Microbiota and Human Milk Oligosaccharides in Premature Infants

Abstract

Gut microbiota plays an important role in infants' health. The prevalence of bifidobacteria in the gastrointestinal tract of term breastfed infants has been associated with reduced infection rates compared with formula-fed infants. However, few studies evaluated microbiota in premature infants. In an observational study of 577 preterm newborns born below 32 weeks gestation, gut microbiota was not driven by bifidobacteria but could be classified into six different clusters with regard to the most abundant bacteria present. Clusters were related to infants' maturity, perinatal determinants, and were associated with short- and longterm outcome. In another study, the effects of caesarean birth on infant gut microbiota could be alleviated by human milk oligosaccharides (HMOs) in mothers' milk. In addition, 58 infants fed with a formula enriched with 2 HMOs had microbiota closer to breastfed infants than 63 infants receiving the same formula without HMOs. The question then arose of the benefit of HMO supplementation for microbiota in premature infants. Thus, a multicenter randomized controlled intervention study of the effect of a liquid supplement containing 2 HMOs was set up. Ongoing data analysis will evaluate gastrointestinal tolerance parameters, intake of HMOs from human milk, long-term growth outcomes, fecal microbiota, and fecal biomarkers of gut maturation and immunity. It is now well recognized that gut microbiota plays an important role in the health of infants. Breastfed term infants have a gut microbiota that is dominated by bifidobacteria, whereas formula-fed infants have a more heterogeneous composition, with comparatively lower levels of bifidobacteria [1, 2]. Diet composition significantly impacts the development of term born infants' gut microbiota [3]. The prevalence of bifidobacteria in the gastrointestinal tract of term breastfed infants has been associated with reduced infection rates and less allergy manifestation as compared with formula-fed infants [1, 4]. However, few data are available to support microbiota composition and its impact on premature and very premature infants' health. In the EPIFLORE study, a prospective observational cohort study that included stool sample collection within the fourth week after birth in 577 preterm newborns born below 32 weeks' gestation, Rozé et al. [5] investigated the associations between neonatal intensive care unit (NICU) policies, gut microbiota, and outcomes at 2 years of age. In that study, gut microbiota was determined by 16S ribosomal RNA gene sequencing and classified with regard to the most abundant bacteria present in the stool sample. Six clusters were identified (Fig. 1): cluster 1 was driven by Enterobacter, cluster 2 by Clostridium, cluster 3 by Escherichia coli, cluster 4 by Enterococcus, cluster 5 by Staphylococcus, and a sixth cluster included non-am- plifiable samples owing to low bacterial load. Perinatal determinants were indeed associated with microbiota: clusters 4, 5, and 6 were significantly associated with lower gestational age (26.7 ± 1.8 weeks in average) and cluster 3 with higher mean gestational age (29.4 ± 1.6 weeks, p < 0.001). Therefore, cluster 3 appears to correspond to more mature microbiota, and thus was chosen as the reference cluster. Birth by cesarean delivery was associated with increased risk of being in clusters 1, 5, and 6. No assisted ventilation on day 1, direct breastfeeding, and skin-to-skin practice were associated with a more mature cluster. Also, late-onset sepsis occurred significantly more often in clusters 4-6 (56.5, 78.5, and 82.1%, respectively) than in clusters 1-3 (37.7, 14.7, and 21.7%, respectively). In addition, in a subset of the study population, looking for the risk of necrotizing enterocolitis [6], microbiota analysis performed in 16 cases and 78 controls showed an association between Clostridium neonatale and Staphylococcus aureus (OR: 5.5 [95% CI, 1.4, 20.1] and OR: 7.1 [95% CI, 2.0, 25.0], respectively) [6]. Finally, clusters 4, 5, and 6 were significantly associated with 2-year non-optimal outcome defined by death and/or neurodevelopmental delay using a Global Ages and Stages questionnaire score (aOR, 6.1 [95% CI, 1.526.0]; aOR, 4.5 [95% CI, 1.0-20.1]; and aOR, 5.4 [95% CI, 1.4-21.6], respectively) [5]. Overall, the EPIFLORE study shows that gut microbiota of very preterm newborns is not dominated by bifidobacteria in early life, but the development is related to infants' maturity. It is associated with perinatal determinants, NICU policies such as breastfeeding and skin to skin practice, and may be related to short- and long-term outcome.

Among the factors influencing microbiota, birth mode has been shown to significantly impact microbiota [7]. In a study about maternal secretor genotype of galactoside 2-alpha-L-fucosyltransferase, Korpela et al. [8] showed that a higher level of 2'fucosyllactose (2'FL) in the milk of 76 secretor mothers as com-pared to 15 non-secretor mothers, may alleviate the effects of caesarian birth on term-infant gut microbiota [8].

In a randomized double-blinded controlled trial, 63 healthy infants received infant formula (control) and were compared to 58 infants fed the same formula with added two human milk oligosaccharides (HMOs), 2'FL and lacto-N-neo- tetraose (LNnT) (test group). Thirty-five breastfed infants served as a reference group [9]. At 3 months of age, microbiota composition in the test group appeared closer to that of breastfed neonates, and the infants were significantly less likely to require antibiotics within their first year of age [9]. In a study of 500 samples of milk from 25 mothers breastfeeding very preterm infants (<32 weeks of gestational age, <1,500 g of birth weight) and 28 mothers breastfeeding term infants, Austin et al. [10] showed that at equivalent postmenstrual age, the con-centrations of a number of HMOs, such as 2'FL, were significantly lower in pre-term compared to term milk. Finally, in their observational cohort, Gabrielli et al. [11] followed 63 mothers who delivered preterm infants and showed that both 2'FL and LNnT tended to drop through the first month of lactation: compared to preterm colostrum, preterm mature milk generally contained less HMOs, and 2'FL levels were significantly different between values found on day 4 (concentration ranging from 7.23 to 7.36 g/L) and day 30 (concentration ranging from 4.41 to 5.85 g/L, p < 0.01).


In summary, prematurity may have a negative impact on gut microbiota and immune and gut functions. HMOs have important physiologic functions in early newborn development. Data support the potential benefit of HMOs in modulating microbiota and suggest that HMOs may alleviate microbiota dysbiosis and reduce the risk of late-onset sepsis and necrotizing enterocolitis in premature infants. Also, prematurity might be associated with lower levels of HMOs in breastmilk. The question then arises of the benefit of HMO supplementation for microbiota and beyond in very premature infants.

To address that question, clinical safety and efficacy data are required. Thus, a multicenter randomized controlled intervention study of the effect of a liquid supplement containing 2 HMOs (2'FL and LNnT) in preterm infants, 27-33 weeks' gestation with a birth weight below 1,700 g, was set up. Proposed HMO dosage distribution (2'FL: LNnT = 10:1) is reflective of the relative amounts found in preterm and term breast milk.

The primary objective of the study is to demonstrate feeding tolerance among preterm infants measured by non-inferiority in days to reach full enteral feeding (FEF) in the HMO versus the placebo group. Shorter time to reach FEF would be an indication of adequate gastro-intestinal tolerance and help to provide sufficient nutrition to assure growth and impact the outcome. Secondary outcome includes growth parameters, gastrointestinal symptoms, any adverse events up to 12 months corrected age, fecal microbiota, gut maturation/immune biomarker outcome and breastmilk HMO composition.

The study design is a multicenter, prospective, randomized, double-blind, controlled trial in 27-33 weeks' gestation infants. Early administration of the products occurs after 24-h trophic feeding and within the first 5 days of postnatal age (Fig. 2). The experimental group receives liquid supplement containing the 2 HMOs (2'FL and LNnT at 0.34 and 0.034 g/kg/day, respectively) in a colorless and odorless solution of260 mOsm/kg, in addition to regular feeding. The control group receives liquid placebo containing glucose only (0.14 g/kg/day) matched to the experimental product for energy intake and comparable in color, odor, and viscosity. FEF is defined as both enteral feeding volume of at least 150 mL/kg/day and parenteral nutrition discontinuation.

Forty-three infants were included in each group. Gestational age was (mean ± standard deviation) 29.7 ± 1.4 versus 30.2 ± 1.4 weeks in the experimental versus control group, respectively. Both groups were comparable for weight for age z-score at -0.37 ± 0.72 versus -0.56 ± 0.76, length for age z-score at -0.42 ± 0.98 versus -0.46 ± 0.91 and head circumference for age z-score at -0.44 ± 0.91 versus -0.27 ± 1.14, respectively. There was no difference for the sex ratio (42 vs. 44% female) or cesarian section delivery (65 vs. 58%). Data analysis is ongoing and will evaluate the gastrointestinal tolerance parameters, intake of HMOs from human milk, long-term growth outcomes, fecal microbiota, and fecal biomarkers of gut maturation and immunity such as fecal calprotectin, a-1 antitrypsin, and sIgA, as well as adverse events up until 12 months of corrected age.

In conclusion, in premature infants, gut microbiota may show different pat-terns related to NICU practices and maturity and may be associated with mor-bidity and outcome. Microbiota appears to be associated with HMOs in preemies. However, prematurity might be associated with lower level of HMOs in breastmilk and lower intake of human milk in early life. Thus, HMO supplementation, given as soon as possible after birth may help reduce dysbiosis leading to more mature microbiota. An intervention study with ongoing analysis will address that question.

Acknowledgments

We thank Doctors Jean-Marc Jellimann, Marie Chevallier, Catherine Gire, Roselyne Brat, Jean-Christophe Rozé, Karine Norbert, Jelena Buncic-Markovic, Mickael Hartweg, and Claude Billeaud for their participation in the realization of the intervention trial.

Conflict of Interest Statement

J.M.H. received a honorarium for his participation to the NNI96 Workshop.

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