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December 03-04, 2018 | Lisbon, Portugal
Public Health, Women's Health, Nursing and Hospital Management
Joint Event
Journal of Community Medicine & Health Education | ISSN : 2161-0711 | Volume 08
Microvesicles released from ectopic endometrial epithelium foci as
potential biomarker of endometriosis
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 fluid 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 superficial
infiltrating endometriosis of pelvic peritoneum. MVs presence locally in peritoneal fluid and
systematically in blood may be yet unknown mechanism of immune response regulation.
Moreover MVs may have influence 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
fluid 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
fluid. The blood samples were collected day before operation during taking blood sample to
preoperative test. The fluids 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 fluid were obtained: 19 samples
in the test group and 8 samples in the control group. The blood samples and the peritoneal
fluid samples were dispensed into tubes containing anticoagulants and were undergone the
process of getting platelet free plasma (PFP)/ platelet free peritoneal fluid. Thirty minutes
after collection the sample was centrifuged (3000g/15minuts) to isolate MVs from the blood
and peritoneal fluid sample. PFP and platelet free peritoneal fluid 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 flow 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 flow
cytometry (FACSCanto II)
Analysis and Results:
In the study we analyzed the results of flow 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 fluid 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 fluids), while the remaining cases had only
one of the tests. Statistical analysis was performed using the STATISTICA program. The data
generated by flow-cytometers were classified 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 influence, of significant differences in the total number of counts between patients, on the
Magdalena Kajdos
Polish Mother's Memorial
Hospital Research Institute
Poland
Magdalena Kajdos, J Community Med Health Educ 2018, Volume:8
DOI: 10.4172/2161-0711-C7-051