An increased incidence in early embryo loss has been observed in aged mares. Moreover, the first foal born to a mare is lighter than her subsequent foals, with reported impaired placental function at term. Because trophoblast function may be affected from the embryo stage, the aim of this project was to determine the effect of parity in aged mares on gene expression in Day-8.5 embryos. Middle-aged (13.5±2.2 years) nulliparous (never foaled) (ON) or multiparous (1.8±1.6 foals) (OM) Saddlebred, non-nursing mares were inseminated with the semen of one unique stallion. At 8 days post-ovulation (10 days post-hCG), embryos were recovered by uterine flushing and bisected to obtain samples of pure (trophectoderm, TE) or inner cell mass enriched (ICM) trophoblast. Paired end, non-oriented RNA sequencing was performed with Illumina (NextSEqn 500) on 5 and 6 TE and ICM collected from ON and OM, respectively. Differential expression was analysed with DESEqn 2. Embryo size was included in the model and a P<0.05 cutoff was used after false discovery rate correction. Gene set enrichment analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Out of the 13 007 and 12 706 genes expressed in ICM and TE, respectively, only 8 in ICM and 6 in TE were differentially expressed, with 2 genes in common. Nevertheless, 19 gene sets were enriched and 6 depleted in the ICM of ON, whereas 2 gene sets were enriched and 8 depleted in the TE of ON compared with OM. Gene sets involved in ribosomal activity and structure, proteasome, integral component of plasma membrane, and immune response were enriched in ICM from ON embryos, and gene sets linked to sphingolipid metabolism, nucleosome, and constituents of the extracellular matrix (ECM) were depleted. In TE from ON mares, enriched gene sets were involved with ribosomes and depleted gene sets were linked to extracellular matrix, focal adhesion, myosin complex, and sequence-specific DNA binding. Overall, 1 enriched (linked to extracellular matrix) and 1 depleted gene set (involved in ribosomal structure) were common to ICM and TE. Thus, embryos from aged nulliparous mares seem to have higher protein turnover and higher immune response compared with those of OM, whereas the depletion of gene sets associated with extracellular matrix and membrane may indicate differences in cellular organisation into lineages. More work is ongoing to study effects on subsequent development.