Isolation and Characterization of the Extracellular Vesicles Secreted by the Human Endometrium
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|ClinicalTrials.gov Identifier: NCT02797834|
Recruitment Status : Unknown
Verified June 2016 by Felip Vilella, Igenomix.
Recruitment status was: Recruiting
First Posted : June 14, 2016
Last Update Posted : June 21, 2016
|Condition or disease||Intervention/treatment|
|Extracellular Vesicles||Procedure: Endometrial fluid collection|
In the last decades, the emission of membrane enclosed compartments, more commonly regarded as extracellular vesicles, has been established as a new mechanism of communication either between cells of a complex organism, between unicellular organisms or between unicellular organism and host. These vesicles have been described in all body fluids and are currently classified into three types according to the mechanism by which they are formed: exosomes, with sizes ranging from 30 to 150 nm (formed intracellularly); microvesicles, between 100 and 1000 nm (formed from the cell membrane) and apoptotic bodies, with sizes greater than 1 µm (produced by cells undergoing programmed cell death).
Endometrial fluid is a viscous liquid present in the uterine cavity originating from the different cell types that form the uterus and serum exudates. It offers a precise view of the context in which the embryo implants and can be retrieved by a minimally invasive technique.
The main objective of this pilot study is to isolate and characterize the extracellular vesicles secreted by the endometrium to the endometrial fluid, being this the first time that this fact is being described. It is intended to standardize a method for the isolation of the different vesicles populations present within the endometrial fluid (i.e.: apoptotic bodies, microvesicles and exosomes), based on serial differential centrifugations and filtration. Morphological characterization of the isolated vesicles will be done at three levels: study of the external morphology by transmission electron microscopy, characterization of the different vesicles populations surface markers by Western Blot and analysis of the vesicles populations size distributions by Nanoparticle Tracking Analysis.
The second part of the study is directed to assess the content of the extracellular vesicles throughout the menstrual cycle. For this purpose, a canonical menstrual cycle of 28 to 30 days has been classified in 5 phases: phase I (days 0-8), phase II (days 9-14), phase III (days 15-18), phase IV (days 19-24) and phase V (25 to 30). The experimental design for vesicles contents description consists of two levels of comparison: (1) analysis of differential contents of the different populations of vesicles in the same phase of the menstrual cycle and for each of the phases and (2) analysis of differential contents of the same population of vesicles in each phase of the cycle and for each of the vesicular populations. This characterization of the vesicular contents will be made using mass spectrometry techniques to analyze the protein, lipid and other small metabolites composition. Their presence aims to be related both to the moment of the cycle and to a normal physiological function of the endometrium. On the other hand, analysis of DNA and RNA contents of these vesicular populations by massive sequencing will be performed. The objective here is to analyze the transmission of functionalities to target cells as well as the potential predictive usefulness of these molecules to determine the moment of the menstrual cycle.
|Study Type :||Observational|
|Estimated Enrollment :||300 participants|
|Official Title:||Isolation and Characterization of the Extracellular Vesicles Secreted by the Human Endometrium to the Endometrial Fluid|
|Study Start Date :||April 2016|
|Estimated Primary Completion Date :||March 2017|
|Estimated Study Completion Date :||March 2019|
Patients Endometrial Fluid
Endometrial fluid samples from healthy and fertile women in their natural cycles, with ages ranging from 18 to 35 years, normal karyotype, negative for HIV, HBV, HCV and RPR, BMI ranging from 18 to 30 Kg/m2 (both included) and regular menstrual cycle (3-4/28-30 days).
This unique assignment group will be divided into 5 subgroups attending to the moment of the menstrual cycle in which the patient could be classified: phase I (days 0-8), phase II (days 9-14), phase III (days 15-18), phase IV (days 19-24) and phase V (days 25-30).
Procedure: Endometrial fluid collection
- Morphological characterization of the extracellular vesicles [ Time Frame: 3 months ]
Assessment of the morphology of the extracellular vesicles using transmision electron microscopy by either ultrathin cuts and positive staining or deposition and positive staining techniques.
The aim is to differentiate between vesicle populations thanks to their external aspects and size.
- Size distribution profiles characterization of the extracellular vesicles [ Time Frame: 45 days ]
Analysis of the size distribution of the vesicles contained in the different extracellular vesicles populations by using two Nanoparticle Tracking Analysis techniques: ZetaSizer Nano and NanoSight300.
The expected outcome is a set of graphs with size range data of the vesicles in the different populations and vesicle concentration data when using NanoSight300.
- Characterization of extracellular vesicles populations by specific markers [ Time Frame: 45 days ]
Analysis of population-specific markers by Western Blot in order to further differentiate endometrial fluid vesicles populations.
The aim is to differentiate populations based on the presence or absence of these markers.
- Qualitative (names) and quantitative (expression levels) information about the different biomolecules that could be present in the samples: DNA, RNA, proteins, lipids and other small metabolites. [ Time Frame: three years ]
Biospecimen Retention: Samples With DNA
DNA, RNA, proteins, lipids and other metabolits will be analyzed in the different samples of the endometrial fluid.
These samples will be completely exhausted during the analysis.
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT02797834
|Contact: Carlos Gomez, BSc MScfirstname.lastname@example.org|
|Contact: Diana Valbuena, MD PhDemail@example.com|
|Valencia, Spain, 46015|
|Contact: Carlos Simon, MD PhD +34963050900 firstname.lastname@example.org|
|Principal Investigator:||Felipe Vilella, BSc PhD||Igenomix|