Analysis of miR-191-3p in the Spent Blastocyst Culture Media by Droplet Digital PCR
Droplet digital PCR analysis of human embryonic culture media collected in a proof of con-cept study on IVF pregnancy outcome. miR-191-3p was analysed in spent human embryonic culture media of morphologically similar, good quality embryos which after later transfer developed either to reproductively competent embryos and healthy neonates or of those, where miscarriage occurred in early stage of the pregnancy. Methods: Spent culture media at the morula-blastocyst stage (3rd day) were collected from embryos fertilised with ICSI and undergoing embryo transfer. After registered pregnancy outcome 40 samples from the group of reproductively competent embryos, 40 samples from miscarriage and their parallel blank culture media samples were enrolled in miRNA analysis. Isolation and quantitative detection of miRNA from embryonic culture media was carried out on automated droplet digital polymerase chain reaction platform. Results: Quantitative analysis and ANOVA evaluation confirmed miR-191-3p to be present in significantly higher concentration in the 3rd day culture media of reproductively competent human embryos (mean concentration difference=20,478, p=1 × 10-4) than the miscarriage. Control blank culturing media were negative for miR-191-3p.
MicroRNAs (miRNAs), in their mature form, represent the class of19-23 nucleotide size endogenous non-coding, but functional repressors of the molecular mechanism of mRNA translation. They influence the output of regulatory and protein coding genes in target-specific manner through partial sequence complementation on the 3’untranslated regions of their mRNA transcript and inhibit the protein synthesis on the ribosome mRNA complex. Studies confirmed differential expression of miRNA during embryo development from morula to blastocyst stages, and expressional differences between the euploid and aneuploid embryos. Cell free miRNAs also known as secretory miRNAs are an emerging class of miRNAs that are released by cells to the extracellular space via vesicular secretion. Small vesicles-that are called exosomes-protect their nucleic acid content from enzymatic degradation securing their high stability in laboratory detection procedures. Cell-free miRNA have been found to co relate with a wide variety of diseases including cancer and other chronicnon-infectious diseases, infections, tissue injury. They have been detected in various body fluids such as blood, serum, saliva, urine, tear, cerebrospinal fluid, peritoneal-fluid, breast milk, semen, amniotic fluid.
There is a great interest in identifying miRNAs in the culture media of developing embryos. Spent blastocyst culture media was found to be appropriate to represent cell-free miRNA fraction that is originating from embryonic secretion. Some miRNAs of the culture media correlated with embryonic aneuploidy and IVF failure enhancing the possibility of a non-invasive embryo-quality assessment.
Journal of Clinical chemistry and Laboratory Medicne