Uterine histotroph and conceptus development. IV. Metabolomic analyses of uterine luminal fluid reveals regulatory landscapes during the peri-implantation period of pregnancy in pigs†

The peri-implantation period of pregnancy in pigs is characterized by rapid morphological transitions of the conceptus necessitating a precisely regulated uterine environment to support elongation, survival, and implantation. Uterine histotroph, composed of nutrients and signaling molecules secreted by or transported by endometrial epithelia, plays a central role in mediating these events. However, dynamic changes in the metabolic composition of uterine luminal fluid (ULF) during early pregnancy are incompletely defined. In this study, we performed stage-resolved, untargeted metabolomic profiling of ULF collected from cyclic and pregnant gilts on Days 10, 12, 14, and 16 of the estrous cycle and pregnancy (n = 2-6/group). A total of 206 metabolites were identified, with amino acids, fatty acids, and carbohydrates being the dominant classes. Principal component and supervised learning analyses revealed progressive divergence in ULF composition between pregnant and cyclic gilts with the most distinct profiles observed by Day 16. Notably, pregnancy induced substantial increases in amino acids associated with mechanistic target of rapamycin (mTOR) signaling and trophectoderm proliferation, including arginine, glutamine, proline, lysine, and phenylalanine. Kyoto Encyclopedia of Genes and Genomes enrichment analyses identified gestational age-dependent activation of pathways involved in amino acid biosynthesis, nucleotide metabolism, and phospholipid turnover. Metabolites such as phosphorylcholine, succinic acid, and asymmetric dimethylarginine increased markedly in pregnancy, suggesting coordinated regulation of membrane remodeling, energy production, and nitric oxide signaling. Targeted quantification of 19 amino acids revealed both linear and quadratic trends across time and pregnancy status, with distinct differences in glycine and serine trajectories between pregnant and cyclic ULF. Collectively, these findings describe the evolving biochemical landscape of the uterine lumen during early pregnancy and highlight key metabolic pathways that likely support conceptus development and uterine receptivity to implantation.