Comparison of marker levels within exosomes and serum in the non-benign and benign group

The mean age of the benign group was 34.78 ± 11.35 years, while that of the non-benign group was 42.91 ± 10.93years, and this difference was statistically significant (t = 4.441, P < 0.001).To determine whether there were differences in the levels of diagnostic indexes between the benign and non-benign groups, we employed an independent samples t-test. The results indicated that the mean values of serum CA125, HE4, ROMA index, extracellular vesicles CA125, HE4, C5a, and OCS exhibited statistically significant differences between the benign and non-benign groups at a significance level of 0.05. Upon further comparison, it was evident that the levels of all these indexes were higher in the non-benign group compared to the benign group. (Table 1)

Table 1 General information of patientsExosomes and serum protein levels between different benign ovarian tumours

To explore variations in serum CA125, HE4, ROMA index, and Serum sEV levels within the subcategories of benign diseases, we further divided the benign group into four subgroups: ovarian endometriosis group (73 cases), mature teratoma group (48 cases), cystadenoma group (34 cases), and other benign tumors group (including luteal cysts, luteinized cysts, follicular cysts, haemorrhagic cysts, etc., totaling 35 cases). The outcomes of one-way analysis of variance (ANOVA) indicated that age [F (3, 186) = 10.144, p < 0.001] and serum CA125 levels (F(3, 186) =, p < 0.001) exhibited significant differences among the benign disease categories. However, the levels of other indicators did not show significant differences within the benign groups at the 0.05 significance level. Notably, serum CA125 levels were notably higher in the ovarian endometriosis group compared to the mature teratoma group and the other tumor groups, respectively. Additionally, the mean age of the other benign tumor group was greater than that of the ovarian endometriosis and mature teratoma groups (Table 2).

Table 2 General information on patients in each subgroup of the benign and non-benign groupsComparison of serum exosomes and serum protein levels in the non-benign group

Within the non-benign ovarian tumor group, we further subdivided it into the borderline tumor group and the malignant tumor group. Initially, we conducted the Shapiro-Wilk test on the data from both groups. At a significance level of α = 0.05, the data for each index did not adhere to a normal distribution. Consequently, we applied the Mann-Whitney test to both sets of data. The test outcomes revealed that age, serum sEV CA125 level, and serum HE4 level were all significantly higher in the ovarian malignant tumor group compared to the ovarian borderline tumor group (Table 2).

Comparison of serum exosomes and serum protein levels in all groups

We employed ANOVA to make comparisons among the four benign groups and the borderline, malignant, and non-benign groups, respectively. The results of post-hoc multiple tests indicated that, with the exception of the level of HE-4 in extracellular vesicles, the ovarian malignant and non-benign groups exhibited higher mean values for serum CA125, HE4, ROMA index, as well as mean values for CA125, C5a, and OCS in serum sEV, compared to the levels of these indicators observed within the four benign groups.

Correlation and differences in the determination of CA125 and HE4 levels between serum exosomes and serum

As presented in Table 3, we conducted an analysis of the levels of CA125 and HE4 in paired serum exosomes and serum samples from the same patients using the paired t-test. The results of Pearson correlation analysis indicated that, across all data, the benign group, and the non-benign group, there was a positive correlation between serum sEV and serum levels of CA125 (with correlation coefficients of 0.805, 0.697, and 0.749, respectively, all with P < 0.05) as well as HE4 levels (with correlation coefficients of 0.752, 0.281, and 0.713, respectively, all with P < 0.05). It is noteworthy that both in the overall dataset and in the benign and non-benign groups, the levels of serum sEV CA125 and HE4 were lower than those in the serum samples, and this difference was statistically significant at the 0.05 significance level.

Table 3 Comparison of CA125 and HE4 levels in serum sEV and serum pairsDifferential diagnosis of benign and non-benign ovarian tumours by serum exosomes and serum proteins

The sensitivity and specificity of serum CA125, serum sEV CA125, HE4, C5a, and their combination (OCS) for differentiating between benign and non-benign ovarian tumors were as follows: serum CA125 sensitivity was 75%, specificity was 66.8%; serum sEV CA125 sensitivity was 61.4%, specificity was 96.3%; HE4 sensitivity was 77.3%, specificity was 73.7%; C5a sensitivity was 68.2%, specificity was 77.9%; OCS sensitivity was 77.3%, and specificity was 93.2%.

Serum HE4 levels and the ROMA index are influenced by women’s menstrual status. Therefore, we divided the non-benign group into premenopausal and postmenopausal subgroups to evaluate the diagnostic ability of these two indices for distinguishing between benign and malignant ovarian tumors in both groups. In Table 4, it can be observed that in premenopausal women, the sensitivity of the ROMA index (≥ 11.4) and serum HE4 (> 68.96 pmol/L) were 60.7% and 57.1%, respectively, with specificities of 95.3% and 80.6%. In postmenopausal women, the sensitivity of the ROMA index (≥ 29.9) and serum HE4 (> 114.9 pmol/L) was 73.3% and 66.7%, respectively, with specificities of 100% and 85%. Notably, the sensitivity and specificity of serum HE4 and the ROMA index in the differential diagnosis of benign and non-benign ovarian tumors in postmenopausal women were higher than those in premenopausal women.

Table 4 Diagnostic efficacy of various indicators on ovarian cancerComparison of AUC of serum sEV and serum proteins

We evaluated the ability to distinguish between benign and non-benign ovarian tumors using the AUC of CA125, HE4, and C5a within serum sEV, as well as the combined index OCS with serum CA125, HE4, and ROMA indices. The AUC values for serum CA125, HE4, and ROMA index were 0.786, 0.817, and 0.815, respectively. For serum CA125, HE4, and C5a within serum sEV, as well as the combined index (OCS), the AUC values were 0.839, 0.818, 0.744, and 0.871, respectively (Fig. 1). To further assess the diagnostic efficacy of the ROMA index and serum HE4 for distinguishing between benign and non-benign ovarian tumors in pre and postmenopausal women, we subdivided the non-benign group into these two groups. In Table 5, we observed that the AUCs for the ROMA index (≥ 11.4) and serum HE4 (> 68.96 pmol/L) were 0.815 and 0.777 in premenopausal women, and 0.777 and 0.887 in postmenopausal women for ROMA index (≥ 29.9) and serum HE4 (> 114.9 pmol/L).To determine whether there were significant differences in sensitivity and specificity between different methods and indexes, we conducted McNemar tests to compare the differential diagnostic indexes two by two. The results indicated that the discriminative ability of the OCS index and extracellular vesicle CA125 was superior to that of serum CA125 (P < 0.05). Additionally, the discriminative ability of OCS was better than that of extracellular vesicle HE4 and C5a (P < 0.05), while the other two-by-two comparisons did not demonstrate any statistically significant differences (Table 6).

Table 5 ROC curve analysis of ovarian malignancy in the diagnosis of various indicatorsTable 6 Comparison of the AUC of each index for the diagnosis of ovarian tumoursAnalysis of prognostic factors in malignant tumours of the ovary

The median follow-up period was 27.9 months, ranging from 8.1 to 41.9 months. Among the 34 cases, 7 patients passed away, 3 experienced disease relapse, 24 survived without disease progression, and 3 were lost to follow-up. The 1-year progression-free survival rate was 94.1%, and the 3-year disease-free survival rate was 73.5%.We conducted a univariate analysis to examine the impact of various factors, including age, menstrual status, pathological type, stage, extracellular vesicular intravesicular CA125, HE4, C5a, and the combined index OCS with serum CA125, HE4, and ROMA index, on the prognosis of ovarian malignancy using the Kaplan-Meier method. The results indicated that tumor stage and serum HE4 level may be associated with the prognosis of ovarian malignancy at a significance level of 0.05 (Table 7). Subsequently, we performed Cox regression analysis specifically on these two individual factors. The results, as displayed in Table 7, confirmed that tumor stage was an independent risk factor influencing the survival of patients with ovarian malignancy (p = 0.043) (Table 8).

Table 7 Prognostic unifactorial analysis of 34 patients with ovarian malignancyTable 8 Multifactorial analysis of the prognosis of 34 patients with ovarian malignancy