Role of human endometrial extracellular vesicles in embryo implantation

Abstract

Embryo implantation is a critical process marking the onset of pregnancy, during which the blastocyst attaches to and invades the receptive endometrium. This process requires a dynamic and complex interaction between the blastocyst and the endometrium, mediated by a wide variety of molecules. Among these, extracellular vesicles (EVs) transfer proteins, lipids, metabolites, and nucleic acids between cells, playing an essential role in reproduction. The main objective of this Ph.D. dissertation was to describe the role of EVs secreted by the maternal endometrial epithelium in regulating molecular mechanisms within the human blastocyst, as part of the communication system between the two during embryo implantation. To achieve this, EVs were isolated by ultracentrifugation from secretory-phase primary human endometrial epithelial cells obtained from fertile women. The proteomic content of these EVs was identified and characterized, revealing proteins associated with key biological processes involved in endometrial receptivity, embryo implantation, and early embryonic development. This study demonstrated for the first time that human blastocysts can internalize endometrial EVs within the first two hours of in vitro co-culture, a crucial step for their molecular content to exert biological functions. Additionally, the microRNA content of these vesicles was characterized to investigate their role in regulating gene expression in recipient cells. Most of the identified microRNAs had been previously detected in human uterine fluid and described as mediators of embryo implantation, regulating processes such as endometrial receptivity, embryonic attachment, and early embryo development. These microRNAs targeted genes involved in key biological processes for implantation. Finally, a transcriptomic analysis of human blastocysts after the internalization of endometrial EVs was performed to investigate the molecular and functional changes induced by this communication system. Differential gene expression patterns were identified between blastocysts cultured with and without endometrial EVs. The results indicated that these vesicles modulate the global transcriptome of blastocysts, regulating biological processes and molecular pathways that promote early embryonic development and enhance their competence for implantation. Endometrial EVs exerted a significant effect on the trophectoderm, primarily regulating RNA processing and mitochondrial function. Furthermore, most of these differentially expressed genes were targeted by the microRNAs previously identified in these EVs, emphasizing the role of these molecules in endometrial-embryo communication. In conclusion, this Ph.D. dissertation indicates that EVs secreted by the endometrial epithelium are internalized by human blastocysts, carrying proteins and microRNAs that modulate gene expression and molecular pathways in embryos, thereby regulating their development and competence for implantation.