Journal of Community Medicine & Health Education
Open Access

Abstract

Objectives: Angiogenesis is one of the key steps engaged in pathogenesis of endometriosis. The purpose was to investigate the presence of MVs with essential angiogenesis mediators, like vasculo-endothelial growth factor (VEGF) and metalloproteinase-9 (MMP – 9) in peripheral blood and peritoneal fl uid of women aged 25-45 with endometriosis staged as II-IV stage according to AFS. MVs released from cells of endometriosis foci were analyzed in this project. The research was performed in group of participants who were subjected to surgical treatment due to the suspected endometriotic cyst, deep and superfi cial infi ltrating endometriosis of pelvic peritoneum. MVs presence locally in peritoneal fl uid and systematically in blood may be yet unknown mechanism of immune response regulation. Moreover MVs may have infl uence on immune tolerance and growth of endometriosis foci. "Metastatic" nature of endometriosis in some patients may suggest such a scenario. Material and Method: The study was conducted on blood samples and peritoneal fl uid samples collected from women aged 25-45 with endometrial lesion in pelvic organs diagnosed during laparoscopic surgery. Women undergoing laparoscopic surgical treatment due to benign non-hormonal dependent ovarian lesions (teratomas) will be used as a control. Microvesicles (MVs) were determined in samples of 5 ml blood and samples of 5 ml peritoneal fl uid. The blood samples were collected day before operation during taking blood sample to preoperative test. The fl uids were collected from the peritoneal cavity during operation. In the study 30 samples of blood were obtained: 23 samples from women with endometriosis and 7 from women with teratomas and 27 samples of peritoneal fl uid were obtained: 19 samples in the test group and 8 samples in the control group. The blood samples and the peritoneal fl uid samples were dispensed into tubes containing anticoagulants and were undergone the process of getting platelet free plasma (PFP)/ platelet free peritoneal fl uid. Thirty minutes after collection the sample was centrifuged (3000g/15minuts) to isolate MVs from the blood and peritoneal fl uid sample. PFP and platelet free peritoneal fl uid were frozen in -40. In the next step the samples were thawed at the room temperature and centrifuged in 1000g in 5 minutes. Analysis of isolated MVs was performed by fl ow cytometry (FACS) with using annexiny V, antibodies for molecules characteristic for cells from endometriosis foci (keratin 18 (K18), CD105, CD146) and antibodies for intraepithelial vascular growth factor VEGF and metalloproteinase - 9 (MMP - 9). There were double "reading" of the sample using fl ow cytometry (FACSCanto II) Analysis and Results: In the study we analyzed the results of fl ow cytometry of 30 plasma samples (23 samples from the test group of women with endometriosis and 7 from the control group with teratomas), 27 peritoneal fl uid samples (19 samples from the test group of women with endometriosis and 8 from the control group with teratomas). In 10 patients, tests were performed in both samples (plasma and peritoneal fl uids), while the remaining cases had only one of the tests. Statistical analysis was performed using the STATISTICA program. The data generated by fl ow-cytometers were classifi ed by size: All – number of whole objects, 05-1/024 – number of objects larger than 0.24 μm, 05-1/022 - number of objects larger than 0.22 μm but smaller than 0.24 μm. Objects with dimensions from 0.22 μm to 0.24 μm (220-240 nm) and marked by antibodies were subjected to analysis. Three sets of arrangements were made: set1- CK18 + annexin V + VEGF + MMP-9, set 2 - CD105 + annexin V + VEGF + MMP-9, set3- CD146 + annexin V + VEGF + MMP-9. The results were expressed as a percentage of counts of particular type in relation to the superior category (% parent). It allowed to avoid the infl uence, of signifi cant differences in the total number of counts between patients, on the result. Median (as a measure of the average value in each group) and 1st and 3rd quartiles (as a measure of the results spread) were counted for each type of test in sets (set 1, set 2 and set 3) in the subgroups of patients. Comparisons between patients groups were made with the Mann- Whitney test. The test hypothesis assumed that both analyzed samples (test and control) came from the same population (or population with identical medians). We compared each of the fl ow cytometry parameters between the control and endometriosis groups to check which of the cytometry results differ the groups. We were looking for a correlation between plasma and peritoneal fl uid to see if there was a relationship between the amount of microvesicles in these media. In plasma samples the statistically signifi cant differences were observed in three cases: set 3 VEGF + / MMP9 - higher percentage of object marked by those antibodies in the control group, a larger percentage of microvesicles with annexinV in the group with endometriosis and higher percentage of microvesicles with MMP9 in the control group. It was also noticed that tests with annexin V plus another marker (antibodies) gave few object to count whereas tests with VEGF plus another possible marker gave much more objects to count. The same criteria were used to analyze samples of peritoneal fl uid. There was detected one signifi cant difference between test and control group: set 3 VEGF + / MMP9 - higher percentage of objects in the control group. Like in plasma, microvesicles marked by VEGF, also in combination with other markers, were the higher number of objects. Moreover we observed higher percentage of objects marked by CK18 in peritoneal fl uid in both group. There was a try to estimate the logistic regression to see if it was possible to predict, to which of group patients belonged using microvesicles profi le. The logistic regression models ware developed for collected date of plasma and peritoneal fl uid analysis. However the odds ratio estimate was impossible due to the small number of samples. Moreover we tried to cluster patients within the plasma and peritoneal fl uid groups to visualize the overall picture of the cytometry results between the groups. Clustering did not properly separate two groups of patients. The results indicated the heterogeneity of the study group. The heterogeneity was observed in both plasma and peritoneal fl uid samples. The reason could be clinical factors not included in the analyzed data. Conclusion: Results of the study did not confi rm hypothesis that microvesicles with proangiogenic factors (VEGF, MMP9) are produced in higher amount by endometriosis foci. The research revealed presence of MVs in blood and peritoneal fl uid samples in both groups. The higher percentage of MVs with VEGF+/MMP9 and only MMP9 in blood was unexpected result. Those factor are important in angiogenesis. Process, which is more advanced in endometriosis foci, not in teratomas cysts. In peritoneal fl uid analysis we also observed more MVs VEGF+/MMP9 in control group. Moreover in peritoneal fl uid there were noticed a lot of microvesicles marked by CK18 in both grup. It was supsrising result, because cytokeratine 18 was choosen as e specifi c marker for endometrial cells. To sum up, the study indicated single parametrs, which differing group of patients witch endometriosis and control group (patients with teratomas). Most of the measurements did not differentiate the analyzed groups of patients. The tests group was heterogeneity, while the control group was quite small, which made diffi culties to obtain statistically signifi cant results. The reason of heterogeneity in group of patient with endometriosis could be many. In this study information of stage of endometriosis advanced and date of menstrual cycle in which was samples collected were not compared with analyzed data. Perhaps these data had a signifi cant impact on variety results

Biography

E-mail: magdalena.kajdos@gmail.com