Transcriptomic profiling reveals similarities between equine IVF and ICSI embryos.

Overview

• This research compares the gene expression profiles and embryo development outcomes of equine embryos produced by two different fertilization techniques: conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).
• The study finds no significant differences in gene expression between embryos produced by IVF and ICSI, suggesting IVF is a safe and viable alternative to ICSI for equine embryo production.

Background and Objective

• For about 20 years, intracytoplasmic sperm injection (ICSI) has been the primary method used for in vitro production of horse embryos.
• Since 2022, a repeatable conventional IVF protocol has been developed as an alternative fertilization method.
• The influence of the fertilization method on embryo quality and gene expression profiles was previously unclear.
• The objective was to compare transcriptomic profiles and developmental outcomes between equine embryos created by IVF versus ICSI to assess their similarity and safety.

Experimental Design and Methods

• Ten sibling blastocysts were produced in vitro from three different mares.
• Half the blastocysts were fertilized using conventional IVF and the other half by ICSI.
• Full-length single-cell RNA sequencing (FLASH-sequencing) was performed on each blastocyst to analyze gene expression.
• Gene expression data were analyzed to identify differentially expressed genes (DEGs) between the two groups.
• Cleavage rates (early embryo cell division) and blastocyst formation rates were monitored and compared between IVF and ICSI groups.

Key Findings

• A total of 11,518 genes were identified from the RNA sequencing data.
• No statistically significant differentially expressed genes were found between IVF and ICSI embryos, indicating similar transcriptional profiles.
• Cleavage rates were comparable: 55.0% for IVF zygotes and 51.9% for ICSI zygotes; the difference was not statistically significant (P = 0.74).
• Blastocyst formation rates were significantly higher in the ICSI group compared to IVF: 37.0% vs. 22.5% based on collected cumulus-oocyte complexes (COCs), and 71.4% vs. 40.9% based on cleaved zygotes (P = 0.04 and 0.004 respectively).
• The average day of blastocyst development did not differ significantly between the two groups (P = 0.55).

Interpretation and Implications

• The lack of gene expression differences suggests that both fertilization methods produce embryos with similar molecular qualities at the blastocyst stage.
• Despite similar cleavage rates, ICSI yielded higher blastocyst rates, which may impact clinical decisions about the preferred fertilization technique.
• The results support the safety and potential adoption of the new equine IVF protocol for assisted reproduction, providing an alternative to ICSI.
• Since horses share some reproductive features with humans, this work positions the horse as a useful model for studying long-term impacts of assisted reproductive technologies (ART) that might be relevant to human medicine.

Summary

• This study provides the first transcriptomic comparison between equine IVF and ICSI embryos, revealing no differences in gene expression.
• The comparable molecular profiles and developmental timings suggest IVF can reliably substitute ICSI in equine embryo production.
• Higher blastocyst rates with ICSI highlight factors beyond gene expression that influence embryo viability.
• The research opens pathways for further clinical studies on equine IVF and offers insights applicable to ART in other species, including humans.