Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.