Unveiling the equine placental transcriptome: A novel study on ICSI-derived pregnancies

Alterations during the early stages of embryo development have been associated with long-term effects on the fetus, neonate, and adult, but this has not been investigated in horses. In recent years, intracytoplasmic sperm injection (ICSI) has gained in commercial popularity in the equine population. Research suggests an association between ICSI-produced embryos and placental malformations, but there exists little understanding of the physiology involved. Therefore, we aim to produce a complete transcriptomic analysis of chorioallantois and provide potential pathways that may be impacted following pregnancies associated with in vitro-produced equine embryos. To do so, seventeen warmblood mares were bred either naturally to produce in vivo-produced pregnancies that were carried by self (in vivo; n = 8) or with in vitro-produced pregnancies created via intracytoplasmic sperm injection (ICSI) that were transferred to a recipient (in vitro; n = 9). Mares were monitored throughout gestation to ensure the health of the pregnancy, and impending parturition was monitored for progress. Chorioallantois was collected immediately postpartum and placed in RNALater for future extraction. RNA was isolated using Trizol, and RNASeq was performed by Novogene, with 93.3 % total mapping and 40 million read depth. The false discovery rate (FDR) was set to <0.05. When comparing groups (in vivo vs. in vitro-produced embryos), 1589 genes were differentially expressed. This included an upregulation of 626 genes, alongside a downregulation of 963 genes. Impacted gene ontology included aspects of the central dogma of molecular biology, including ribosome biogenesis, RNA polymerase activity, and spliceosome function. Additional biological processes that were impacted included aspects of the immune system relating to auto-immunity and disordered antigen response, such as the IL-17 signaling pathway, rheumatoid arthritis, and lupus. Additionally, pathways relating to hypoxia and ribosome biogenesis were associated with in vitro-produced pregnancies. Overall, it appears that the in vitro production of pregnancies is associated with placental dysregulation during pregnancy, which may be related to poor fetal and neonatal outcomes that have been associated with ART in other species.