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MicroRNA-based signatures obtained from endometrial fluid identify implantative endometrium

ABSTRACT: Improving embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. Implantation process requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial crosstalk. MicroRNAs (miRNAs) and extracellular vesicles (EVs) from endometrial fluid (EF) have been described to be mediators of this communication. Therefore, the molecular analysis of the miRNA content of EF could provide a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. In this work, we optimized EF sample collection procedure and performed a comparative analysis of five different methodologies to enrich miRNA content of EF with the purpose of identifying a simple, sensitive, reproducible and non-invasive method that allow the quick identification of an implantative endometrium. Then, we applied the selected methodology in a set of real samples with different implantation outcome. Our results showed that EVs enrichment protocols permit to detect a higher number of miRNAs. In addition, we obtained two predictive models based on three miRNAs that allow us to differentiate between an implantative and non-implantative endometrium. Model 1 (Area under the curve (AUC)=0.93; p-value=0.0033): hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (AUC=0.92; p-value=0.0002): hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. This work supports the feasibility to analyze EVs and EVs-associated miRNAs from a small volume of EF sample and the development of EV-miRNAs based assays as low-invasive tools for the detection of an implantative endometrium.

ORGANISM(S): blank sample Homo sapiens

PROVIDER: GSE178917 | GEO | 2022/08/18

REPOSITORIES: GEO

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Project description:Objective: To study the uptake by human embryos of extracellular vesicles secreted by the maternal endometrium, and to investigate their miRNA cargo, in order to describe their role in implantation and early embryo development. Design: Prospective descriptive study. Subjects: Healthy women oocyte donors with confirmed fertility and day 5 human blastocysts. Intervention: Endometrial biopsies were collected from healthy oocyte donors undergoing transvaginal ultrasound-guided cyst aspiration for oocyte retrieval. Main Outcome Measures: Extracellular vesicle were isolated from culture media of primary human endometrial epithelial cells by ultracentrifugation. Concentration and size were analyzed by nanoparticle tracking analysis, their morphology visualized by transmission electron microscopy and extracellular vesicle protein markers expression was determined by western blotting. Vesicles were fluorescently labelled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of extracellular vesicles was performed using miRNAseq, target genes of the most expressed miRNAs were annotated and functional enrichment analysis was performed. Results: Extracellular vesicle characterization revealed a size within 100-300 nm, and expression of extracellular vesicle protein markers HSP70, TSG101, CD9 and CD81. Fluorescent microscopy showed an efficient extracellular vesicle internalization by human blastocysts within 1-2h, being the fluorescent signal stronger in the hatched area of the embryo. miRNAseq described 149 annotated miRNAs and top 37 most expressed miRNAs targeted 6,592 genes. Functional enrichment analysis of these targeted genes indicated that they participate in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization or gene expression. Among miRNAs contained in these EVs, hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p and hsa-let-7a-5p were highly implicated in all these biological processes. Conclusion: Data suggest that extracellular vesicles secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Knowledge of the communication system between human endometrium and embryo via miRNA cargo of these vesicles could describe new biomarkers of implantation success and embryo competence.

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MicroRNA-based signatures obtained from endometrial fluid identify implantative endometrium

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