Metabolomic Analyses of Uterine Luminal Fluid Reveals Regulatory Landscapes During the Peri-implantation Period of Pregnancy in Pigs

Abstract 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 or transported by endometrial epithelia, plays a central role in mediating these events. However, dynamic metabolic remodeling of uterine luminal fluid (ULF) during early pregnancy remain poorly understood. In this study, we performed stage-resolved, untargeted metabolomic profiling of ULF collected from cyclic and pregnant gilts from Days 10 to 16 of the estrous cycle and pregnancy. 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 increases in amino acids associated with mTOR signaling and trophectoderm proliferation, including arginine, glutamine, proline, lysine, and phenylalanine. KEGG 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 temporal and physiological status effects, with distinct glycine and serine trajectories between pregnant and cyclic ULF. Collectively, these findings define the evolving biochemical milieu of the uterine lumen and highlight key metabolic pathways likely required for conceptus development and implantation.