivariate analysis identified serum levels of AOPP, calprotectin, and CA125 as independent predictors of endometrioma, while chronic pelvic pain was inversely associated with the presence of endometrioma. Among the markers analyzed, CA125 demonstrated the highest diagnostic accuracy in the ROC analysis, followed by AOPP and calprotectin. These findings highlight the potential utility of combining oxidative stress and inflammatory biomarkers in the non-invasive, preoperative diagnosis of endometrioma, reflecting the underlying pathophysiological mechanisms of the disease. Endometriosis is now increasingly recognized not merely as a localized pelvic condition but as a chronic systemic inflammatory disease with multisystemic manifestations. Traditionally defined by the ectopic presence of endometrial-like tissue outside the uterus, endometriosis commonly involves the ovaries (as endometriomas), peritoneum, and deep pelvic structures, but can also manifest in extra-pelvic sites, highlighting its complex pathogenesis and clinical spectrum [ 2 ]. The etiology of endometriosis remains multifactorial and incompletely understood. Several hypotheses have been proposed, including retrograde menstruation, stem cell implantation, coelomic metaplasia, and genetic-epigenetic interactions, all of which suggest a dysregulated interplay of hormonal, immunologic, and environmental factors in disease onset and progression [ 14 ]. Among these, retrograde menstruation has long been the most widely accepted theory; however, it fails to explain the presence of endometriosis in locations distant from the pelvis or in individuals without a uterus. More recent studies emphasize the roles of bone marrow-derived progenitor cells, altered immune cell function, and microRNA dysregulation, which collectively contribute to lesion survival, systemic inflammation, pain sensitization, and progesterone resistance [ 2 ]. The diagnosis of endometriosis remains a significant clinical challenge. The condition frequently presents with nonspecific symptoms such as chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility—features that are commonly seen in various other gynecological and systemic disorders [ 15 ]. Furthermore, visual inspection via laparoscopy with histological confirmation has traditionally been considered the gold standard. However, recent consensus supports a clinical diagnosis based on symptoms and imaging findings, particularly to avoid delays in initiating treatment. According to Crump et al. (2024), transvaginal ultrasound and MRI have become key non-invasive tools for detecting endometriomas and deep infiltrating disease, although they may still fail to identify superficial lesions. In our study, although age, BMI, and age at menarche were similar among the three groups, patients with endometrioma had significantly lower gravidity. Similarly, in a national population-based study conducted by Farland et al., the rate of nulliparity with endometriosis was also found to be higher [ 16 ]. Notably, Ranjan et al. emphasized that nulliparity is both a risk factor for and a clinical correlate of endometriosis, particularly in the context of infertility-related diagnoses [ 17 ]. In our study, CA125 and CA19-9 levels were found to be significantly higher in the endometrioma group. Among these, CA125 demonstrated the highest diagnostic accuracy, with an AUC value of 0.864. According to the literature, CA125 is a widely used biomarker for the diagnosis of endometrioma, with a reported sensitivity of 53.4% and specificity of 96.1% [ 18 ]. Similarly, it has been reported that CA19-9 is significantly elevated in endometrioma cases and is correlated with disease severity. The combined use of these markers may improve diagnostic accuracy [ 19 ]. Emerging evidence highlights the oncological relevance of endometriomas, suggesting that chronic inflammation and oxidative stress within the endometriotic microenvironment may promote malignant transformation. Recent studies have identified distinct subtypes of endometriosis-associated ovarian cancer (EAOC), differentiating between endometriosis-correlated and incidental carcinomas based on the presence of transitional atypical lesions, as well as recognizing mesonephric-like adenocarcinoma as a newly defined, aggressive histotype frequently associated with endometriosis [ 20 ]. These findings underscore the importance of careful long-term surveillance in women with endometriomas, particularly those with large, recurrent, or atypical cysts, given their potential risk for oncogenic progression [ 21 ]. Calprotectin is an inflammatory marker belonging to the S100 protein family and has been shown to be elevated in chronic inflammatory diseases, particularly rheumatoid arthritis. Calprotectin also acts as a link between intestinal and pelvic inflammation, as gut microbiome alterations in women with endometriosis may contribute to immune dysregulation and estrogen-dependent lesion growth [ 22 ]. Serum and plasma levels have been reported to correlate with disease activity indices and to decrease in response to treatment, supporting its role as a sensitive indicator of systemic inflammation [ 23 ]. In our multivariate analysis, CA125, calprotectin, and AOPP were identified as independent predictors of endometrioma, consistent with the literature emphasizing the role of inflammation and oxidative stress in the pathogenesis of endometriosis. Elevated CA-125 levels are often seen in endometriosis due to peritoneal inflammation, but similar increases may occur in benign conditions [ 24 ]. Iavarone et al. (2023) likewise reported high CA-125 levels in Meigs syndrome, a benign adnexal pathology mimicking ovarian cancer [ 25 ]. This supports that CA-125 and oxidative stress–related markers can rise in non-malignant inflammatory states, as shown in our study. Similarly, the high levels of calprotectin observed in our study suggest that it may be an indicator of neutrophil-related inflammation in endometriosis. It has been reported that serum calprotectin levels are significantly higher in PCOS patients than in healthy individuals, and this elevation may be related to inflammatory processes contributing to the disease’s pathogenesis [ 26 ]. Plasma calprotectin levels were also found to be significantly higher in patients with endometrial carcinoma, with this increase being associated with disease stage and survival, suggesting that calprotectin may serve as a potential biomarker linked to tumor progression through inflammation [ 27 ]. In this study, serum AOPP levels were identified as an independent predictor of endometrioma (aOR = 1.238, p = 0.001). Previous studies have also reported that AOPP is an oxidative stress marker implicated in endometriotic pathologies. AOPP has been shown to promote proliferation and inhibit apoptosis by activating mitogenic pathways such as ERK and p38 MAPK in rat-derived endometrial epithelial cells [ 28 ]. These findings indicate that inflammation and oxidative stress play a significant role in the pathogenesis of endometriosis. AOPP may serve as a potential biomarker, warranting further investigation, as evidenced by an AUC of 0.732. Our results are consistent with previous studies reporting significantly elevated AOPP levels in the peritoneal fluid of women with endometriosis. In that study, the increase was particularly notable in cases of deep infiltrative endometriosis (DIE) with intestinal involvement [ 29 ]. Similarly, it was shown that AOPP levels were higher in the follicular fluid of infertile women with endometriosis compared to the control group [ 12 ]. These findings support that AOPP may be associated with disease severity and systemic oxidative burden. In addition, recent studies have explored the potential of circulating microRNAs detected through liquid biopsy as novel non-invasive tools for early diagnosis. Ronsini et al. reported that dysregulated miRNAs such as miR-145, miR-21-5p, and miR-200c show promising diagnostic accuracy, suggesting that molecular assays could complement traditional biochemical markers in identifying early or subclinical endometriosis [ 30 ]. Calprotectin also stood out as an independent marker in our study, although its discriminatory power (AUC: 0.693) was slightly lower than that of AOPP. This finding aligns with previous evidence indicating that calprotectin, a neutrophil-derived inflammatory mediator, may contribute to the inflammatory processes in endometriosis [ 23 ]. Although a recent cross-sectional study reported no correlation between calprotectin levels and disease stage, the elevated serum levels in our study suggest its potential relevance, particularly in cases of symptomatic or active disease [ 31 ]. Limitations of this study include the relatively small number of patients and the fact that it was a single-center study. It should also be noted that biomarker levels may be affected by variables such as menstrual cycle phase and other inflammatory conditions. The lack of confirmatory analyses at the tissue level also limits biological interpretation. Although all participants underwent laparoscopic evaluation to exclude visible endometriotic lesions, and histopathological confirmation was performed in cases with simple ovarian cysts, the presence of microscopic or subclinical foci cannot be completely ruled out. This study revealed that AOPP and calprotectin levels may have potential diagnostic value, but further validation in larger, multicenter cohorts is needed to confirm their clinical utility. CA125 had the highest diagnostic performance (AUC = 0.864), while AOPP also demonstrated a notable diagnostic accuracy (AUC = 0.732). Although calprotectin showed a lower AUC (0.693), the analysis suggested that it may have an independent predictive role. These findings support the role of inflammation and oxidative stress in the pathogenesis of endometriosis and indicate that these biomarkers may aid in its noninvasive diagnosis. The strengths of this study include its prospective design, histopathological confirmation of all cases, and the inclusion of both benign and healthy controls, which ensured accurate group classification and reduced diagnostic bias. Furthermore, it is one of the few studies to simultaneously assess AOPP and calprotectin, providing novel evidence on their combined diagnostic potential. However, larger sample sizes and multicenter studies are required to validate these results.

Endometriosis is a common chronic gynecological disease that affects 6–10% of women of reproductive age and is characterized by the growth of endometrial stroma and glands outside the uterine cavity [ 1 ]. Typical locations include the ovaries, pelvic peritoneum, uterosacral ligaments, and rectouterine sac, but may also involve many parts of the body [ 2 ]. Affected women often present with chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility; however, the clinical symptoms of endometriosis can be very heterogeneous [ 3 ]. To date, intraoperative visualization is considered the most accurate method for diagnosing endometriosis [ 4 ]. Non-surgical diagnostic approaches, such as blood tests, transvaginal sonography, and pelvic MRI—although frequently used—cannot replace surgery, which remains the gold standard for diagnosing endometriosis, due to their limited sensitivity and specificity [ 5 ]. Many studies have shown that the mean time between the onset of symptoms and a definitive (surgical) diagnosis is approximately 10.4 years [ 6 ]. Since endometriosis is a leading cause of disability and reduced quality of life in women and girls, shortening the time to diagnosis will clearly improve endometriosis management and enhance the cost-effectiveness of treatment [ 7 ]. Therefore, there is an urgent need for the development and implementation of a rapid, non-invasive test with high sensitivity and specificity. Recently, many serum and urinary markers have been studied for this purpose, but a test that can be used widely and reliably in daily clinical practice has not been developed yet [ 8 , 9 ]. Studies have revealed that endometriosis involves an inflammatory process and increased oxidative stress in affected patients. Based on these findings, some literature suggests that antioxidant treatments may be beneficial [ 10 ]. AOPPs are dityrosine-containing protein cross-linking products formed by the action of chlorinated oxidants, particularly hypochlorous acid and chloramines, generated by activated neutrophils and monocytes under oxidative stress conditions. They are considered reliable markers of protein oxidation and serve as mediators of oxidative stress–induced inflammation by stimulating NADPH oxidase and triggering downstream signaling pathways such as NF-κB, ERK1/2, and p38 MAPK [ 11 ]. Gynaecological conditions like endometriosis, uterine fibroids, and polycystic ovary syndrome have been linked to elevated AOPP levels. In gynecologic disorders, elevated AOPP concentrations have been reported in the serum, peritoneal, and follicular fluids of women with endometriosis, correlating with disease severity and infertility [ 12 ]. According to recent in vitro and in vivo research, AOPPs can activate the ERK and P38 MAPK signalling pathways, causing endometrial epithelial cells to proliferate and migrate while preventing apoptosis [ 13 ]. These findings suggest that AOPPs not only reflect systemic oxidative stress but may also actively contribute to the pathogenesis of endometriosis through enhanced cellular proliferation, migration, and apoptosis resistance. In the current study, we aimed to evaluate the diagnostic accuracy of serum levels of AOPP, calprotectin, CA-125, and CA19-9 in patients with endometrioma.