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Desarrollo de un modelo de adhesión celular "in vitro" para el estudio de la adhesión trofoblástica al endometrio humano

AuthorsImbaud, Juan Ignacio
AdvisorCastrillo, José Luis CSIC
Issue Date2006
PublisherUniversidad Autónoma de Madrid
AbstractThe low efficiency of embryo implantation is one of the major limiting factors in human reproduction. One of the initial events in this process is the adhesion of trophoblast cells to the uterine epithelium. In latest years, researchers made several trials in order to decipher signalling mechanisms occurring during this stage. In this way, numerous factors have been identified as potential markers of endometrial receptivity. Nevertheless, the molecular mechanisms underlying embryo implantation are still poorly understood. Several systems have been described in the literature for the measurement of embryonic adhesion and studying the roles of potential implantation factors. However, most of them can not be practiced in any laboratory, are difficult to beginners or requires sophisticated and expensive equipment. In this work, we have developed a new method for studying trophoblast to uterine epithelial cells adhesion forces in humans and, furthermore, analyse environmental and molecular factors surrounding this initial stage of embryo implantation. The goal of our adhesion trophoblast-endometrium model was the introduction of the EGFP gene in trophoblast type cells (JAR and JEG-3) by retroviral gene transfer, to generate cells that stably express green fluorescent protein (JAR/G and JEG-3/G). Stable expression allowed a rapid detection, analysis and selection of fluorescent cells by flow cytometry after co-culture onto endometrial epithelial monolayers (RL95-2 and HEC-1A). Trophoblast adhesion was quantified at several times. This new trophoblast-endometrial adhesion model also allowed temporal-spatial analysis of several factors involved during embryo implantation, as well as the study of genes and proteins at different cell-cell interaction times. Using this model, we have performed gene expression analysis of pre-selected genes in order to identify those that are specifically upregulated or downregulated during the initial adhesion process. We have identified three differentially expressed genes in endometrial cells and two in JAR/G trophoblasts after 42 hours co-culture. Mucin MUC-1 and UBA-2 showed to be upregulated in RL95-2 cells while IGFBP7/MAC-25 was repressed. In JAR/G trophoblast cell line the tissue specific transcriptional factor PLA-1 was upregulated and TEF-5 slightly downregulated in response to 42 hours interaction with RL95- 2 endometrial cells. Protein level analyses developed by Western Blot and Immunofluorescence demonstrated a narrow correlation with mRNA levels previously quantified by real time RT-PCR. We have also performed microarray analyses of endometrial (RL95-2 and HEC-1A) and trophoblast (JAR/G) cell lines before and after co-culture. Data analysis allowed us to identify several genes upregulated and downregulated in endometrial (“adherent” vs. “non adherent cells”) and trophoblast cells, probably involved in human embryo implantation, placentation and early pregnancy maintenance. As normal implantation and placentation are critical for successful pregnancy, a better understanding of the molecular mechanisms responsible for these processes will improve clinicians’ ability to treat disorders as infertility, early pregnancy loss or preeclampsia, and also to increase embryo implantation rates in assisted reproduction techniques as in vitro fertilization.
DescriptionTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 10-03-2006
Appears in Collections:(CBM) Tesis

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