Longitudinal gene expression profiling of the equine amniotic membrane in normal pregnancy

The equine amniotic membrane directly interfaces with the fetus and provides physical protection during gestation. It also actively responds to infection and inflammation during placentitis, contributing to preterm birth and abortion. However, it remains unknown how it participates in fetal development and preparation for parturition through signaling pathways. Therefore, our objective is to characterize amniotic membrane gene expression across distinct gestational stages, providing insights into its roles in fetal development and labor. Eighteen pregnant mares were euthanized at 4, 6, 9, and 10 months of gestation. Amniotic membrane samples were collected at each time point and submitted for RNA sequencing. Using dynamic gene expression trend analysis and differential gene expression (DEG) analysis, we identified significant patterns of change throughout gestation. DEG analysis revealed 1,580 DEGs during late-gestation (9 and 10 months) compared to mid-gestation (4 and 6 months). Trend analysis identified 3,970 genes in eight expressional direction pathways. Functional annotation suggests a shift in gene expression from a profile associated with active proliferation and structural reorganization toward a pro-inflammatory profile, accompanied by concurrent expression of anti-inflammatory regulatory genes, which may reflect a counterregulatory mechanism that balances inflammation as parturition approaches. Our findings show that the amniotic membrane shifts gene expression throughout gestation, supporting fetal development, nutrient transport, amniotic fluid homeostasis, and parturition signaling.