Early pregnancy associated changes in systemic lipid profiles of mares revealed by high resolution mass spectrometry

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Early pregnancy in the mare presents a risk of embryo loss, with little known of the systemic lipid dynamics that contribute to a successful or failed pregnancy. Observations in horses and other species suggest that pregnancy is characterized by an anabolic (i.e. fat deposition) lipid profile initially, followed by a catabolic (lipid mobilization) profile toward the end of gestation. There have been no investigations of lipid metabolism in the very early stages of pregnancy, leading up to and during the time of maternal recognition. High resolution lipidomics presents an unprecedented opportunity to map the changes in lipid profiles of pregnant mares, to examine metabolic pathways contributing to establishment of pregnancy and to identify biomarkers for detection and monitoring of early pregnancy and pregnancy loss. Blood samples were collected from Thoroughbred mares in a commercial breeding operation at day 7 and day 14 following ovulation. Mares were live-covered, and pregnancy tested by transrectal ultrasound at day 14, hereby designated pregnant or non-pregnant. High resolution lipidomics analysis was performed on isolated plasma to compare profiles of pregnant (7P, 14P) and non-pregnant (7NP, 14NP) mares at day 7 and day 14 (total n = 72, split evenly in groups). Lipids were extracted using ice-cold butanol/methanol (1:1 v/v) with 10 mM ammonium acetate containing internal standards.LC separation was performed on a Phenomenex Kinetex C18 Column (2.6 µm, 100 Å, 100 × 2.1 mm) using 5 µL injection volume. The samples were analyzed in data dependent mode using Zeno EAD IDA on the ZenoTOF 7600 system. All data were analyzed using MS-DIAL 5.1. The analysis yielded a total of 10034 unique lipid species across all samples. A total of 277 and 1722 lipids were significantly altered (p<0.05) in pregnant vs non-pregnant animals at day 7 and day 14, respectively. Phosphatidylcholines (PC) were significantly increased in 7P vs 7NP, and phosphatidylethanolamines (PE) were significantly decreased in 7P vs 7NP, with similar trends observed at day 14. Lysophosphatidylcholines (LPC) were increased in 14P vs 14NP. PC and LPC concentrations also increased from day 7 to day 14, but only in pregnant animals. Collectively, these observations point to a major role for phospholipid metabolism in early pregnancy and are consistent with PE being a substrate for methylation to PC, which is a key source of choline crucial for early embryonic development. The roles of PC biosynthesis and supplementation in early equine pregnancy warrant further investigation. Furthermore, alterations in the lipid profile of mares at day 7 post-ovulation challenge the current paradigm that no systemic changes occur in the pregnant mare prior to classical maternal recognition and luteal maintenance.