Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

Fecha de publicación: 05/07/2023 Fecha Ahead of Print: 05/07/2023

Autores de IIS La Fe

Marina Segura

Autor
Alba Bas Rivas

Autor
Elena Juarez Barber

Autor
Maria Cristina Carbajo García

Autor
Amparo Faus Esteve

Autor
María José De Los Santos Molina

Autor
Antonio Pellicer Martínez

Autor
Hortensia Ferrero Chafer

Autor

Grupos

Biología Reproductiva y Bioingeniería en Reproducción Humana

Leader

Nicolás Garrido Puchalt
Biomarcadores, Medicina Genómica, Estadística y Análisis masivo de datos en Reproducción humana asistida

Investigador Principal

Marcos Meseguer Escrivá
Medicina Reproductiva

Investigador Principal

José Morales Roselló

Abstract

STUDY QUESTION: Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? SUMMARY ANSWER: Data suggest that EVs 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. WHAT IS KNOWN ALREADY: Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. STUDY DESIGN, SIZE, DURATION: A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. MAIN RESULTS AND THE ROLE OF CHANCE: EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight 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 as master regulators of the biological processes. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. WIDER IMPLICATIONS OF THE FINDINGS: This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Datos de la publicación

ISSN/ISSNe:: 0268-1161, 1460-2350
Tipo:: Article
Páginas:: 1547-1559
DOI:: 10.1093/humrep/dead138
PubMed:: 37407281
Factor de Impacto:: 1,993 SCImago ℠
Cuartil:: Q1 SCImago ℠

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Filiaciones

Segura-Benítez M:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain

Departamento de Pediatría, Obstetricia y Ginecología, Universidad de Valencia, Valencia, Spain
Bas-Rivas A:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
Juárez-Barber E:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
Carbajo-García MC:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain

Departamento de Pediatría, Obstetricia y Ginecología, Universidad de Valencia, Valencia, Spain
Faus A:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
De Los Santos MJ:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain

IVIRMA Valencia, Valencia, Spain
Pellicer A:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain

IVIRMA Rome, Rome, Italy
Ferrero H:: Instituto de Investigación. Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain