Keywords

1 Introduction

For mammals, following birth, the source of their entire nutrition is milk. Milk provides a transition between placental nutrition and an adult diet. Weaning is the stage of moving onto an adult diet. Evolutionary pressures in mammals have led to milk which performs many complex functions, displays dynamic characteristics and enables molecular signalling to occur. Thus, breast milk can optimally support the infant’s growth needs [1]. The nutritional environment at this early stage creates epigenetic modifications which may programme metabolism in a way that predisposes individuals in the longer term to metabolic disorders [2]. Detailed research in recent years has shown that micro-RNA (miRNA) transcripts may function in cell-to-cell molecular signalling [3,4,5]. These transcripts consist of RNA sequences with a typical length of around 20 nucleotides. They are highly conserved within phyla. A miRNA sequence binds to a matching messenger RNA (mRNA) sequence, preventing the mRNA being translated into the protein for which it encodes [6]. A new approach makes use of miRNA as a diagnostic probe, allowing identification of targets and visualisation [7]. Milk is secreted by lactocytes in the mammary gland as a fluid containing an unusually large volume of both RNA and miRNA [8]. The miRNAs are encased within an exosomic protective vesicle encased by a lipid bilayer and measuring around 100 nm in diameter. This extracellular vesicle is excreted by the lactocyte [9]. miRNA is thus transported and may be taken up into the infant’s cells by the process of endocytosis [10,11,12]. The exosomes within both breast milk and milk from other mammals, such as cows, act therefore to permit genetic material to enter the body of the infant or whoever else consumes the milk.

It is regrettable that the epigenetic modification that can be achieved by breast milk through transfer of miRNA is not possible for infants fed using artificial baby milk. However, there is some contamination of the food chain caused by human consumption of cows’ milk, and it seems that the efforts to increase the amount of milk cows can produce for human consumption is also exposing consumers to greater amounts of bovine miRNA [13, 14]. This ongoing exposure of human consumers to bovine genetic signals may risk inducing epigenetic modifications that predispose to severe chronic metabolic problems including obesity and diabetes mellitus type 2.

2 Milk Exosomes

Exosomes contained in breast milk are amongst the most essential ways for the mother–infant dyad to communicate during breastfeeding [15]. The initial isolation and characterisation of exosomes in human colostrum and breast milk was undertaken by Admyre et al. [16]. Following this study, milk exosomes have been noted to occur in the colostrum and mature milk of a number of mammals apart from humans, in particular the cow, buffalo, goat, pig, Macropus eugenii (a wallaby, i.e. marsupial mammal) and rodents [17,18,19]. Exosomes in milk either enter milk directly from lactocytes or enter via the cytoplasmic crescents within the fat globules, which appear to contain exosomes [20]. The miRNA that occurs at the highest level, miRNA-148a, is a significant ingredient in skimmed milk, both as globules and within exosomes [21,22,23].

3 How Well Do Milk Exosomes Survive Passage Through the Gut?

There is increasing evidence to show that milk exosomes are not degraded within the gut. The exosome performs an important function in protecting miRNA and ensuring it can be delivered in a stable form to the infant. Exogenous, artificially produced miRNA is broken down by the unfavourable conditions within the gut, but miRNAs performing immune functions and protected within exosomes survive much better [21]. When commercially produced cows’ milk was examined, the milk miRNA remains stable when subjected to acid exposure, RNAse degradation or freezing, but did break down when detergents were applied or bacteria were permitted to ferment the milk [22, 24].

4 Biological Availability of miRNA Derived from Milk

The bioavailability of miRNAs derived from cows’ milk was at the level of biological significance when consumed at a level typical of a normal diet by human volunteers, as evidenced by the altered genetic expression within circulating polymorphonucleocytes [25]. In another study, piglets were fed either colostrum or mature sow milk. Colostrum contained higher levels of miRNAs of immunological significance and piglets receiving this type of milk had higher circulating levels of the miRNAs in question. Thus, bioavailability depends on dose [20]. In newly born wallabies (M. eugenii) the circulating levels of particular miRNAs known to be secreted in milk are significantly higher than in adult animals, which supports the idea the miRNAs in the circulation have been absorbed from ingested milk [26]. A study which combined genomics with a computerised analysis concluded that transfer of miRNAs from milk to the plasma probably occurred [27]. When mice were force-fed each day with cows’ milk containing exosomes labelled by a fluorescent probe, the label was detectable both in the ileal epithelium and in the cells of the spleen [28]. In a recent paper, Manca et al. demonstrated the bioavailability of bovine milk exosomes tagged with a fluorescent probe, using a murine model [29, 30]. A significant finding was that some of the exosomes were absorbed without any change in their packaging. There was hepatic and splenic accumulation of the milk-derived exosomes. In research investigating the fate of exosomes in the diet using a murine model, exosomes in bovine milk containing RNA to which a red-coloured GLOW label was attached were noted in central nervous, renal, pulmonary and hepatic tissues, leading the authors to the conclusion that RNA in the diet can ultimately reach tissues at distant sites in the body [29, 30].

5 Artificial Baby Milk Is Low in miRNAs

Cows’ milk which had undergone sterilisation and evaporation to render it suitable for feeding infants first entered the market in 1929. Doctors in the 1920s saw breast milk as a food just like any other, and many mothers soon took up the option to feed their infants formula rather than rely on breastfeeding [31, 32]. Until more recently, it was unknown that breast milk could function to pass miRNAs from the mother to the neonate. At present, artificial milk lacks the majority of miRNAs found in breast milk. Thus, formula milk does not offer the protective benefits that breast milk does in terms of immunity and programming of the metabolism [12]. When Chen et al. compared artificial baby milk with untreated bovine milk, they noted the formula milk contained only a tenth as many miRNA-148a sequences as the unpasteurised milk [33]. This finding of depleted miRNAs in powder-form artificial baby milk was also noted by Golan-Gerstl et al. [34].

6 Homologous Features of miRNA Shared by Different Species

There is considerable preservation of miRNA sequences and seed sequences between different mammalian species, which is especially clear in the case of miRNA-148a-3p, as has been demonstrated in recent research [35]. Thus, the miRNA signals in mammals appear to go back far into the evolutionary past. The seed sequences for miRNA-148a-3p are the same in both cows and humans. The base pairing between the heptameric seed sequence and the complementary mRNA sequence on the DNMT1 messenger RNA transcript is very strong. If a miRNA sequence binds weakly to an mRNA sequence, inhibition of mRNA translation occurs, but if the base pairs match up exactly, the mRNA is disassembled by the cellular machinery [4]. It seems likely that consumption of miRNA-148a-3p leads to downregulation of DNMT1 mRNA expression and thus its gene product.

The p53 transcription factor performs such an important role that it is sometimes referred to as a genomic guardian [36]. There is close control on how the p53 gene (TP53) is expressed both at the level of transcription and after the mRNA has been translated [28, 37, 38]. A number of miRNAs present in large quantities in milk [20, 21, 24] act to downgrade mRNA transcribed from the TP53 gene, namely miRNA-125b, miRNA-30d and miRNA-25 [28, 37, 38]. The function of miRNA-125b in regulating p53 exhibits preservation of molecular interactions for all members of the subphylum Vertebrata [24]. Rather surprisingly, there are no differences between humans and cows in the seed sequence of this particular miRNA.

7 How Genes Involved in Development Are Activated Through CpG Demethylation of DNA

7.1 FTO

Since milk provides the entire nutritional requirements of the developing infant, it is to be expected that it will contain some mechanism capable of directing the transcribing and translating of appropriate genes. There is indirect evidence suggesting that breast milk can activate the FTO enzyme (fat mass and obesity-associated protein), which controls transcription. It can also activate translation under the control of mTORC1 (mechanistic target of rapamycin complex 1). FTO is an enzyme which catalyses the demethylation of N6-methyladenosine (m6A) contained in messenger RNA sequences. Messenger RNA bearing m6A is found in many cellular processes and greatly influences the composition of the transcriptome in eukaryotic cells. It regulates the splicing, export, addressing, translation and stabilisation of mRNA. M6A methylation/demethylation has a role in many processes under the control of RNA, such as development, circadian cycles and resetting of the programmed cellular function. Typically, RNA bearing m6A is thought to inhibit the expression of particular genes and thus formation of the gene product. The m6A modification is most common on stop codons as well as on the three prime untranslated regions. These sequences are targeted by members of the human YTH domain family 2 (YTHDF2) in the course of selecting which mRNAs should be degraded. The modification also prevents mRNA from attaching to human antigen R (HuR) which increases the stability of mRNA [27, 39,40,41,42,43,44,45,46,47].

A mutated allele for m6A demethylase FTO resulting in a non-functional protein causes humans with the mutation to have delayed growth following birth. Similarly, when the FTO gene was silenced in a murine model, the animals put on less weight, exhibited metabolic changes and had delayed growth. On the other hand, when the FTO gene was overexpressed, also in a murine model, the mice gained weight and laid down more fat in proportion to the degree of overexpression. These changes were related to the animals overfeeding and eventually becoming obese. Thus, it seems clear that growth following birth and energy balance fall under the vital control of FTO. In mice with the overexpressed FTO gene, the numbers of RNA sequences with an m6A modification were reduced overall. There are already associations known between SNPs within the initial intron of the FTO gene and raised BMI, fat deposition and development of diabetes mellitus type 2. The FTO protein is overactive both in individuals with specific SNPs and in cases where there has occurred an epigenetic modification of the gene as a result of CpG demethylation at certain points within the initial intron. Certain miRNAs found within exosomes in milk, specifically miRNA-148a, miRNA-152, miRNA-21 and miRNA-29 s, potentially fulfil a key function in epigenetic modification of the FTO gene by blocking CpG demethylation at key points on the gene. This then means FTO expression goes up and more of the mRNA can be transcribed [48,49,50,51,52,53,54,55,56,57,58,59,60].

7.2 Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)

The actions of FTO occur in close conjunction with those of mTORC1, a kinase whose function alters depending on nutrient levels. mTORC1 increases translation and anabolism in response to milk. It becomes activated in the presence of leucine, in particular, through the action of another enzyme, leucyl-tRNA synthase. FTO participates in the activation. In particular, the activity of mTORC1 in response to high levels of amino acids can only occur due to FTO-related demethylation events. Thus, the fact that components of milk modify how the FTO gene is expressed through epigenetic modifications means that this mechanism also has downstream effects on the activity of mTORC1.

Furthermore, there are other epigenetically mediated ways in which milk affects the activity of mTORC1, including translation of mRNA. Another transcription factor, the behaviour of which is controlled epigenetically, is nuclear factor erythroid 2-related factor 2 (NRF2). When the DNA methyltransferase enzyme is inhibited, NRF2 activity rose, both in terms of mRNA transcripts produced and gene products synthesised. This effect was mediated through NRF2 demethylating DNA. NRF2 can directly activate transcription of the MTOR gene. This gene encodes the core protein contained within the mTORC1 and 2 molecular complexes. NRF2 also stimulates RagD, a low molecular weight G-protein that makes mTORC1 more active. Furthermore, miRNA-29 production goes up under the influence of NRF2, resulting in a downgraded level of DNMT3B. This last event acts as positive feed forward for epigenetic modifications enhancing the expression of NRF2 [36, 61,62,63,64,65,66,67,68,69,70].

7.3 The Insulin Gene (INS)

Insulin plays a major role in stimulating anabolic metabolism through signals regulated by PI3K-mTORC1. Following consumption of milk, the circulating insulin level rises. Transcription of the INS (insulin) gene is controlled by methylation of the DNA sequence encoding for the gene. According to Kuroda et al., the demethylated INS is expressed in pancreatic beta cells, whilst methylated copies of the gene remain unexpressed [66, 68]. In cells synthesising insulin, the INS promoter region in particular is demethylated. Indeed the CpG demethylation of this region of the gene appears essential for the beta cell to fully differentiate and for insulin to be expressed only within a certain tissue. Accordingly, miRNAs which downregulate the activity of DNMT, namely miRNA-148a, miRNA-21 and miRNA-29s and which originate in milk may exert control over the activity of mTORC1 on translation and anabolism (see Fig. 2). This suggestion is supported by recent evidence showing that, in mice, miRNA-29a positively regulates the release of insulin in vivo [69].

7.4 Insulin-like Growth Factor-1 (IGF1)

Insulin-like growth factor-1 (IGF1) acts in a similar manner to insulin. It is the most potent factor controlling growth and acts by stimulating the mTORC1 signalling pathway [65,66,67,68,69,70,71,72,73]. A diet containing milk results in a significant rise in the circulating IGF1 level. Children who drink bovine milk attain a greater height. Ouni et al.[72] undertook a study investigating the effect of CpG methylation on the promoter regions for IGF1 (i.e. P1 and P2), since they already knew that this gene is responsible for growth following birth. They wondered if demethylation could account for the different levels of IGF1 seen in the plasma of children as they grow. Results showed that, where six particular CpGs, which occur proximally in the P2 region, were methylated, the circulating IGF1 level and the child’s growth were both lower. The level of transcription occurring at the P2 region in the circulating polymorphonucleocytes of patients who are administered growth hormone has a negative association with the degree of CpG methylation in that region of the gene [65,66,67,68,69,70,71,72,73].

The cumulative evidence from transcriptomic studies points to the conclusion that miRNAs in milk play a normal role in increasing the activity of signals which themselves activate mTORC1, namely mTOR, FTO, insulin and IGF1. The miRNAs achieve this through influencing epigenetic modification by (de)methylation.

7.5 Caveolin 1 (CAV1)

This molecule is a protein embedded in a microdomain of the plasma membrane. It can modify signals depending on the context in which they occur. CAV1 has interactions with the insulin and IGF1 receptors (IR and IGF1R), which encourages these signals to be transduced. When CAV1 is bound to low density lipoprotein receptor-related protein 6 (LRP6), the complex takes on a signalling function and can activate both the IGF1 receptor and IR, leading to strengthening of signals by Akt-mTORC1. CAV1 is, surprisingly, strongly expressed following demethylation of the initial exon and intron of the gene. This change is noted to occur when adipocytes are maturing. One possibility is that miRNAs derived from milk and targeting DNMTs actually strengthen signalling by insulin, IGF1 and mTORC through their epigenetic modificatory effects on the CAV1 gene, which mean CAV1 synthesis increases. The consequence of this is then that exosomes in milk are absorbed in higher quantities [74,75,76,77].

7.6 FOXP3

FOXP3 exerts overall control over transcriptional events in regulatory T-lymphocytes (Tregs). These lymphocytes play a key role in ensuring the immune system does not target self-antigens (i.e. does not cause autoimmunity to occur). They also prevent immunoreactivity against innocuous allergens in the environment, including in the diet. Thus, they are anti-allergenic. It has been demonstrated that when milk-derived exosomes are presented to circulating polymorphonucleocytes, the levels of Tregs expressing FOXP3 rose. The presence of this protein is sufficient to indicate that the T lymphocytes expressing it possess a unique role in immunosuppression. FOXP3 is expressed at a fixed rate when certain epigenetic switches are set on Tregs in the Treg-specific demethylated region (TSDR) of the FOXP3 gene. This region is rich in CpG sequences. Indeed, FOXP3 is only steadily expressed where this section of the DNA is demethylated. In recent studies, it has even been noted that the extent of demethylation within the TSDR region correlates directly with the level of FOXP3 in the cell. Accordingly, the TSDR is considered a key location for epigenetic control of FOXP3. If the epigenetic modifications do not occur here, Treg numbers may fluctuate widely. On the other hand, if the FOXP3 gene region contains an excessive level of methylation, Tregs fail to prevent allergic responses from developing. It is noteworthy that patients with a tendency to allergic disorders have fewer Tregs in which the FOXP3 region is demethylated [78,79,80,81,82,83,84,85,86,87,88].

DNMT1 and DNMT3b both act on the FOXP3 gene in CD4+ T lymphocytes. When DNA methyltransferase action was prevented by the enzyme inhibitory agent decitabine, the TSDR region remained relatively demethylated and FOXP3 was expressed steadily and at high levels. This result suggests that expression of FOXP3 can be reliably regulated at the epigenetic level by preventing DNA methyltransferase activity. This then results in adequate numbers of well-functioning Tregs. MiRNAs capable of downgrading DNMT1 and DNMT3b (i.e. miRNA-148a and miRNA-21, and miRNA-148a and miRNA-29b, respectively) and entering the body via milk exosomes are accordingly believed to participate in the epigenetic regulation of FOXP3 and thus are of major importance in stabilising the generation of competent Tregs. The evidence is growing that miRNAs found in milk exosomes play a key part in epigenetic modulation and thus influence the immunological behaviour of the gut and the body as a whole [92–96].

A study that was recently undertaken in the Netherlands examined the relationship between hypermethylated DNA generally and allergic sensitivity to bovine milk. Children with and without an allergy to milk were compared. The TSDR of FOXP3 was more highly methylated in those cases where an allergic response to milk mediated through IgE was known to occur than in children who no longer exhibited an active allergic response or in whom allergy to milk had never occurred. In a rodent model involving atopic very young rats, the rat pups were given either rat milk or artificial formula. The rat milk contained the normal level of miRNAs, unlike the formula milk. When the mesenteric lymph nodes were examined for FOXP3 expression, the pups fed rat milk had higher levels. The circulating IgE specific for beta-lactoglobulin was also lower in the rats fed rat milk. Seemingly, miRNAs have the effect of inhibiting DNA methylation and thus ensuring FOXP3 is expressed at higher levels. In such a situation, Tregs can efficiently induce immunotolerance. Thus, all evidence points to miRNAs in milk exosomes functioning to regulate FOXP3 expression at the epigenetic level, which then has a key role in halting the development of autoimmune and allergic reactions. It also seems reasonable to suppose that breast milk, through its effects on Tregs, lays the ground for appropriate immunotolerance of non-self-innocuous food antigens when the child is eventually weaned [89,90,91,92,93,94,95].

7.7 NRA4

There are a number of receptors within the NR4A (nuclear receptor subfamily 4a) grouping where the endogenous ligand is unknown. This subfamily is contained within the nuclear receptor superfamily. These receptors all function as transcription factors and can switch gene expression on and off, thereby regulating an intricate system of interacting pathways by which signals are transduced within the cell. A number of members of the NR4A subfamily, notably NR4A1, NR4A2 and NR4A3 have all been implicated in modulating the growth of Tregs via an effect upon FOXP3. These receptors have a direct action on the promoter region of FOXP3. When NR4A members are stimulated artificially, Tregs begin to be produced, hence they may be considered ‘nursing factors’ in the growth of this cellular population [96,97,98,99,100,101].

Milk has a potential role in regulating immunological development in the infant through an epigenetic mechanism and may also regulate how the NR4A subfamily is expressed. When the promoter region underwent CpG demethylation and the surrounding histones where hyperacetylated, the result was a rise in the synthesis of the luteinising hormone receptor (LHR). When the promoter region of NR4A3 was methylated, the gene stopped being expressed, but demethylation of the same region leads to upgraded synthesis. Moreover, the effect of deactivating the HDAC1 and 3 histone deacetylases is to increase the transcription of the NR4A3 gene. DNMT1 has been frequently associated with the histone deacetylases and there are interactions between these enzymes. An important way in which methylated regions of DNA are silenced is by the interaction of methyl CpG binding protein 2 (MeCP2) with sections of methylated CpGs. In part this restriction of transcription by MeCP2 relies on the synergistic activity of histone deacetylases. To take an example, the H19 imprinting control region is not transcribed when MeCP2 and a histone deacetylase act jointly on it. If miRNA-148a from milk exosomes can downgrade the activity of DNMT1, neither MeCP2 nor the histone deacetylase can function and the histone will be hyperacetylated. This hyperacetylated state upgrades transcription of genes involved in development, in particular the NR4A orphan receptors [102,103,104,105,106,107,108].

7.8 Nuclear Factor Kappa B (NF-κB)

Milk importantly influences the health of the gastrointestinal system in newborns. Breastfed premature babies are at lower risk of necrotising enterocolitis (NEC) than bottle-fed infants. There is an association between NEC and a raised level of certain pro-inflammatory cytokines (especially interleukin 1 (IL1) and tumour necrosis factor alpha (TNFα)) in the peripheral circulation. These effects occur through the action of Nuclear Factor Kappa B (NF-κB). In a study involving a tissue culture model of NEC, the supernatant from breast milk was applied to plates cultured with Caco-2 gut epitheliocytes. The effect of doing so was to prevent interleukin-1β, IL-6 TNFα from being expressed. Expression of these cytokines is stimulated by NF-κB and promotes an inflammatory response. One of the key molecules with the ability to deactivate NF-κB is IκBα. It achieves this inhibition by blocking signals that precede migration of NF-κB into the nucleus. Instead, the proteins remain within the cytoplasm and lack activity. Additionally, NF-κB is prevented from attaching to DNA, a vital step in its function. IκBα is expressed more strongly by epithelial cells of the gut which lack DNA methyltransferase activity than in those where these enzymes are highly active. The extent to which the promoter region of NfκBI is CpG methylated strongly affects how the NfκB functions in gut lining cells. A high degree of demethylation results in IκBα being highly expressed, whilst extensive methylation has the opposite effect. Epithelial cells without DNMTs exhibit less activity by NFκB. Since miRNAs derived from milk decrease the level of DNMT synthesis, the anti-inflammatory effect of breast milk may occur because a lower level of demethylation occurs. This then means IκBα is relatively more active and NF-κB less active as a result. The actions of miRNAs in breast milk thus resembles that of glucocorticoids, which exert their effects through enhancing IκBα activity [109, 110].

7.9 The Lactase Gene (LCT)

For virtually all other mammals than humans, lactase is no longer synthesised within the gut once the young have been weaned onto adult food. Humans are unusual in this regard, namely by virtue of showing lactase persistence. The mechanism by which the expression of LCT declines so massively with age in certain humans, but not all, is not understood currently. However, the latest evidence suggests that the varying levels of mRNA transcripts of LCT expressed in different people, different cell populations or different species are accounted for by an epigenetic mechanism. It appears highly probable that the reason some adults continue to transcribe mRNA from LCT as adults is thanks to epigenetic regulation of the LCT gene. The ongoing expression of LCT in European adults has a strong association with a particular SNP in MCM6, a gene located next to the LCT region. The SNP consists of 13910C>T within intron 13. The effect of the substitution is to promote expression of the LCT gene. There are 7 different sites where epigenetic control of LCT occurs, including that at the 13910 location. Individuals who do not exhibit lactase persistence are found to have a greater extent of methylation within intron 13, the opposite being the case for those individuals demonstrating lactase persistence and therefore continuing to express a significant amount of lactase [110,111,112,113,114,115].

One possible explanation of how lactase remains available for the duration of breastfeeding is that miRNAs regulating expression of DNMTs thereby ensure intron 13 remains relatively demethylated and thus actively transcribed while breastfeeding occurs. Once breastfeeding ceases, lactase levels then would fall since miRNAs no longer prevent the silencing of the LCT gene, and it is no longer needed. In the Neolithic period, humans may have continued milk consumption after weaning and this then meant a selective pressure favouring those individuals with the 13910C>T mutation. This mutation might ensure that intron 13 remains demethylated and thus the LCT gene continues to be translated into lactase.

8 Conclusion

The earlier medical view of breast milk as a straightforward source of nutrition has altered to one in which milk is seen as a complex medium carrying both nutrients and key communicative signals between mother and child. These signals are the way the youngest children’s metabolism is programmed. A major part of the signalling occurs through transfer of miRNAs via exosomes and fat globules in milk. These miRNAs are secreted by the lactocytes of the breast. Exosomes are capable of avoiding degradation in the gut and are endocytosed by the gut lining, eventually reaching the systemic circulation of the child. The miRNA expressed in the highest amount is miRNA-148a, noted in both human and bovine milk exosomes and fat globules, and known to downgrade the activity of DNMT. This enzyme plays a pivotal role in epigenetic control. miRNA-125b is a further miRNA with major effects and it regulates p53, a protein responsible for the integrity of genomic DNA. P53 has multiple effects on different processes. The fact that exosomes containing miRNAs are largely lacking in artificial baby milk but present in bovine milk, which may continue to be consumed long after infancy, raises some concerns about potential ill-effects on the health of people over the longer term [116, 117].

The current era, in which genomic studies are becoming prevalent, offers the chance that individuals at risk of specific disorders can be identified before the condition itself manifests. Genetics alone does not explain predisposition, however, since there are epigenetic modifications and environmental causes to consider. The influence of the environment is especially strong during in utero life and in the neonatal period. It is hypothesised that changes to the metabolism at this early stage in life produce effects at much older ages. One key environmental effect to consider is diet. An essential component of avoiding diseases linked to lifestyle at later ages is for neonates and infants to be breastfed. There is a steadily accumulating evidence base showing that being breastfed at this stage reduces the likelihood of the individuals becoming obese, hypertensive, dyslipidaemic or exhibiting insulin insensitivity as they grow older. Nonetheless, for a complete understanding to emerge, there need to be well-controlled studies for long periods, so that the benefits of breast milk can be properly appreciated [116, 117].