Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86–91%) and snoRNA (9–14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.
Supplementary Fig 1. Receiver operating characteristic (ROC) and area under the curve (AUC, 95% confidence interval) of predict model.
Supplementary Table 1. Quality control of RNA from EVs. Quantification of total RNA of EVs by the RiboGreen assay. G1 (n =73): V-EB: viable with early blastulation; G2 (n =68): V-EB: Non-viable with early blastulation; G3 (n =61): V-LB: Viable with late blastulation (V-LB) and G4 (n =52): NV-LB: Non-viable with late blastulation. SD: Standard deviation.
Supplementary Table 2. Growth of individually embryo cultured in vitro until day 11. Blastocyst diameter, developmental stage and embryo quality were recorded at day 7.5 of in vitro development, using criteria described in IETS manual (Stringfellow and Givens, 2010) and Bó and Mapletoft (2013). Different letters within a column means statistical differences with p<0.05. EB: Early blastulation; LB: Late blastulation; SD: Standard deviation.
Supplementary Table 3. Parameters of EVs secreted for bovine embryo according to post-hatching viability (day 11). Different letters within a column means statistical differences with p<0.05. EB: Early blastulation; LB: Late blastulation; SD: Standard deviation.
Supplementary Table 4. MiRNAs with significantly conserved expression in EVs of viable versus non- viable embryos.
Supplementary Table 5. Differentially expressed snoRNAs in EVs of viable embryos.
Supplementary Table 6. Parameters from logistic regression of Model 1 to predict embryo viability.
Supplementary Table 7. Parameters from logistic regression of Model 2 to predict embryo viability.