Abstract
Preterm birth remains a significant global health concern, affecting one in ten infants and represents a major early life insult with long-term health implications. Preterm birth interrupts normal developmental processes, which leaves the neonate anatomically and metabolically underprepared for extrauterine life. Among the organs most disrupted by the shortened gestation is the liver, which is crucial for metabolism, detoxification and homeostatic maintenance. While the early clinical signs like neonatal cholestasis (hepatobiliary and/or metabolic dysfunction characterised by conjugated hyperbilirubinemia in the newborns and young infants) are well documented, the molecular mechanisms underlying hepatic dysfunction remain poorly characterised.
This project aimed to examine the effect of preterm birth on hepatic molecular alterations and metabolism at 24hours, and to determine whether these changes contribute to long-term hepatic reprogramming. Using a well-established preclinical guinea pig model, time-mated pregnant dams were randomised to give birth at term (approximately 69 (67-72) days of gestation) or preterm (90% of gestation; 62 days; 30-32 weeks human equivalence) via pharmacological induction. Offsprings were culled at 24 hours (to assess immediate molecular and metabolic function), 7 days (to examine adaptations at term-equivalent age, TEA), and 40 days (to evaluate long-term metabolic programming and disease susceptibility at late childhood, human equivalence).
Whole blood and liver tissue fatty acid methyl esters were quantified, and differential protein expression was analysed using Perseus and Ingenuity Pathway Analysis (IPA), with validation by Western blotting. Statistical analyses were conducted using a two-way ANOVA with prematurity and sex as factors. Significance was set at P < 0.05.
At 24 hours, preterm pups demonstrated a significantly lower plasma glucose and insulin resistance indices, reduced absolute liver and visceral fat weights, and lower liver- and fat-to-body weight ratios compared to term controls. Blood analysis revealed significantly higher concentrations of free fatty acids, including tetradecanoic, hexadecanoic, icosanoic, oleic, palmitoleic, and γ-linolenic acids, as well as elevated total monounsaturated fatty acids. In contrast, hepatic tissues of preterm pups contained significantly lower levels of several fatty acids, such as pentadecanoic, heptadecanoic, icosanoic, vaccenic, and total trans-monounsaturated species. Bioinformatics analyses indicated upregulation of energy generation pathways such as fatty acid β-oxidation and oxidative phosphorylation and downregulation of LXR/RXR activation, with increased expression of ACADM, ACSL6, SLC27A5, and mitochondrial complexes I, IV, and V. Western blotting confirmed significant elevation of complex IV abundance at the 24hour timepoint. Sex differences were observed in levels of liver vaccenic and gamma-linolenic acids, as well as ACADM and acyl-CoA synthase expressions
At TEA, preterm pups exhibited some metabolic adaptations, including higher plasma glucose but lower saturated and polyunsaturated fatty acids, with elevated monounsaturated fatty acids. Hepatic fatty acid levels were largely normalised, except for persistently elevated docosanoic, gondoic, and nervonic acids. Proteomic profiling revealed activation of fatty acid β-oxidation I, LXR/RXR, xenobiotic metabolism (AHR signalling), and NRF2-mediated oxidative stress pathways, alongside inhibition of mitochondrial dysfunction.
By late childhood (40days), preterm offspring demonstrated significant postnatal recovery despite lower plasma glucose, insulin and insulin resistance indices, as well as significantly lower levels of blood icosanoic and total saturated fatty acids. Males (both term and preterm) had heavier livers and liver-to-body weight percentages. Adipocyte morphology (count and size) demonstrated a significant main effect of sex. Both term and preterm females exhibited a higher percentage of lipid droplets, as well as higher icosanoic and gondoic acid, with lower levels of alpha-linolenic acid and total omega-3 fatty acid than males. While the earlier lipid and hepatic proteomic dysregulation had largely resolved, upregulation of resistin and transcription factors CCAAT/enhancer-binding protein (C/EBP) and SREBP-1c persisted, suggesting a reprogramming of lipid regulatory and inflammatory pathways. In conclusion, at late childhood, preterm offspring demonstrate a similar metabolic and molecular phenotype to term counterparts, with minor persistent alterations and sex specific implications.