Introduction:Endometriosis is common benign gynecological disorder, with a particularly high incidence in patients experiencing fertility problems. The diagnosis of endometriosis on ovaries (endometriomas) or deep infiltrative endometriosis can generally be diagnosed with transvaginal ultrasound, while peritoneal endometriosis needs to be proven through surgery - laparoscopy. The desire for non-invasive diagnostics is the basis for our research work. Previous studies of non-invasive biomarkers have focused on glycoproteins, inflammatory and non-inflammatory cytokines, cell adhesion molecules, growth factors, and angiogenesis factors. However, no individual biomarker or panel of biomarkers has shown adequate characteristics for non-invasive diagnosis. The discovery of reliable biomarkers that can aid in diagnosis would shorten the time to disease detection, enable earlier treatment, reduce pain, and improve fertility. The objectives of this PhD dissertation are: to identify proteins present in altered concentrations in the peritoneal fluid and blood of patients with endometriosis and patients with idiopathic infertility (without endometriosis) using a proteomic approach, and to determine the correlation between the concentration of specific proteins in plasma and peritoneal fluid, as well as to elucidate how differences in protein concentrations reveal processes associated with endometriosis pathophysiology.
Methods: The study was designed as a clinical prospective investigation involving subjects and control individuals. Based on inclusion criteria, patients with endometriosis were included in the study group. The control group consisted of individuals who underwent diagnostic laparoscopy as part of primary infertility management, with no other pathology discovered during the laparoscopic surgery. Plasma and serum samples were collected before surgery, and peritoneal fluid samples were collected during the surgery. The research was conducted in three parts. In the first part (1), peritoneal fluid samples were analyzed. The first part of the study was divided into two phases - a discovery phase and a validation phase. In the second part of the study (2), proteins identified as possible biomarkers in the peritoneal fluid in the first part of the research were analyzed and validated in blood samples. In the third part of the study (3), we analyzed markers of oxidative stress in blood and peritoneal fluid.
The first part of the research (1) began with the discovery phase, involving 6 patients with endometriosis and 6 patients in the control group. In the validation phase, we included 26 patients with endometriosis in the experimental group and 20 patients in the control group. The discovery phase utilized the Sciomix protein microarray method, while the validation phase employed the conventional ELISA immunological method.
In the second part of the research (2), during the discovery phase, we examined differences in the concentration of two potential biomarkers using the ELISA method in plasma samples from the cohort of patients from the first part of the study, as well as differences in the level of CA 125. In the validation phase of the second part of the research (2), we validated differences in the concentration of TGFBI using the ELISA method and CA 125 in a sample of 237 participants (166 patients with endometriosis and 61 individuals in the control group).
The third part of the research (3) involved the analysis of markers of oxidative stress in blood and peritoneal fluid using the RANDOX and RANSEL investigative methods, as well as the ELISA method. We enrolled 86 women with primary infertility( case group - 57 patients with endometriosis, control group - 29 patients with unexplained primary infertility).
Results: (1) We identified 16 proteins whose concentrations significantly differed between the group of patients with endometriosis and the control group. All 16 proteins were upregulated in the group of patients with endometriosis compared to the control group. Angiotensinogen (AGT), transforming growth factor-β-induced protein ig-h3 (TGFBI), cartilage oligomeric matrix protein (COMP), and angiopoietin-4 (ANGP4) have not previously been described in association with endometriosis. In the validation phase, we confirmed significantly elevated concentrations of TGFBI (1.7-fold) and COMP (1.3-fold) in the group of patients with endometriosis (p < 0.0001). AGT was elevated 1.9-fold in patients with endometriosis with a p-value of 0.0199. ROC curves demonstrated that TGFBI and COMP have diagnostic potential (area under the curve (AUC) of 0.84 and 0.78, respectively). The analysis showed a sensitivity of 88.5% and specificity of 70% for TGFBI, and a sensitivity of 95% and specificity of 54.3% for COMP. (2) With the analysis of blood plasma, we did not find differences in the concentration of COMP, while the concentration of TGFBI was higher in the group of patients with endometriosis (p = 0.0007). As expected, we also confirmed higher levels of CA-125 in the serum samples of patients with endometriosis (p < 0.0001). ROC curve analysis showed the diagnostic potential of TGFBI with an AUC of 0.77, sensitivity of 58%, and specificity of 84%. In the validation phase, we analyzed TGFBI and CA-125 in a larger number of samples and confirmed the diagnostic potential of TGFBI, determining an AUC of 0.69, sensitivity of 61%, and specificity of 74%. Research-wise, and especially clinically, TGFBI proved to be a good biomarker in cases of minimal and mild endometriosis (rASRM I-II). TGFBI was significantly elevated in the plasma samples of patients with minimal and mild endometriosis in comparison to the control group (p < 0.0001). ROC analysis showed the potential for differentiation between patients with endometriosis and control subjects with an AUC of 0.74, sensitivity of 61%, and specificity of 67%. ROC curve analysis of different types of endometriosis showed that TGFBI performed best in cases of peritoneal endometriosis, with an AUC of 0.76, sensitivity of 58%, and specificity of 89%.
(3) In comparing the group of patients with laparoscopically confirmed endometriosis to the control group of patients with idiopathic infertility without endometriosis (and without other pelvic pathology), we did not find statistically significant differences in the levels of glutathione peroxidase (GPX), superoxide dismutase (SOD), or hexanoyl lysine (HEL) in blood samples (serum and erythrocytes) and peritoneal fluid. Further analysis did not reveal statistically significant differences between the selected markers of oxidative stress and dysmenorrhea, dyspareunia, and chronic pelvic pain. Additionally, the levels of oxidative stress markers were not associated with the severity of endometriosis. The ROC curve analysis also did not show predictive value for the selected markers of oxidative stress.
Conclusions: Proteomic analysis of peritoneal fluid revealed 16 proteins with significantly different concentrations in patients with endometriosis compared to the control group. AGT, TGFBI, COMP, and ANGP4 have not been previously described in association with endometriosis. Based on the results of the validation study, COMP and TGFBI are potential biological markers for endometriosis. Through further analysis of peripheral blood, we found that the protein TGFBI, as well as the combination of TGFBI and CA-125, represent potential non-invasive biological markers for endometriosis in blood samples. The potential of TGFBI as a non-invasive biological marker is most pronounced in cases of minimal and mild endometriosis and isolated peritoneal endometriosis, which represents the greatest clinical potential in the future. Importantly, the same trend was confirmed in the validation, which is rare in other published studies on biological markers.
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