Lipidomics of allantoic fluid collected by allantocentesis indicates a possible mechanism underlying partial litter loss in multifetal pregnancies of ewes

Abstract Fetal mortality significantly impacts reproductive efficiency in sheep, with approximately 30% of fetuses failing to survive until lambing. In multifetal gestation, partial litter loss (PLL) occurs in 28% to 37% of cases. The biochemical composition of allantoic fluid (ALF), which plays a key role in metabolic regulation and fetal waste clearance, has been shown in other species to reflect fetal development and maturity. However, ALF metabolomics in sheep, especially in relation to PLL, remains unexplored. This study investigated late-gestation fetal mortality in multifetal sheep pregnancies by analyzing maternal metabolic status, placental gene expression, and ALF composition to identify potential metabolic compounds associated with PLL. The study was conducted on 25 ewes, of which 14 experienced PLL, and 11 had vital litters (VL). Plasma analysis revealed that VL ewes have higher β-hydroxybutyrate concentrations than PLL ewes, whereas plasma insulin and cortisol levels are higher in PLL ewes. Analysis of gene expression in the placental cotyledon demonstrated increased androgen receptor expression in PLL pregnancies and trends toward elevated solute carrier family 2 member 1, and reduced nuclear receptor subfamily 3 group C member 1 expression. Insulin-like growth factor 2 expression tended to be higher in VL placentas. Allantocentesis was performed on 25 ewes with multifetal pregnancies (11 VL and 14 PLL). ALF samples were collected from 74 fetuses at 140.4 ± 2.0 days in pregnancy, and 41 samples were selected for metabolomic profiling. Using genomic analysis, 25 ALF samples were confidently matched to individual lambs. Untargeted lipidomics of ALF from 14 lambs in PLL (7 born alive vs. 7 stillborn) revealed 24 differentially abundant lipid compounds. Stillborn lambs exhibited lower phosphatidylglycerol (PG) 15:0_20:1, but higher levels of sphingomyelin (SM), lysophosphatidylcholine (LPC), ether-linked phosphatidylethanolamines (PEs), lysophosphatidylethanolamines (LPEs), and monogalactosyldiacylglycerols (MGDGs). Collectively, PLL in multifetal ewe pregnancies is linked to maternal metabolic imbalance, elevated cortisol levels, dysregulated placental gene expression, and altered ALF lipid profiles in stillborn fetuses. Elevated SM and LPC, and lower PG, suggest immature lungs. These molecular and metabolic signatures may provide new insights into the mechanisms underlying PLL in late-gestation multifetal pregnancies.