Human milk

Human milk (HM) remains the gold standard for feeding infants, as it is proved as the most complete and natural feeding for the newborn. Deoni et al11 showed in a longitudinal study that early nutrition influences developmental myelination and cognition in term infants. They followed up a cohort of infants with brain MRI and cognitive assessment tools until 5 years of age and they found that exclusively breastfed infants had an overall increase in myelin content by 2 years of age that persisted throughout childhood when compared with exclusively formula-fed infants.11 In particular, breastfeeding duration has been associated with increased myelination in several areas of the brain, related to cognitive and behavioural performances.12

Obviously, the extremely positive effects of mother’s milk derive not only from its components but also from the mother–child interaction that breast feeding involves and from other social and environmental aspects. For this reason, studies evaluating for beneficial effect of HM on neurodevelopmental outcome should always consider adjusting for confounding.13

Nowadays, HM is considered the best choice even for preterm infants,14 but the latter beneficial effects are difficult to demonstrate in the preterm population, which differs significantly from the term population regarding nutritional needs, feeding practices and neurodevelopmental risk. Some large cohort studies have yielded inconsistent results on the association between HM intake and neurodevelopment. A systematic review with meta-analysis including those trials found that inconsistency between results of studies evaluating the effect of HM on preterm infants could be driven by methodological limitation (eg, lack of data on dose and duration of HM intake, adjustment for confounders).15

Nevertheless, studies conducted in the last decade confirmed that HM received during NICU stay is positively associated with a better brain development on MRI at term equivalent age, with a correspondingly better neurodevelopmental outcome.

Specifically, Belfort et al demonstrated that preterm infants fed with more than 50% of breast milk for the longer period in the first month of life had a greater grey matter volume at term equivalent age and better IQ, academic achievement, working memory and motor function at 7 years of age.16 Later studies in the last 5 years confirmed the differences in brain structure of preterm infants fed with breast milk: Ottolini et al found a larger total brain, amygdala–hippocampus and cerebellum volumes, with greater white microstructural organisation17; Parikh et al found a decreased risk of diffuse white matter abnormality18; Blesa et al showed a better developed white matter tract and improved structural brain connectivity, with a dose–effect relation.19 Lastly, in 2022, a large longitudinal cohort study confirmed that higher breast milk intake during hospital stay after preterm birth is associated with higher IQ, reading and math scores, fewer attention deficit hyperactivity disorder (ADHD) symptoms at 7 years of age.20 The association was stronger for infants born at lower gestational ages (eg, <30 weeks’ gestation) and was dose dependent as the children gained 1 point on the reading scale per each additional 25 mL/kg of HM received daily, which means 6–7 points gained by infants on exclusive HM feeding (160–180 mL/kg/day).20

The specific mechanism of action of breast milk on neurodevelopment has long been studied by experts, to understand which factor has the highest effect on brain maturation. Of particular interest are HM oligosaccharides (HMOs), which are the third most abundant breast milk component, after lactose and lipids. They are non-digestible complex carbohydrates, which could be divided in three main families (non-fucosylated neutral HMOs, fucosylated HMOs and sialylated HMOs) depending on the group added to the core structure, with a high diversity into them, counting over 200 different HMOs. Their concentration and composition in breast milk vary between age of gestation at delivery and time of lactation, with colostrum and premature milk having the highest concentration.21 Moreover, the concentration and composition of HMOs in mother’s milk depend on the expression of specific genes (‘Le gene’ and ‘Se gene’), depending on which women are defined as ‘secretors’ (Se+) or ‘non-secretors’ (Se−) or Lewis positive (Le+) or Lewis negative (Le−). The ‘secretors’ produce a specific enzyme which permits the synthesis of 2’-FL (2’-fucosyllactose), instead the ‘Lewis positive’ synthesises the LNFP-II (lacto-N-fucopentaose II). Around 60–70% of women are ‘secretors’ and their milk is richer in HMOs,22 among which the dominant one is 2’-FL.

Roles of HMOs are several: they have an antimicrobial effect (binding pathogens or blocking them from entering gut epithelium), a prebiotic role driving microbiome development and, together with post-biotics, they regulate the immune system development and lastly modulate the brain maturation (through the gut–brain axis).21 A recent narrative review found that in breastfed term infants, neurocognitive development at 18 and 24 months of age is positively associated with HMO exposure, while it is clear the influence of maternal secretor status on neurodevelopmental outcomes.23 Only one study until now investigated the correlation between the exposure to HMOs contained in mother’s milk and neurodevelopment at 2 years of age in a cohort of exclusively breastfed very preterm infants.24 Rozè et al included 137 preterm infants and found that neither total HMOs nor any individual HMO correlated with neurodevelopmental score at discharge and at 2 years except for LNFP-III exposure from Se+/Le+ mothers.24

Finally, numerous studies have been conducted over time to assess the benefits of donated breast milk in the case of lack of milk from the mother’s own milk for preterm infants. The most recent studies, including an important randomised controlled trial (RCT) published in JAMA and including 483 extremely preterm infants, have shown that fortified donated milk does not confer better neurodevelopmental outcomes at 22 and 26 months of corrected age when compared with formula for preterm infants (cognitive, language and motor scores on the Bayley Scales of Infant and Toddler Development, neurodevelopmental impairment and cerebral palsy incidence were the same in the two groups).25 The same result was obtained by the 2018 Cochrane systematic review, which concluded with moderate evidence that the data did not show an effect of donor milk on neurodevelopment.26

HM fortification

Although HM has been recognised as the best food for the newborn, its macronutrient, micronutrient and energy content are insufficient to meet the nutritional needs of most preterm infants, particularly when using donated breast milk. For this reason, the use of multicomponent fortifiers derived from cow’s milk has entered common practice in the NICU and it is recommended by scientific societies in order to promote better growth in young patients.14 Indeed, the available evidence shows a positive effect on growth in weight, length and head circumference, while it is insufficient to demonstrate an effect on long-term neurodevelopment as the trials included in the recent meta-analysis were generally small and methodologically weak.27 Therefore, there is an urgent need for well-designed clinical trials to assess potential benefits of tailored HM fortification on neurodevelopmental outcomes, especially with the potential of novel quantitative brain MRI techniques.28

Recently, HM-derived fortifiers have been marketed, but further high-quality studies are needed to recommend the routine use and to evaluate their effect on preterm infants’ neurodevelopment.14

Preterm formula

Thanks to the advancement of technologies and the increase in our knowledge regarding the benefits of breast milk (fresh, expressed or donated), formulas for artificial feeding are produced in such a way as to imitate HM as much as possible. Despite this, formulas with different nutritional characteristics are available on the market.

Evidence coming from studies conducted in 1990s first demonstrated that providing preterm infants with formulas enriched with macronutrients and micronutrients was beneficial on weight gain, head growth, brain size and later neurodevelopmental outcome in infancy and at school age when compared with standard formula.29 In particular, Lucas et al showed in series of randomised prospective trials how preterm infants fed with enriched formula had better motor scores and social maturity at 18 months and lower incidence of IQ <85 at 7–8 years of age when compared with standard formula.30 From these publications, it became clear that undernutrition during vulnerable periods of brain growth could have long-term neurodevelopmental consequences, and preterm formulas began to be fortified to meet the high nutritional needs of the preterm infant during hospitalisation.

Qualitative and quantitative differences in brain MRI have been demonstrated between infants who received formula with enriched composition.11 In particular, formulas with higher levels of long-chain polyunsaturated fatty acids (LCPUFAs) (docosahexaenoic acid (DHA) and arachidonic acid), choline and sphingolipids were associated with increased levels of myelin development, corresponding to better cognitive performance. The role of these nutrients in myelin composition and development was already been proposed by previous clinical and preclinical studies.31 Nevertheless, a recent RCT failed to demonstrate any improvement in neurodevelopmental outcomes in a cohort of preterm and term infants at risk of neurodevelopmental impairment (intrauterine growth restriction, IVH, WMI, hypoxic-ischaemic encephalopathy) when supplemented with DHA, choline and uridine-5-monophosphate, even though mean cognitive and language scores were higher in the treatment group, as the adaptative behaviour score.32

In addition to multicomponent enriched formulas, several studies evaluated more specifically the effect of single components of the preterm formula on neurological outcomes.

The beneficial effect of LCPUFA-supplemented formula on visual and neurodevelopmental and physical growth was not confirmed by a Cochrane meta-analysis conducted in 2016 and including 17 trials with 2260 preterm infants.33 Moon et al did not find any significant effect of supplementation on Bayley Scales of Infant Development at 12 months and 18 months, nor on visual acuity or on physical growth. The overall quality evidence was low and the heterogeneity between trials was consistent, and this could have influenced the pooled results. Moreover, the infants enrolled in the trials were relatively mature and healthy as the median gestational age was 30 weeks with a median birth weight of 1300 g. The same result was obtained by considering term infants34 and the absence of effect of DHA supplementation was confirmed also on VLBW infants followed up at 8 years with brain MRI and parenteral report on behavioural problems.35 At the same time, Tam et al demonstrated instead an association between early postnatal red blood cell DHA levels and improved microstructural white matter development on MRI and decreased IVH incidence, corresponding to better neurodevelopmental scores at follow-up of a cohort of 60 preterm newborns.36 This increases the focus on the timing of DHA administration, giving importance to prenatal and perinatal supplementation in order to influence developmental processes and early injuries such as IVH.

Another class of components studied were HMOs. It has been demonstrated that supplementing infant formula with HMOs (in particular with 2’-FL), the composition and effects of the formula become similar to those of breast milk (better growth, reduced number of respiratory infections, better gut microbiome composition, reduced plasma levels of proinflammatory cytokines),37 but no clinical study has so far demonstrated a direct effect on neurocognitive development, although this can be presumed from preclinical studies (improved neuronal connectivity, long-term memory and potentiation).21 Until now, only one study analysed the effect of HMO supplementation on preterm infants: Hascoët et al38 conducted an RCT which included 86 preterm and VLBW infants (43 received HMO supplement with 2’-FL and lacto-N-neotetraose; 43 received glucose-based placebo) and reported a significant better growth in length and head circumference in the HM group. No neurodevelopmental outcomes were reported.

Micronutrients

It is well-known that undernutrition and in particular macronutrient deficiencies in early infancy could lead to poor neurodevelopmental outcomes. Instead, there is still a debate around the role that micronutrient deficiencies could have on later neurocognitive functioning.39 Micronutrient could be given to the newborn infant through breast milk, composition of which may be sensitive to dietary intake of mothers, or through infant formulas or micronutrient supplementation.

Iron is an essential micronutrient for growth and particularly for brain development. In fact, it is essential not only for blood oxygen-carrying capacity but also for the functioning of numerous enzymes and cellular processes involved in myelination, neurotransmitter production, neuronal energy status and thyroid regulation. A deficiency of this micronutrient in the neonatal period can alter hippocampal development and therefore cognitive functions such as learning and memory capacity, processing speed and socioemotional regulation in term infants.8 Confirming this, a beneficial effect on neonatal and infant neurodevelopment has been demonstrated in case of prenatal or postnatal iron supplementation for maternal and neonatal anaemia.40

The preterm infant is at increased risk of anaemia and impaired brain development; thus, scientific societies recommend a routine supplementation starting from 2 weeks of age for VLBW infants.14 Indeed, iron supplementation is associated with reduced incidence of anaemia and need for transfusion, but evidence is scarce regarding possible long-term beneficial effects on neurodevelopment and optimal timing and dosing.

In an RCT conducted on preterm infants in the 90s, early iron supplementation (14 days of life vs 61 days of life) seemed to be associated with fewer long-term neurological abnormalities on physical examination and a trend to higher mental processing composite score at 5 years, even if the study was underpowered to demonstrate a statistically significant effect.41 Two other studies evaluated the use of iron supplementation on cognitive outcomes. Friel et al found no differences on Griffiths’ Development Assessment at 3, 6, 9 and 12 months between neonates given higher or lower iron supplementation.42 Similarly, Ohls et al found no differences at neurodevelopmental and cognitive functions between infants supplemented with erythropoietin and iron compared with iron alone.43 Recently, a post-hoc analysis of the randomised Preterm Erythropoietin Neuroprotection Trial including 692 extremely preterm infants demonstrated a positive association between cognitive scores at 2 years of age and cumulative enteral iron intake at 60 days of life. Difference in motor and language component was not significant for different iron intake at 60 days. Any difference was found in the three components (cognitive, motor and language) for different enteral iron intake at 90 days.44 Actually, an RCT (NCT04691843) is ongoing evaluating whether individualised iron supplementation using higher doses of oral iron (3–6 mg/kg/day) started earlier (starting from 7 days of life) could improve neurodevelopmental outcomes of extremely premature infants when compared with standard iron dose (4 mg/kg/day) started at least at 2 weeks of life.

Lutein is another important micronutrient for brain and vision development. It belongs to the carotenoid family and its antioxidant properties are particularly important for premature babies, who are exposed to OS and free radical production during their stay in the NICU and have reduced antioxidant defences.45 The retina and brain are organs that are particularly susceptible to the formation of free radicals due to their active metabolism and richness in polyunsaturated fatty acids46 and so susceptible to OS damage. Lutein is transferred to the fetus transplacentally (principally in the last trimester) and to the newborn through breast milk. For this reason, preterm infants are exposed to lower levels of this important antioxidant that can help in preventing retinal damage due to OS. In particular, thanks to its molecular shape, lutein can integrate into cellular membranes with two orientations (either perpendicularly or parallel in the bipolar membrane), protecting neural tissues from OS and inflammation by neutralising free radicals, modulating the inflammatory response, absorbing high-energy short wavelengths and influencing gap junctional communications.45

Even if better cognitive performances have been demonstrated in children and adults who have received higher doses of lutein through the diet, to date, there are no precise indications of supplementation of this micronutrient in preterm infants as the current evidence on this population is considered insufficient.14

Iodine, in the form of thyroxine and triiodothyronine, influences the development of neural tissue prenatally and the myelination process postnatally. Considering that 10–40% of infants born before 28 weeks of gestation could present with transient hypothyroxinaemia, two RCTs enrolling 1394 preterm infants (<32 weeks’ gestation) compared the effect of iodine enteral or parenteral supplementation versus placebo on mortality and neurodevelopmental assessment at 2 years (Bayley score). Analyses found no effect and a Cochrane meta-analysis has concluded that there is currently no convincing evidence of beneficial clinical effects of iodine supplementation of preterm infants.47

Lastly, choline is also a vital micronutrient for brain development. It is a precursor of acetylcholine (neurotransmitter which regulates neuronal proliferation and maturation, synapse formation), a substrate of phosphatidylcholine and sphingomyelin (fundamental for myelination) and a source of methyl groups (essential for methylation and thus gene expression).48 Maternal supplementation during pregnancy has been demonstrated to significantly accelerate infant information processing speed and visuospatial memory in the first year of life in term born infants.48 Thus, despite the paucity of studies, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition recommends that preterm formulas allow a daily intake of choline of 8–55 mg/kg/day, while they do not recommend supplementation in breastfed preterm infants.14

Components with an effect through immune system and microbiota

Given that hypoxia and inflammation are the major determinants of brain and particularly white matter lesions, it is clear that any nutritional supplementation that reduces these two mechanisms could indirectly lead to an improvement in neurodevelopment.

In the last decade, the strong interconnection between gut microbial structure and brain development has been extensively studied. There is now evidence of the so-called gut–brain axis, according to which intestinal microorganisms and the metabolites produced by them (together called ‘gut microbiome’) can regulate the inflammatory response and OS, mechanisms responsible for neurological damage of preterm infants.49 The perinatal period is the most important window for the action that the microbiota may have on brain development. The maturation of the gut microbiome proceeds hand in hand with brain development: it begins in utero, through the placenta and the digestion of amniotic fluid, and continues after birth through feeding.50

The premature infant after birth is in an unfavourable situation both neurologically (increased vulnerability and increased risk of damage) and intestinally (use of antibiotics, lack of breast milk, prolonged fasting, mechanical ventilation), characterised by delayed colonisation, lower degree of bacterial diversity (dysbiosis) and an imbalance of bacterial species to the detriment of non-pathogenic ones (Bifidobacterium and Bacteriorides).51 Dysbiosis could lead to an imbalanced neuroactive metabolite production such as short-chain fatty acids (SCFAs), tryptophan metabolites, glutamate, GABA and dopamine. These could then cross the blood–brain barrier and influence the neurodevelopment, increasing the risk of developing behavioural psychiatric conditions in older age (eg, ADHD, autism spectrum disorders).51 Consistently, a recent multicentre prospective observational study made on the EPIPAGE-2 cohort in France demonstrated that specific bacterial intestinal patterns at week 4 of VLBW infants are significantly correlated with worse neurological outcome at 2 years of age.52

The exact mechanism of microbiota metabolites on the brain is not so well-known. Studies have shown that SCFAs can reduce inflammatory processes by modulating innate and adaptive immune cells.49 In the animal model, SCFAs could attenuate white matter impairment in neonatal hypoxic-ischaemic rat models favouring the differentiation of oligodendrocytes or inhibiting the production of specific proinflammatory chemokine.53

Considering this, it is reasonable thinking that adding prebiotics (microbial nutrients) to preterm infants’ feeds could lead to an improvement of intestinal microbiota promoting the growth of beneficial bacteria and at least improving neurological outcomes. Nevertheless, evidence is still lacking on this point. In fact, if it has been well demonstrated that they can improve the intestinal growth of beneficial microbes, no studies still proved an effect on the preterm brain.54 Two recent studies failed to demonstrate a significant protective effect of prebiotic supplementation on neurodevelopmental outcomes of preterm infants.55 56

Similarly, probiotics in Se (such as Lactobacillus, Saccharomyces and Bifidobacterium) could influence brain development both through microbiome–gut–brain axis and by improving feeding tolerance, peristalsis, absorption and lastly, growth. Again, the available literature is conflicting. Two recent systematic reviews with meta-analyses including, respectively, 50 and 30 trials on preterm infants showed that probiotics may have little or no effect on neurodevelopmental outcome.57 58 They attributed the lack of effect to variability throughout the studies included in type of probiotic strain, dose and duration of the supplementation, age and methods of neurodevelopmental assessment. Moreover, there was a concern in the sample size, as only few of the trials included the follow-up for neurological outcomes (five trials). Lastly, the certainty of the evidence was assessed as low.

Nutritional intake

Early nutritional intake has been studied and recommendations have been changed through the years. Recent evidence showed that higher caloric and lipid intake (with lipids providing one-third of total energy) during the first 2 weeks of life of a preterm infant is associated with less severe brain injury and dysmaturation on brain MRI at term equivalent age.59 Additionally, the same intake predicts a better brain development, both in grey matter (with a higher total brain, basal nuclei and cerebellum volumes) and white matter (with accelerated microstructural maturation), corresponding then to better neurodevelopmental outcomes at 18 months’ corrected age.60 Consistently, a small RCT showed a better white matter maturation (that appears as lower mean diffusivity at brain MRI) in VLBW infants treated with an enriched diet.61

In addition to a preventive nature, nutrient intake also appears to play a role in cases where brain damage has already established itself. Dabydeen et al conducted an RCT to verify if high-energy and protein diet (120% of recommended intake) was better than average recommended intake in preterm infants with perinatal brain damage. The trial was terminated after the inclusion of just 16 patients because of the statistically significant difference in the occipitofrontal head diameter, weight, length and greatest growth of corticospinal axonal diameter. Unfortunately, they did not analyse neurodevelopmental outcomes.62