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.

Media Contact: 
Allison Grey 
Journal Manager 
Journal of Clinical chemistry and Laboratory Medicne