Extracellular Vesicles Clinical Trial
Official title:
Isolation and Characterization of the Extracellular Vesicles Secreted by the Human Endometrium to the Endometrial Fluid
The main aim of this study is to describe the morphology, size distributions and specific markers of the different vesicles populations present in the human endometrial fluid. To further characterize the vesicles populations, contents of these membrane compartments (i.e.: DNA, RNA, proteins, lipids and other small metabolites) will be assessed by using mass spectrometry and next generation sequencing techniques. The objective here is to relate a specific vesicles composition to the moment of the cycle, to a normal physiological endometrial function and to the transmission of functionalities from the mother to the embryo.
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.
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Observational Model: Case-Only, Time Perspective: Prospective
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