Difference in human milk oligosaccharides (HMO) composition in breast milk may be one explanation why some preterm infants develop necrotizing enterocolitis (NEC) despite being fed exclusively with breast milk. The aim of this study was to measure the concentration of 15 dominant HMOs in breast milk during the neonatal period and investigate how their levels correlated to NEC, sepsis, and growth in extremely low birth weight (ELBW; <1000 g) infants who were exclusively fed with breast milk. Milk was collected from 91 mothers to 106 infants at 14 and 28 days and at postmenstrual week 36. The HMOs were analysed with high-performance anion-exchange chromatography with pulsed amperometric detection. The HMOs diversity and the levels of Lacto-N-difucohexaose I were lower in samples from mothers to NEC cases, as compared to non-NEC cases at all sampling time points. Lacto-N-difucohexaose I is only produced by secretor and Lewis positive mothers. There were also significant but inconsistent associations between 3′-sialyllactose and 6′-sialyllactose and culture-proven sepsis and significant, but weak correlations between several HMOs and growth rate. Our results suggest that the variation in HMO composition in breast milk may be an important factor explaining why exclusively breast milk fed ELBW infants develop NEC.

Lactobacillus reuteri DSM 17938 supplementation reduces morbidities in very low birth weight infants (<1500 g), while the effect on extremely low birth weight infants (ELBW, <1000 g) is still questioned. In a randomised placebo-controlled trial (ClinicalTrials.gov ID NCT01603368), head growth, but not feeding tolerance or morbidities, improved in L. reuteri-supplemented preterm ELBW infants. Here, we investigate colonisation with the probiotic strain in preterm ELBW infants who received L. reuteri DSM 17938 or a placebo from birth to postmenstrual week (PMW) 36. Quantitative PCR was used on 582 faecal DNA samples collected from 132 ELBW infants at one, two, three, and four weeks, at PMW 36, and at two years of age. Human milk oligosaccharides were measured in 31 milk samples at two weeks postpartum. At least 86% of the ELBW infants in the L. reuteri group were colonised with the probiotic strain during the neonatal period, despite low gestational age, high antibiotic pressure, and independent of infant feeding mode. Higher concentrations of lacto-N-tetraose, sialyl-lacto-N-neotetraose c, and 6′-sialyllactose in mother’s milk weakly correlated with lower L. reuteri abundance. Within the L. reuteri group, higher L. reuteri abundance weakly correlated with a shorter time to reach full enteral feeding. Female sex and L. reuteri colonisation improved head growth from birth to four weeks of age. In conclusion, L. reuteri DSM 17938 supplementation leads to successful colonisation in ELBW infants.

Background: Preventing neurodevelopmental impairment after extremely preterm birth remains challenging. While breast milk feeding is linked to better neurodevelopment, the underlying mechanisms are unclear. This study explored the association between individual human milk oligosaccharides (HMO) and neurodevelopment at two years of age in extremely preterm children. Methods: Milk samples from mothers of 76 extremely preterm infants collected at two weeks after birth were analyzed for 15 dominant HMOs. Register data from examination and Bayley-III neurodevelopmental assessment at two years’ corrected age was retrieved and categorized into levels of impairment. An exploratory analysis examined associations between the HMO composition and neurodevelopment. Results: Bioinformatic volcano plots revealed associations between specific HMOs and outcomes: 3FL with less neurodevelopmental impairment, LSTb with higher Bayley-III cognitive scores, and LSTa with worse neurodevelopmental impairment outcomes. Spearman correlations indicated LSTa was linked to more neurodevelopmental impairment (p = 0.018), lower language (p = 0.009), and motor (p = 0.02) scores, whereas 3FL correlated with less neurodevelopmental impairment (p = 0.02). Dichotomized analysis showed LSTa was associated with more neurodevelopmental impairment and lower language scores (p < 0.05), 3FL with milder neurodevelopmental impairment (p < 0.05), and LSTb with better cognitive (p < 0.01) and language (p < 0.05) scores. No significant associations were found for HMO diversity, total sialic acid content, or secretor/Lewis patterns. Conclusions: In this explorative hypothesis-generating study, certain HMOs appeared to be associated with both potentially beneficial and adverse neurodevelopmental outcomes in extremely preterm infants. However, these findings should be interpreted with caution, as they do not constitute evidence but rather serve as a preliminary foundation for future hypothesis-driven research.

Aim: To assess neurodevelopment and growth at 2 years corrected age in children born extremely preterm, supplemented with Limosilactobacillus reuteri or placebo. Methods: Design: Secondary analysis of register data after randomised double‐blind placebo‐controlled trial. Setting: Inclusion at two neonatal intensive care units in Sweden. Local follow‐up per national guidelines. Subjects: 134 infants born at 23–27 weeks' gestation with a birth weight < 1000 g. Intervention: Daily enteral supplementation with placebo or Limosilactobacillus reuteri DSM 17938 (2.5 × 10 ⁸ colony forming units), from within 72 h after birth to postmenstrual week 35–36. Main outcome measures: Bayley‐III developmental assessment and auxology at 24 ± 3 months corrected age. Results: Follow‐up data were available for 110 of 117 surviving participants, and 85 children were assessed with Bayley‐III. Mean language score after supplementation with L. reuteri was 90 (95% confidence interval: 85–95) compared to 83 in the placebo group (95% CI: 78–88), p = 0.04. No other significant differences in neurodevelopment or growth were observed. Conclusion: Supplementation with L. reuteri in extremely preterm infants was not associated with altered growth nor general neurodevelopmental outcomes at 2 years while language index was improved. This is interesting given previously reported early improved growth of head circumference.

Preterm birth is associated with a high risk of long-term neurological deficits. Although research underscores the role of the gut microbiome in the gut-brain axis, the mechanisms of neurodevelopmental impairment remain elusive. In this prospective observational study (PROPEL), we evaluated whether early gut microbiota development and Limosilactobacillus reuteri supplementation to extremely preterm infants with extremely low birth weight (EPT-ELBW) are associated with neurodevelopment at 2 years. Gut microbiota was characterized by 16S sequencing, and neurodevelopment was assessed by Bayley-III score. Microbiota composition constellations and lower microbial diversity, but not single bacteria, are associated with impaired neurodevelopment. Microbial maturation over the first month was discriminative for motor development, with higher abundance of E. coli and Enterococcus relative to Cutibacterium associated with impairment. L. reuteri supplementation did not seem to affect neurodevelopment via the gut microbiome. In conclusion, dynamics of gut microbiota maturation during early life may impact neurodevelopment in EPT-ELBW infants.

Preterm birth yields survivors with heightened susceptibility to long-term neurological deficits, with severity being linked to gestational age. The mechanisms behind neurodevelopmental impairment remain elusive, although research underscores the role of the gut microbiome in regulating the gut-brain axis. In this prospective observational cohort study (PROPEL-study follow-up) we evaluated whether modulation of the gut microbiota by Limosilactobacillus reuteri supplementation to 105 extremely preterm infants with extremely low birth weight (EPT-ELBW), as well as their microbiota development during the first month of life, are associated with neurodevelopment at two years of age. The gut microbiota was characterized with 16S amplicon sequencing, and neurodevelopment was assessed with clinical examination and the Bayley-III score. Our results indicate that specific microbiota composition constellations are associated with impaired neurodevelopment, as indicated by alpha- and beta-diversity being discriminative for neurodevelopment, but not a single bacterium, with increased bacterial diversity being associated to normal neurodevelopment. Microbial maturation over the first month of life was also discriminative for neurodevelopment, where higher abundance of E. coli and Enterococcus in relation to Enterobacter was associated to impaired neurodevelopment. In conclusion, the dynamics of gut microbiota maturation during early of life may have an impact on neurodevelopment in EPT-ELBW infants.