Predictive Value of Serum YKL-40, Interleukin-37, and Cancer Antigen 125 Panel in Noninvasive Staging of Endometriosis

Endometriosis is an enigmatic progressive disease which is defined as implantation of functional endometrial tissue including both stromal cells and glands outside the uterine cavity.[] The gold standard diagnosis of endometriosis is laparoscopic visualization and staging of lesions and histological confirmation. Thus, the exact diagnosis, staging, and then treatment of endometriosis are often delayed.[] Endometriosis is considered a chronic inflammatory condition with a malignant tumor-like behavior including growth, recurrence, adhesion, and invasion to the adjacent tissues; this can lead to various difficulties during surgical procedure such as injury to the bowel, bladder, and pelvic vessels and organs.[] Therefore, it is critical to accurately evaluate the degree of adhesion and determine the severity of endometriosis before operative procedures for proper selection of surgical method and adequate preparation to reduce the risk of organ damage during operation and guidance of the follow-up treatment.[] There has been much speculation about the role of immune mediators in the pathogenesis of endometriosis, especially cytokines. This was based on the presence of different cytokines in both endometriosis tissue, peritoneal fluid, and high levels of cytokines in serum of endometriosis patients.[,] Many serum markers had been so far investigated and have shown to be associated with endometriosis. Yet, these markers alone are neither sensitive nor specific enough to diagnose and stage endometriosis.[] Cancer antigen 125 (CA 125) is a tumor marker and is the most commonly used biomarker in the diagnosis and follow-up of endometriosis despite its limited sensitivity and specificity as it is elevated in various conditions such as ovarian cancer, endometrial cancer, and pelvic inflammatory disorders.[,] YKL-40, also known as chitinase-3-like protein 1 (CHI3 L1). YKL-40 is a 40 kD plasma glycoprotein and belongs to the mammalian chitinase-like proteins. YKL-40, an inflammatory biomarker, is released by neutrophils and activated macrophages in different tissues and cells with inflammation.[] Several cytokines are known to regulate YKL-40 expression, including interleukin (IL)-6, IL-13, IL-17, and interferon (IFN)-γ.[] Serum concentrations of YKL-40 are elevated in certain conditions compared to healthy subjects such as rheumatoid arthritis,[] osteoarthritis,[] psoriasis,[] and inflammatory bowel disease.[] IL-37 is subclassified to be one of the IL-1 families. It is considered an anti-inflammatory cytokine and is expressed in immune cells such as circulating monocytes, macrophages, dendritic cells, plasma cells, and tonsil B-cells.[] Levels of IL-37 are elevated in response to pro-inflammatory cytokines such as IL-1, TNF-α, IL-18, and IFN-γ. Studies have shown that IL-37 has a role in controlling the inflammatory process in various inflammatory diseases.[] Moreover, some authors reported that the immunostaining score of IL-37 was significantly higher in the eutopic endometrium and ectopic endometrium of women with ovarian endometriosis in comparison to controls without endometriosis.[] The aim of this work is to explore the potential use of serum YKL-40, IL-37, and CA125 for noninvasive staging of endometriosis for proper intervention and the most optimal management of the condition.

Ethical consideration This study was approved by the Ethics Committee of the Faculty of Medicine, Tanta University. All included patients provided written informed consent for the use of biological specimens for research purposes (approval code 36264PR297/8/23). The attached copy of ethical approval was included. Study type About 90 women were selected for this study, who had undergone surgery either laparoscopic or laparotomy surgery due to suspected pelvic endometriosis, infertility, and chronic pelvic pain between January 2021 and January 2023. They were admitted to the Gynecology and Obstetrics Department of Tanta University Hospitals. The diagnosis of endometriosis in selected patients was suspected by clinical findings (infertility or pelvic pain symptoms such as dysmenorrhea and dyspareunia) and imaging findings (transvaginal ultrasonography, computed tomography, or magnetic resonance imaging). Type of sampling and reasons of selection The study was performed on blood samples and postoperative tissue samples. Endometriosis was diagnosed and staged according to the revised American Fertility Society classification by visual inspection of the pelvis by laparoscope or laparotomy and confirmed by histopathological examination afterward. The study was conducted in three groups: Group 1 (initial endometriosis): 30 patients were diagnosed as minimal/mild stage (Stages I–II) of endometriosis Group 2 (advanced endometriosis): 30 patients diagnosed as moderate/severe stage (Stages III–IV) of endometriosis Group 3: 30 women used as controls, free of endometriosis after laparoscopic and histopathological examination. General information such as age, gravidity, parity, body mass index (BMI), existence and duration of infertility, and existence of dysmenorrhea were collected and evaluated. Inclusion criteria Inclusion criteria were as follows: (1) absence of any previous ovarian surgery and (2) absence of any endocrine and autoimmune diseases. Exclusion criteria Exclusion criteria were as follows: (1) presence of pregnancy, (2) any suspicion of malignant ovarian disease, (3) history of oral contraceptive use or any other hormone therapy in the past 3 months, and (4) presence of any nonendometriotic ovarian cyst/mass. Blood samples for serum YKL-40, IL-37, and CA125 assay were taken from all subjects at admission for laparoscopy or laparotomy. All cases were subjected to histopathological evaluation. After confirmation of the diagnosis, the selected cases were immunostained for YKL-40, IL-37, and CA125. Informed consent The authors certify that they have obtained all appropriate consent forms from the subjects. In the form, the subjects have given their consent/assent for clinical and other information to be reported in the journal. The subjects understand that their name and initials will not be published, and due efforts will be made to conceal their identity. Assessment of serum YKL-40, interleukin-37, and cancer antigen 125 levels using enzyme-linked immunosorbent assay Five milliliters of peripheral venous blood was collected from all participants before surgery under complete aseptic conditions into plain sterile tubes. Samples were then centrifuged at 4000 rpm for 10 min. Separated serum was aliquoted and stored at −80°C until enzyme-linked immunosorbent assay (ELISA) assay. YKL-40, IL-37, and CA125 levels were measured in serum samples using sandwich-type ELISA in accordance with the manufacturer’s instruction of each kit. The ELISA kits used were provided by Quidel, CA, USA, for YKL-40 assay, Elabscience, USA, for IL-37 assay, and Calbiotech, USA, for CA125 assay. Histopathological evaluation After operative laparoscope or laparotomy, all excised samples were evaluated histopathologically, tissue biopsies were fixed in formalin, embedded as paraffin blocks, and sections were cut at 4 μm, stained with hematoxylin and eosin. Immunohistochemistry procedure Paraffin-embedded sections were immunostained for YKL-40, IL-37, and CA125. After dewaxing, inactivating endogenous peroxidase activity and blocking cross-reactivity with normal serum. An overnight incubation was done in a humidity chamber with anti-human YKL-40 mouse monoclonal antibody (AT4A3 ac86428, Santa Cruz; dilution 1:100), anti-human IL-37 mouse monoclonal antibody (Abcam, USA; dilution 1/1000), and anti-CA125 (clone M11 diluted 1:50 DAKO: M3520), followed by washing in Phosphate-buffered saline (PBS). Sections were then covered with 4–5 drops of Ultra Vision biotinylated goat anti-polyvalent secondary antibody, incubated at room temperature for 10 min, and then washed in PBS. Finally, sections were counterstained with hematoxylin. As positive controls, sections from colonic tissue (for YKL-40), sections from a lymph node (for IL-37), and sections from a case of endometrial carcinoma (for CA125) were used. Negative controls were prepared by primary antibody with PBS and normal mouse or rabbit serum. Staining analysis Light microscopy examination of all tissue sections was done by three different pathologists. The immunostaining was evaluated according to the following criteria: for all YKL-40, IL-37, and CA125 expressions, according to Kakimoto et al.[] that cytoplasmic staining in endometrial epithelial and stromal cells was considered positive. The staining of all studied markers was categorized as negative (0%) or positive and scored as an overall proportion of stained cells (weak, 50%); quantitative calculation in 10 high-power fields at 400 magnification of tumor tissue was performed using ImageJ analysis software. Statistical analysis of the data Data were fed to the computer and analyzed using IBM SPSS software package version 20.0 (Armonk, NY, USA: IBM Corp). Categorical data were represented as numbers and percentages. Chi-square test was applied to compare between two groups. Alternatively, Monte Carlo correction test was applied when more than 20% of the cells have an expected count <5. For continuous data, they were tested for normality by the Shapiro–Wilk test. Quantitative data were expressed as range (minimum and maximum), mean, standard deviation, and median for normally distributed quantitative variables. Student’s t-test was used to compare two groups while F-test (analysis of variance) was used to compare between more than two groups and post hoc test (Tukey). On the other hand for not normally distributed quantitative variables. Kruskal–Wallis test was used to compare between more than two studied groups, and post hoc (Dunn’s multiple comparisons test) for pairwise comparisons. Receiver operating characteristic curve (ROC) it is generated by plotting sensitivity (TP) on Y axis versus 1-specificity (FP) on X axis at different cutoff values. The area under the ROC curve denotes the diagnostic performance of the test. Area more than 50% gives acceptable performance, and area about 100% is the best performance for the test. The ROC curve allows also a comparison of performance between two tests. The significance of the obtained results was judged at the 5% level.

About 60 women with endometriosis (30 patients with initial endometriosis as Group 1 and 30 patients with advanced endometriosis as Group 2) and 30 healthy women were included in this study. The clinicopathological characteristics of both endometriosis and nonendometriosis groups are shown in Table 1. The median age of the studied groups was 34 for Group 1 and 31.5 years for both Groups 2 and 3. Regarding parity, 24 women with advanced endometriosis in Group 2 (80%) were nulliparous, while only 1 and 2 nulliparous women (3.3% and 6.7%) were included in Groups 3 and 1, respectively. The median BMI of the studied groups was 24.5, 26, and 25.5 kg/m2 for Groups 1, 2, and 3, respectively. The serum levels of YKL-40, IL-37, and CA-125 were significantly higher in Group 2 (median: 195 ng/mL, 222 pg/mL, and 99.5 U/mL, respectively) than Group 1 (median: 99.5 ng/mL, 112 pg/mL, and 32.5 U/mL) and Group 3 (median: 37 ng/mL, 53.5 pg/mL, and 14 U/mL), respectively. All these findings were statistically significant between the three studied groups (P < 0.05) except for age and BMI (P value: 0.724 and 0.350, respectively). The comparison between studied endometriosis Groups 1 and 2 is illustrated in Table 2. According to recurrence, only 7 cases (23.3%) were included in Group 1 while 22 cases (73.3%) were in Group 2. Out of the studied 30 patients in Group 1, 28 patients (93.3%) had laparoscopic surgery; however, 24 patients (80%) in Group 2 had laparotomy due to extensive pelvic adhesions. Complaint of pelvic pain was noticed in 22 (73.3%) and 25 (83.3%) patients in studied Groups 1 and 2, respectively. Regarding site of endometriosis, ovarian endometriosis was the most common site for Group 1 (13 [43.3%] unilateral and 9 [30%] bilateral), while deep infiltrating endometriosis was commonly for Group 2 (21 patients [70%]). The percentage of YKL-40, IL-37, and CA125 expression was higher in Group 2 (median: 58.7%, 60.1%, and 47.21%, respectively) in comparison to Group 1 (median: 20.3% and 22.1%, respectively). Besides, the score of intensity for YKL-40 expression and IL-37 expression was mainly +3 in Group 2 (each 76.7%) while the CA125 expression was mainly +2 in Group 2 (56.7%). On the other hand, the score of intensity for studied markers was mainly +1 for Group 1 (70% for both IL-37 and CA125 and 63.3% for YKL-40) [Figure 1]. The serum levels of YKL-40, IL-37, and CA125 were significantly higher in Group 2 than Group 1. All these findings were statistically significant between the three studied groups (P < 0.001) except for pelvic pain (P = 0.347). The ROC curve analysis was performed to assess the power of the studied serum markers in discriminating between initial endometriosis (Stages I–II) and advanced endometriosis (Stages III–IV), as illustrated in Table 3 and Figure 2. The cutoff values for serum YKL-40, IL-37, and CA125 for predicting moderate-severe endometriosis were more than 109 ng/mL, 129 pg/mL, and 34 U/mL, respectively. As regards the validity of the studied markers, the validity of serum YKL-40 was with a sensitivity of 80%, specificity of 76.67%, positive predictive value (PPV) of 77.4%, and negative predictive value (NPV) of 79.3% (P < 0.001; area under curve [AUC]: 0.823). For serum IL-37, their validity was with a sensitivity of 80%, specificity of 80%, PPV of 80%, and NPV of 80.1% (P < 0.001; AUC: 0.834). The validity of serum CA125 was with a sensitivity of 60%, specificity of 53.33%, PPV of 56.2%, and NPV of 57.1% (P < 0.096; AUC: 0.625). The validity of the combined use of serum YKL-40 and IL-37 was with a sensitivity of 93.33%, specificity of 96.67%, PPV of 96.55%, and NPV of 93.55% (P < 0.001; AUC: 0.983). Meanwhile, the validity of the combined use of all serum markers together was with a sensitivity of 96.67%, specificity of 100%, PPV of 100%, and NPV of 96.77% (P < 0.001; AUC: 0.982).

Endometriosis is a chronic, progressive condition, and its incidence among infertile women at childbearing age is as high as 40%.[] The diagnosis of advanced endometriosis remains challenging, as even the recommended standard, namely laparoscopy, has drawbacks such as invasiveness and high costs.[] This motivates many researchers to find inexpensive, noninvasive biomarkers for appropriate endometriosis staging. The development of the disease is associated with complex inflammatory processes, the immunological determinants of which may be detected in peripheral blood.[] Some inflammatory cytokines may directly or indirectly support the implantation and progression of endometrial tissue implants.[] Thus, a well-defined panel of inflammatory biomarkers could be a crucial tool to early detect women with advanced endometriosis. In this study, we investigated the potential utility of serum YKL-40, IL-37, and CA-125 as noninvasive staging tools in endometriosis patients. Our findings revealed that serum YKL-40, IL-37, and CA-125 levels were significantly higher in patients with endometriosis than healthy controls. These findings were in agreement with other studies.[] As regards the relationship between the studied markers and the degree of severity of endometriosis, we observed a significant increase in serum levels of YKL-40 in patients with moderate-severe endometriosis (Stages 3–4) as compared to those with minimal-mild endometriosis (Stages 1–2). In the same way, we also found that YKL-40 expression in endometriotic lesions using immunohistochemistry was significantly higher in advanced endometriosis than initial endometriosis. Supporting these results, a previous study reported that higher levels of serum YKL-40 were positively correlated with the severity of endometriosis.[] Kim et al.[] showed a significant association between YKL-40 overexpression immunohistologically and the severity of peritoneal endometriosis. YKL-40 is a nonspecific inflammatory marker and is released by neutrophils and activated macrophages in various tissues during inflammation.[] The significant association between YKL-40 and the stage of endometriosis could be hypothesized to the elevation of YKL-40 during inflammation, as pelvic inflammation exclusively stands as one of the major pathological features of endometriosis.[] On the other hand, a study by Kokot et al.[] reported that lower concentrations of serum YKL-40 in Stages III and IV of endometriosis were observed; however, they were insignificant. The discrepancies in YKL-40 concentration values obtained in various studies could be attributed to the differences in the tests used, including their sensitivity and specificity. In the present study, serum levels of IL-37 as well as its immunohistochemical expression were significantly increased in patients with advanced endometriosis compared to patients with early endometriosis. These findings were consistent with Ragab et al.[] and Kaabachi et al.[] who reported that IL-37 serum levels and IL-37 mRNA expression in peritoneal fluid cells were significantly elevated in late-stage endometriosis than early stages. These findings altogether with significantly low IL-37 concentrations in healthy subjects contrary to both endometriosis groups may suggest that IL-37 concentration slowly increases from the beginning of the disease (early stages) and reaches the maximum level when endometriosis is regarded as severe.[] The above observation indicates that IL-37 may be a good marker of disease progression. Nevertheless, the hypothesis needs to be confirmed by future studies. IL-37 is mainly an anti-inflammatory cytokine and a natural suppressor of inflammatory conditions.[] Past studies reported the association of high IL-37 expression with chronic inflammatory conditions, autoimmune disorders, and cancers.[] The anti-inflammatory role of IL-37 might explain its significant association with the severity of endometriosis as IL-37 attempts to suppress the inflammatory process in the disease. As a result, it is possible that IL-37 could play a potential role in the immune response that occurs in endometriosis.[] CA125 is the most commonly used and studied biomarker for endometriosis. However, it was reported that its specificity and sensitivity were not sufficient enough for screening and follow-up of the disease.[] Our study revealed increased serum CA125 levels in moderate-severe endometriosis cases compared to minimal-mild cases. This was in accordance with previous studies.[,,] In contrast, Tuten et al.[] reported no significant association between serum level of CA125 and the severity of endometriosis. This difference may be due to different inclusion criteria in their study. Using ROC curve analysis, serum YKL-40, IL-37, and CA125 were assessed and compared for their ability to predict endometriosis stage and severity. Serum YKL-40 had a sensitivity of 80.0% and a specificity of 76.67%. Serum IL-37 had shown a sensitivity of 80.0% and a specificity of 80.0%. However, serum CA125, as a single marker, had the lowest sensitivity and specificity (60.0% and 53.33%, respectively) compared to YKL-40 and IL-37. Furthermore, the double combination panel of serum YKL-40 and IL-37 had a sensitivity of 93.33% and a specificity of 96.67%. The integration of the three studied markers increased the validity in order to predict the severity of the endometriosis. Indeed, the triple combination panel of serum YKL-40, IL-37, and CA125 had the highest sensitivity and specificity (96.67% and 100.0%, respectively) in our study to determine the severity of endometriosis.

The current study demonstrated that serum levels of YKL-40, IL-37, and CA125 were significantly higher in patients with endometriosis than controls. Moreover, YKL-40, IL-37, and CA125 levels in serum and their immunohistochemical expression were significantly associated with the severity of endometriosis. A triple combination panel of serum YKL-40, IL-37, and CA125 was found to have the best sensitivity and specificity compared to any single serum marker alone or double combination panel in prediction of advanced endometriosis. This triple panel could be a promising clinical tool in the future for noninvasive staging and predicting the degree of pelvic adhesion in endometriosis patients, providing a practical reference for making better-informed decisions on the best management plan for the condition. Rationale of the study The diagnosis of advanced endometriosis remains challenging with considerable limitations in the diagnosis of retroperitoneal and deep infiltrating lesions. Thus, a well-defined panel of inexpensive, noninvasive inflammatory biomarkers could be a crucial tool for appropriate staging for women with suspected endometriosis. Outcomes of the study Serum levels of YKL-40, IL-37, and CA125 were significantly higher in patients with endometriosis than controls YKL-40, IL-37, and CA125 levels in serum and their immunohistochemical expression were significantly associated with the severity of endometriosis Triple combination panel of serum YKL-40, IL-37, and CA125 was found to have the best sensitivity and specificity compared to any single serum marker alone or double combination panel in prediction of advanced endometriosis Triple panel could be a promising clinical tool in the future for noninvasive staging and predicting the degree of pelvic adhesion in endometriosis patients, providing a practical reference for making better-informed decisions on the best management plan for the condition.

of the study This work is limited to the use of triple combination panel of serum YKL-40, IL-37, and CA125 in noninvasive staging of endometriosis in the studied patients; however, further studies are needed on larger scale to guarantee their application in all suspected cases with endometriosis before any intervention. Data availability statement All data underlying the results are available as part of the article and no additional source data are required. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

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