Characterization of human pre-implantation embryonic exosomes

Abstract

To isolate and characterize exosomes secreted from human embryos. Prospective experimental study Spent IVF culture media was collected prospectively and pooled from 50 embryos at two different stages of early embryonic development on day 3 and at blastocyst. The 4 groups assessed were: control media without protein supplement (plasmanate), control media with plasmanate, day 3 embryo media, and blastocyst media. Exosome size and number were measured using nanosight tracking analysis (NTA). Characterization of embryonic exosome cargo was performed through mass spectrometry proteomic analysis. Exosomal protein cargo unique to embryos was submitted to bioinformatic analysis for molecular pathways and biological processes. NTA identified exosomes in control media with plasmanate, pooled day 3 embryo media, and pooled blastocyst media, which had significantly higher concentrations compared to control media without plasmanate. The particle size distribution for all 3 groups showed isolated peaks in the known exosome size range of 50-150 nm. Moreover, day 5 embryo media had significantly higher protein amount per mL media and per exosome compared to day 3 media and control media with plasmanate. Mass spectrometry proteomic analysis identified 273 distinct proteins in the control with plasmanate, day 3, and day 5 embryonic exosomes, of which 193 were shared by all groups. We identified 30 unique proteins with increased expression in day 3 and/or day 5 exosomes. These proteins were enriched in components of the WNT and p53 pathways, which have been shown in mouse and human models to play vital roles in embryonic development and implantation. Importantly, 11 of these genes have been shown previously via knockout models to play roles in blastocyst development, implantation and early embryogenesis. Furthermore, 10 of these unique embryonic proteins are involved in biological processes of immune cell activation and vesicle mediated exocytosis, which further supports their potential immune function and packaging of these proteins into exosomes. By analyzing spent IVF media, we have shown that pre-implantation embryos secrete exosomes with unique cargo that may serve potential roles in implantation, early embryogenesis and immune regulation. Future directions include molecular functioning studies with isolated embryonic exosomes as well as correlating exosome proteomic profile to embryonic outcomes such as blastocyst morphology and aneuploidy.