Clinical value of preoperative serum calcium level in predicting deep infiltrative endometriosis among ovarian endometrioma

Endometriosis, affecting approximately 5–15% of women of reproductive age, is characterized by endometrial tissues abnormally situated outside the uterine cavity [ 1 ]. Lesions are classified into three types: ovarian endometriosis, deep infiltrating endometriosis (DIE), and superficial peritoneal endometriosis [ 1 , 2 ]. Clinical manifestations can vary widely, including dysmenorrhea, chronic pelvic pain, and gastrointestinal symptoms [ 3 ]. No correlation has been established between the lesion type and the specific symptoms [ 4 ]. Surgical treatment is one therapeutic strategy for patients with endometriosis. The rationale for surgical treatment of endometriosis is to achieve the complete removal of all lesions in a one-step surgical procedure, aiming for promising long-term outcomes for pelvic pain, recurrence rate, and infertility. In such contexts, a precise preoperative assessment of concurrent DIE becomes crucial for optimizing surgical strategies and minimizing surgery-related complications. Serum carbohydrate antigen 125 (CA 125) remains the most commonly used biomarker for endometriosis. Although serum CA 125 levels increase in endometriosis, it has limitations in primary diagnosis and does not indicate the invasion depth. Therefore, it is crucial to explore more effective non-invasive preoperative evaluation biomarkers. Calcium, an essential component for various physiological processes [ 5 ], has its intracellular flow regulated by ion channels and pumps—altered expression of which is a feature of tumorigenesis [ 6 ]. Although endometriosis is a benign condition, it exhibits tumor-like biological behaviors. The association between serum calcium and endometriosis, however, remains unclear. Consequently, this study aims to elucidate the clinical utility of serum calcium in the diagnosis of ovarian endometrioma and predicting the presence of DIE among Chinese women.

The patient selection process was detailed in Figure 1 . This study incorporated a total of 2,557 women, of whom 987 (38.60%) were pathologically confirmed with endometriosis, and 1,570 (61.40%) with other benign adnexal diseases such as ovarian teratoma, simple cyst, corpus luteum cyst, serous cystadenoma, and mucinous cystadenoma. As presented in Table 1 , no differences were found in mean age, parity, nationality, or marital status between women with endometriosis (EM) and those without endometriosis(non-EM). Compared with the non-EM group, the EM group had a higher proportion of women with a tertiary education level and a higher proportion of women working in offices. The mean age of patients with endometriosis was 32.5 ± 7.2 years, while the mean age of patients without endometriosis was 32.3 ± 8.3 years, with no significant difference ( p  > 0.05). Flowchart detailing patient selection. rAFS: revised American fertility society (rAFS). Characteristics of included women with benign ovarian tumor. EM: endometriosis; DIE: deep infiltrating endometriosis; BMI: body mass index; OCP: oral contraceptive pills; CA: carbohydrate antigen. Out of the 987 women with endometriosis, 34 (3.44%) were classified as Stage I, 28 (2.84%) as Stage II, 458 (46.40%) as Stage III while 467 (47.32%) were diagnosed as Stage IV of the revised r-ASF classification. The majority of patients (666/987, 67.48%) had DIE, while the remaining 321 (32.52%) had solely ovarian endometriosis. The mean age of patients with DIE was 31.7 ± 6.6 years, which was significantly younger than the mean age of patients without DIE, at 34.1 ± 8.2 years ( p  < 0.001). As shown in Table 1 , the mean CA 125 level of patients in EM group was significantly higher than in patients in non-EM group. The mean adjusted calcium level in the EMs group was 2.28 ± 0.09 g/L, which was significantly lower than in the non-EMs group (2.30 ± 0.09 g/L) ( p  < 0.001) ( Table 1 ). The overall associations between serum adjusted calcium, CA 125 and endometriosis risk are shown in Table 2 . Adjusted serum calcium was inversely correlated with the risk of endometriosis, whereas CA 125 demonstrated a contrasting trend. These associations remained statistically significant even after further adjustments for other important confounding factors, such as age, parity, nationality, marital status, occupation, education, occupation, BMI, serum albumin, and the presence of dysmenorrhea symptom ( Table 2 ). The association of serum adjusted calcium, CA 125 and risk of endometriosis. EM: endometriosis; DIE: deep infiltrating endometriosis; BMI: body mass index; CA: carbohydrate antigen; OR: odds ratio; 95% CI: 95% confidence interval. To assess the accuracy of diagnosing endometriosis, we analyzed the ROC curves for different risk factors and their combinations. Figure 2 showed the ROC curve of adjusted serum calcium, CA 125, and combined marker (derived by multiplying the albumin-adjusted calcium level with the CA125 level). The analysis indicated that the combined marker had the highest diagnostic accuracy for predicting endometriosis (AUC = 0.810, p  < 0.001) ( Table 3 ). Distribution of DIE in endometriosis patients of different age groups. DIE: deep infiltrating endometriosis. AUC and p -value of risk factors for predicting endometriosis. DIE: deep infiltrating endometriosis; CA: carbohydrate antigen; AUC: area under receiver operating characteristic curve; 95% CI: 95% confidence interval. The characteristics of the patients with endometriosis are outlined in Table 1 . No significant differences were observed between the two groups in terms of smoking habits, nationality, occupation, oral conceptive pills (OCP) use, and history of endometriosis. However, patients with DIE exhibited higher proportions of nulliparity, severe dysmenorrhea compared to those without DIE ( Table 1 ). Additionally, the preoperative adjusted calcium level was significantly lower in patients with DIE than in those without DIE. However, no significant difference was observed in the mean CA 125 level between the two groups. Multivariate logistic regression analysis adjusted serum calcium was inversely correlated with the risk of DIE (OR = 0.234, 95% CI: 0.056–0.980, p  = 0.047), which remained statistically significant even after further adjustments for other important confounding factors, such as age, parity, nationality, marital status, occupation, education, occupation, BMI, serum albumin, and the presence of dysmenorrhea symptom ( Table 4 ). The association of serum adjusted calcium, CA 125 and risk of DIE. EM: endometriosis; DIE: deep infiltrating endometriosis; BMI: body mass index; OCP: oral contraceptive pills; CA: carbohydrate antigen; OR: odds ratio; 95% CI: 95% confidence interval. As demonstrated in Table 1 , patients with DIE had a mean age of was 31.7 ± 6.6 years, significantly lower than the 34.1 ± 8.2 years observed in patients without DIE ( p  < 0.001). Multivariate logistic regression analysis identified age as an independent risk factor for DIE (OR = 0.970, 95% CI: 0.944–0.996, p  = 0.024), accounted for potential confounding variables, such as parity, nationality, marital status, occupation, education, OCP use, BMI, serum albumin, serum adjusted calcium, the presence of dysmenorrhea symptom, and history of endometriosis surgery ( Table 5 ). Further analysis involved stratifying endometriosis patients by age groups. The results indicated a negative correlation between age and the prevalence of concurrent DIE, with the prevalence decreasing across age groups (Gamma value = −0.181, p  < 0.001) ( Figure 2 , Table S1 ). The association of age and risk of DIE. DIE: deep infiltrating endometriosis; CA: carbohydrate antigen; OR: odds ratio; 95% CI: 95% confidence interval; OCP: oral contraceptive pills. Adjusted for parity, nationality, marital status, occupation, education, OCP use, BMI, serum albumin, serum adjusted calcium, the presence of dysmenorrhea symptom, and history of endometriosis surgery.

We conducted a retrospective, observational, non-interventional cohort study, including all patients with benign ovarian tumor who underwent surgery and were identified through the medical record management system at the Affiliate Hospital, Sun Yat-sen University, between January 2020 and March 2024. The study was approved by the ethics committee of the Third Affiliated Hospital, Sun Yat-sen University (II2024-315-01). The study protocol included a waiver of informed consent for patients enrolled between 1 January 2020, and 31 March 2024. For patients admitted between 1 January 2020 and 28 February 2022, the waiver was granted due to the loss to follow-up among the majority of patients. Additionally, informed consent for the use of medical data in clinical research had not been obtained at the time of admission. Given that the data used in this retrospective study were fully anonymized and non-traceable to individual patients, posing extremely low risk to the patients, the Third Affiliated Hospital of Sun Yat-sen University’s Ethics Committee approved the waiver of informed consent for the inclusion of these patients. For patients admitted between 1 March 2022, and 31 March 2024, the waiver of informed consent was granted because all patients had already provided their written informed consent for the use of their medical data in clinical research at the time of admission. The study population consisted of reproductive-age women (15–50 years old). Women with a suspicion or pathologically confirmed diagnosis of pregnancy, leiomyoma, adenomyosis, pelvic inflammatory disease, diabetes, hypertension, kidney disease, cancer, autoimmune system disease, thyroid dysfunction, or those who had taken calcium supplements 3 months prior to their surgery were excluded. Women pathologically confirmed to have endometriosis were assigned to the endometriosis group, while those with other benign adnexal diseases were placed in the non-endometriosis group (which included conditions such as teratoma, serous cystadenoma, and mucinous cystadenoma). Women in the EMs group, according to the revised American Fertility Society (rAFS) staging standard established by the American Society for Reproductive Medicine [ 7 ], were further categorized into stage I, II, III, and IV. This research strictly adheres to the principles of the Declaration of Helsinki. Data collection, conducted by the study investigator, included age, parity, marital status, occupation, nationality, smoking habit, BMI (body mass index), dysmenorrhea symptom, pathological results, stage of rAFS, the presence of DIE, serum albumin, serum calcium, and CA 125. Results of the analysis of peripheral venous blood samples, collected before surgery, were also recorded. The expected serum calcium level for normal female adult falls between 2.15 and 2.50 mmol/L [ 8 ]. An accurate estimation of biologically active calcium relies on serum albumin concentration. Each gram per deciliter of albumin binds 0.2 mmol of calcium [ 9 ]. To correct for albumin, the standard equation was used, Albumin-adjusted calcium = Calcium + 0.02*(40-Albumin) [ 10 ]. Based on previous findings [ 11 ], mean albumin levels vary across different age groups. Therefore, in this study, if the woman was under 45, we subtracted 0.9 g/L from the albumin; otherwise, we made no adjustments [ 12 ]. Statistical analysis of data was conducted through SPSS version 27.0 (IBM Professional Concurrent User). Categorical variables were presented as frequencies and percentages, whilst continuous variables were given as means (±standard deviation). An independent sample t-test was utilized to compare mean age, serum levels of calcium, albumin, or CA125 across groups. Multivariate logistic regression model was used to identify the independent risk factors for DIE. Receiver operating characteristic (ROC) curve was used to assess the power of risk factors for DIE. A two-sided p value less than 0.05 was considered statistically significant.

In our study, we had observed a statistically significant correlation between preoperative adjusted calcium level and endometriosis, and DIE. The correlation remained significant after adjusted for potential confounding variables, such as parity, nationality, marital status, occupation, education, OCP use, BMI, serum albumin, and the presence of dysmenorrhea symptom. This research included only women who had undergone surgery and had a pathological diagnosis from both the EM group and the non-EM group, constituting one of the study’s strengths. Accurate preoperative assessment of endometriosis and concurrent DIE is critical for optimizing surgical strategies and minimizing surgery-related complications. To date, there is no clinically pertinent biomarker available for either screening or patient stratification. Serum CA125 is yet the most commonly used biomarker in diagnosing and monitoring endometriosis. Study found that the production and secretion of serum CA125 originate from the endometrium and peritoneum [ 13 ]. Therefore, different type of endometriosis may correspond to varying levels of elevated CA125. In our study, the average CA125 level in the EM group was significantly elevated in comparison to the non-EM group, aligning with prior study [ 14 ]. However, we found no statistically significant difference in CA125 level between patients with DIE and those without DIE, which was consistent with Pietro et al.’s study [ 15 ]. Given that DIE presents the greatest challenge during surgery, these findings render CA125 less optimal as a biomarker for endometriosis and concurrent DIE. Endometriosis exhibits tumor-like biological behaviors. Studies revealed that oxidative stress contributes to the initiation and progression of endometriosis [ 15 , 16 ]. It’s known that the altered expression of calcium ion channels is a feature of tumorigenesis in certain organs. Links have been established between intakes of dairy foods and dietary calcium and a countering influence on inflammatory stress [ 17 ]. The association between serum calcium and endometriosis, however, remains unclear. This study found a statistically lower serum calcium level in women with endometriosis compared to the non-endometriosis group. Additionally, women with DIE showed a prescribed lower serum calcium level. Previous large-scale studies have observed an inverse relationship between food-derived calcium intake and endometriosis [ 18 ], a finding echoed in other studies [ 19 , 20 ]. Considering these results, we can speculate that calcium might be an important mineral element in the pathogenesis and growth of endometriosis, even though the precise mechanism warrants further exploration. Our research found that the EMs group and non-EMs group differed in CA125 and adjusted serum calcium levels. We developed a combined marker by multiplying the adjusted calcium level by the CA125 level, and used ROC analysis to assess the performance of CA125, adjusted calcium, and the combined marker. The combined marker achieved the highest AUC value in diagnosing endometriosis. However, we also acknowledge that the AUC value is not exceptional. While the combined marker of CA125 and adjusted calcium may be a potential biomarker for diagnosing endometriosis, its added value is relatively small and should be interpreted with caution. The practical advantage of these markers is that they can be easily obtained through routine blood tests without additional cost or further testing. Yet, their diagnostic performance is limited. Regarding adjusted serum calcium levels, although lower levels might carry predictive value for concurrent DIE in endometriosis patients, this finding should also be viewed with caution and requires further validation in larger and more diverse cohorts. Information regarding the relationship between the severity of endometriosis and patient age is valuable and may enhance understanding of the risk of disease progression when left untreated over time. However, the literature on the phenotypic changes of endometriosis with age remains limited and controversial. Some studies suggest that the severity of endometriosis remains relatively stable in adulthood [ 21 , 22 ], while others propose that endometriotic lesions undergo changes over time [ 23 ]. Our study found that the diagnosis rate of DIE increased from ages 20 to 29, peaking at ages 25–29, before declining after age 30. This trend supports the idea that deep endometriotic nodules evolve until at least age 30 [ 21 ], which could aid practitioners in making informed clinical decisions. Our study included 987 endometriosis patients and 1570 non-endometriosis patients, with all of them receiving a pathological diagnosis, presenting a strength of this study. Nonetheless, there are a few limitations. The hospital-based setting of the study might limit the extent to which our findings can be generalized to encompass all women with endometriosis. Moreover, the fact that our sample was drawn from a single institution could introduce potential bias. Further research involving more diverse sample size is required to confirm the clinical utility of our findings.

In conclusion, this study has shown that the level of albumin-adjusted calcium was significantly lower in patients diagnosed with endometriosis compared to those with other benign ovarian tumors. The combined use of albumin-adjusted calcium and CA125 may potentially offer enhanced diagnostic utility for EM, though further validation is required. Moreover, a lower serum level of albumin-adjusted calcium was associated with the presence of DIE among endometriosis patients.