OC98 Host-microbiome and immune-related protein interactions in the preterm milk fortification in neonates (PUFFIN) study

Most infants born before 32 weeks gestation will need fortification to meet nutritional requirements.1 2 Fortifiers can be bovine milk-based (BMF, usually powdered) or human milk-based (HMF, previously liquid) and uncertainties remain about potential benefits of HMF.3 Powdered HMF preserves maternal milk volume in the infant diet and may offer greater immunomodulatory and less inflammatory benefits compared to BMF.

Within a randomised clinical trial of powdered BMF (PBMF) vs powdered HMF (PHMF) in 28 infants <32weeks gestation otherwise receiving only human milk we compared: faecal microbiota, stool and urinary secretory immunoglobulin A (sIgA), and stool cytokines from first fortification (commenced at 150 mls/kg/day of milk feeds) until discharge. 129 stool and 109 urine samples were analysed from infants of median gestation 27 weeks. Faecal samples were analysed for sIgA (ELISA), 39 immune proteins (MSD V-PLEX), and microbiome (16S rRNA sequencing), while urine samples were assessed for sIgA (ELISA). While stool sIgA levels appeared higher in the intervention group at day 21, the difference was not statistically significant. However, when assessing sIgA progression across all time points until discharge, levels were significantly higher in the PHMF group (p = 0.04). The fortifier type did not directly impact other measures.

Gestational age at birth and unit of care (two recruited units) had a significant impact on microbiome and cytokine profile (figure 1, Panel A and Panel B). One NICU using Bifidobacterium­-based probiotics had higher relative abundance of this genera (p ≤0.05) and distinct immune related proteins profiles.

Multi-omic analysis revealed a positive correlation between sIgA and Enterobacteriaceae (p ≤0.05), with additional correlations among Staphylococcus, Bifidobacterium, Escherichia, and specific immune proteins. No significant differences in microbiota composition or cytokine profiles were found between PHMF and PBMF, unlike previous studies with liquid fortifiers.4 PHMF appeared to increase stool sIgA levels longitudinally until discharge, but no significant differences were detected at individual time points (figure 1, Panel C). The clinical significance of this finding remains uncertain. Covariates, including gestational age and routine probiotic use play a role in shaping microbiota composition and cytokine profiles, potentially masking smaller impacts of fortifier type. These findings highlight the complex interactions between clinical variables, gut-health, microbiome, and immune responses in preterm neonates. Further research in larger cohorts is needed to evaluate the clinical implications, efficacy, and cost-effectiveness of fortifiers.