Introduction: Approaches for non-invasive embryo selection would be a major advance that would increase efficiency and reduce both cost and risks associated with Assisted Reproductive Treatments. Exosomes are a particular subtype of extracellular vesicles (EVs) that are secreted from a wide range of cells, including placental and endometrium cells. The aim of this study was to characterize the exosomes and their miRNA content in embryo-conditioned media.
Methods: Embryo-conditioned (ECCM) used to culture embryos was obtained from the Queensland Fertility Group In-vitro fertilization embryology laboratory. Exosomes were characterised using electron microscopy, western blot (CD63 and TSG101), and size distribution (NanoSight). Total exosomal RNA was isolated by miRNAeasy (Qiagen) according to manufacturer’s' instructions. Illumina TrueSeq Small RNA kit was used to construct a small RNA library. The resulting sequencing FASTQ file was analysed using miRDeep2, a program specifically designed to identify both known and novel microRNA’s. Gene target identification for miRNAs was performed using the CyTargetLinker application in Cytoscape. A total of 3 miRNA gene target databases (MicroCosm, miRTarBase and TargetScan) were allocated to Cytoscape for analysis.
RESULTS: The exosome fractions displayed particle sizes ranging from 30 to 150 nm in diameter (with mean of 89 ± 58 nm). The presence of exosomal markers CD63 and TSG101 was confirmed. Electron Microscopy showed a typical cup-shaped morphology of exosomes from ECCM. A total of 207 miRNA were identified in exosomes. The top 20 high abundance miRNA were: hsa-miR-22-3p, hsa-miR-21-5p, hsa-miR-143-3p, hsa-miR-10b-5p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-let-7i-5p, hsa-miR-27b-3p, hsa-let-7f-5p, hsa-let-7a-5p, hsa-miR199a-3p, hsa-miR-199b-3p, hsa-miR-181a-5p, hsa-miR-423-5p, hsa-miR-26a-5p, hsa-miR-3168, hsa-miR-100-5p, hsa-miR-30a-5p, hsa-miR-21-3p and hsa-miR-424-5p. A total of 240 genes were identified to be regulated by at least two of the top 20 miRNAs, and are detected within at least two miRNA-gene target databases.
CONCLUSIONS: We suggest that exosomes released by the embryo may be used to identify a predictive biomarker from spent media, and therefore, at no risk to the embryo, would be a powerful non-invasive innovation.