P-155 MicroRNA-155 (miR-155) expression profile and developmental characteristics of bovine early embryos are altered when cultured in group versus individually
Authors: Di Berardino T , Werry N , Angle M , Yu L , LaMarre J 
Published: June 2023

Abstract

Study question
Culturing bovine preimplantation embryos in group versus individually has an impact on the expression profile of miR-155 and developmental characteristics.

Summary answer
MiR-155 was significantly greater in its expression when embryos were cultured in groups versus singly. Developmental characteristics of blastocysts were similar regardless of culture condition.

What is known already
MiRNAs are short non-coding RNAs that play important roles in many biological pathways including embryogenesis and are potent effectors of post-transcriptional gene silencing. It has been estimated that approximately 60% of all proteins-coding genes are regulated by miRNAs. MiR-155 is involved in early events of embryogenesis; however, it has not been characterized in bovine embryos and this knowledge has not yet been clinically translated. Existing literature indicates that group culture yields superior embryo development as embryos can benefit from autocrine signaling and paracrine communication but embryos in groups may be exposed to negative effects of dying or delayed embryos.

Study design, size, duration
The study consisted of two groups: zygotes cultured to blastocysts in group and those cultured individually. Each sample consists of 3 blastocysts. Droplet digital PCR (dd-PCR) was used to examine the expression of miR-155. Gene Set Enrichment Analysis (GSEA) of miRNAs was carried out to identify functional Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are enriched for the targets of one or more miRNAs.

Participants/materials, setting, methods
Oocytes were aspirated from antral follicles of bovine ovaries and matured in vitro. Twenty-four hours post retrieval, matured oocytes were inseminated with sperm from similar bull. Presumptive zygotes were cultured either in groups or individually until blastocyst stage, Blastocyst were collected and purified for RNA. Analysis for KEGG and GO Biological Process, Cellular Component and Molecular Functions were carried out on the identified target genes. Results were plotted according to level of enrichment for miR-155.

Main results and the role of chance
Five hundred and seventy-five oocytes were retrieved from 120 bovine ovaries. Cleavage rate and blastocyst formation after IVF were compared between those blastocysts cultured in groups versus individually. There was no significant difference among the two groups with regards to cleavage rate (69% vs. 54%) and blastocyst formation (42% vs.35%). ddPCR assessment showed significantly greater MiR-155 expression in those blastocysts cultured in groups (p = 0.002). The highest level of miR-151 expressed was 20 copies/sample. Every sample, regardless of the culture conditions, had detectable miR-155 level. The interaction of predicted miR-155 target clusters based on the GO Biological Processes shows that miR-155 plays a role in DNA binding, transcription factor binding, regulates transcription and affects cell adhesion. KEGG analysis revealed an association between miR-155 to steroidogenesis and controls apoptosis pathways.

Limitations, reasons for caution
The number of copies expressed for this miRNA was relatively low, which could suggest that miR-155 may not have a functional role at the blastocyst stage as the effectiveness of a miRNA depends on the relative number of binding site and target molecules. Blastocysts were pooled based on morphological assessment.

Wider implications of the findings
MiRNAs offer a novel mean to assess embryonic fitness. Further validation and more complex study designs are required to implement the profiling of embryonic miRNAs as a diagnostic test for embryo selection. Group culture increased miR-155 expression, possibly reflecting differences in cellular behavior between group cultured embryos and individually ones.

Trial registration number
Not applicable