Transcriptomic profiling in equine IVF and ICSI embryos

In vitro production of equine embryos has been performed using intracytoplasmic sperm injection (ICSI) for the last two decades. Since 2022, a repeatable protocol for conventional in vitro fertilization (IVF) provides a successful alternative. However, little is known about the influence of the fertilization method on embryo quality and the transcriptomic profile. In this study, we aimed to examine differentially expressed genes (DEGs) between ICSI and IVF embryos in the horse. Therefore, ten equine sibling blastocysts, produced in vitro by either ICSI or IVF from three different mares, were subjected to Full-Length Single-Cell RNA-Sequencing (FLASH-sequencing). As such, 11,518 genes were identified, with only 1 DEG between ICSI and IVF embryos. Interleukin 4 induced 1 (IL4I1), a protein coding gene related to sperm cell death, was upregulated in IVF embryos (adjusted P-value = 0.01, FC = 2.84). Cleavage rates, calculated on collected COCs, of IVF zygotes (55.0%) were similar to those of ICSI zygotes (51.9%; P = 0.74), but blastocyst rates were higher following ICSI (37.0% vs 22.5% calculated on collected COCs and 71.4% vs 40.9% calculated on cleaved zygotes; P = 0.04 and 0.004, respectively). The average day of blastocyst development did not differ (P = 0.55). In conclusion, gene expression was similar for the two fertilization techniques, aside from one sperm related DEG, supporting the safety of equine IVF for further clinical studies. Overall, the horse provides a valuable model to study long-term effects of assisted reproductive technologies with potential extrapolation to human medicine. Keywords: Transcriptome, FLASH-seq, embryo, horse, IVP, fertilization Overall design: Equine sibling embryos were created using OPU-ICSI or OPU-IVF from three mares (R, V, T) and a single stallion. Blastocysts were individually frozen (5 replicats). Library preparation was performed using the FLASH-seq method.