1. Introduction
Breastfeeding is the natural and best feeding type for infants, providing not only nutrition but improving the general health of the mother-infant dyad [
1]. Besides the perfect balance of nutrients and water in an amount matching the infant’s needs, human milk contains a myriad of bioactive components, including immunoglobulins, hormones, oligosaccharides, and others [
2]. Human milk oligosaccharides (HMOs) are a complex group of free glycans synthesized by the lactating mammary gland composing the third major solid fraction of human milk, after lactose and lipids [
3,
4]. Emerging evidence has shown that HMOs act as prebiotics [
5,
6,
7], antimicrobials [
8], prevent pathogen binding and infections [
9,
10,
11], modulate the immune system [
12] and also may support brain development [
13,
14].
HMOs are composed of the monosaccharides glucose (Glc), galactose (Gal),
N-acetylglucosamine (GlcNAc), fucose (Fuc) and
N-acetylneuraminic acid (Neu5Ac). So far, about 150 individual HMOs have been identified [
15], yet approximately 90% of the HMOs fraction is composed of less than 20 different structures [
16,
17]. Nearly all HMOs contain lactose in the reducing end, which can be elongated by the addition of GlcNAc and Gal to form type 1 (Galβ1-3GlcNAc) or type 2 (Galβ1-4GlcNAc) chains in β1-3 or β1-6 linkages, producing core structures. Additionally, Fuc and Neu5Ac can be attached to the HMO core or directly to the lactose reducing end [
18]. According to the monosaccharides present in the structure, HMOs can be classified into three main groups: 1. neutral core, containing Glc, Gal and GlcNAc; 2. neutral fucosylated, containing one or more Fuc units; and 3. acidic, containing one or more Neu5Ac units.
The composition and concentrations of HMOs are unique in the milk of each woman and strongly depend on the activity of the Secretor (
Se) and Lewis (
Le) genes in the mammary glands.
Se and
Le genes encode the enzymes α1-2-fucosyltransferase (FUT2) and α1-3/4-fucosyltransferase (FUT3), respectively, involved in the biosynthesis of fucosylated HMOs [
19,
20]. Mutations on the
Se gene inactivate FUT2, and consequently, milk from non-secretor (Se−) women contain no or only traces of α1-2 fucosylated HMOs. Mutations on the
Le gene inactivate FUT3, and consequently, milk from Lewis-negative (Le−) women contain no or only traces of α1-4 fucosylated HMOs [
21]. Based on the activity of the FUT2 (
Se) and FUT 3 (
Le) enzymes in the lactating woman, HMOs composition can be classified into four phenotypes: 1. Se+Le+, the most common, containing α1-2 and α1-4 fucosylated HMOs, such as 2’-fucosyllactose (2’-FL) and lacto-N-difuco-hexaose I (LNDFH I); 2. Se−Le+, which contain α1-4 fucosylated HMOs, such as lacto-N-difuco-hexaose II (LNDFH II), but does not contain α1-2 fucosylated HMOs, such as 2’-FL, lacto-N-fucopentaose I (LNFP I), difucosyllacto-N-hexaose c (DFLNH c) and LNDFH I; 3. Se+Le−, which contain α1-2 fucosylated HMOs, such as 2’-FL and LNFP I, but does not contain α1-4 fucosylated HMOs, such as DFLNH c, LNDFH I and II and; 4. Se−Le−, the least common phenotype, containing neither α1-2 nor α1-4, but only α1-3 fucosylated HMOs, such as 3’-fucosyllactose (3’-FL) and difucosyl-para-lacto-N-neohexaose (DFpLNnH), which occur in all the four
SeLe groups, since their synthesis apparently is not influenced by the
Se and
Le genes [
22,
23]. Besides the influence of the
Se and
Le genes in the composition of HMOs, great differences in HMOs concentrations occur in the milk of women with the same genetic background [
24]. The variability within
SeLe groups indicates that other factors besides the activity of
Se and
Le genes may be involved in HMOs biosynthesis influencing their concentrations in human milk. However, little is known about the influence of non-genetic factors on HMOs composition and concentrations and only a few studies have addressed this question. There is some conflicting evidence about the influence of gestational age and lactation time on HMOs concentrations [
23,
25,
26]. Recently, maternal factors such as parity and body mass index (BMI), as well as environmental factors such as geographic location have been associated with HMOs concentrations [
27,
28,
29].
The understanding of the influencing factors on HMOs composition and concentrations is important since some of the HMOs effects on infant health have been related to specific structures and usually in a dose-dependent manner. For example, infants whose mother’s milk had low concentrations of α1-2 fucosylated HMOs presented a higher incidence of Campylobacter, calicivirus and moderate-to-severe diarrhea than those whose mother’s milk contained higher concentrations of α1-2 fucosylated HMOs [
11]. Higher concentrations of α1-2 fucosylated HMOs also have been related to a lower risk of allergy at 2 and 5 years of age in infants with high hereditary allergy risk [
30]. A lower total HMOs concentration and a higher proportion of 3’-sialyllactose (3’-SL) have been correlated with higher HIV transmission in Zambian infants [
31]. Higher total HMOs concentrations were also associated with reduced mortality in HIV-exposed uninfected Zambian infants [
32]. Furthermore, disialyllacto-N-tetraose (DSLNT), an acidic HMO, prevents necrotizing enterocolitis (NEC) in rats and lower amounts of DSLNT in human milk may predict the risk of NEC in preterm infants [
33,
34]. Furthermore, infant weight, body composition and nutritional status also have been associated with concentrations of specific HMOs [
35,
36].
Bearing this in mind, we developed this cross-sectional, observational study to measure absolute HMOs concentrations in a cohort of Brazilian mothers, characterize HMOs profiles and identify maternal and infant factors associated with HMOs concentrations.
4. Discussion
There are currently a few studies reporting HMOs absolute concentrations obtained by state-of-the-art analytical methods—such as LC-MS—in well-defined human milk samples [
47]. Knowing the exact amount of the most representative HMOs is important to access the daily intake of HMOs by the infants and the biological effects of these molecules, since some HMOs may act in a dose-dependent manner [
31,
32,
34,
48,
49]. In this study, we presented absolute concentrations of 16 representative HMOs measured by LC-MS from 78 full-term, mature human milk samples classified according to the
SeLe phenotype and investigated associations between maternal and infant characteristics and HMOs composition/concentrations.
The frequency of the
SeLe phenotypes varies among different ethnic populations. In Caucasians and Asians, the Se+Le+ phenotype (determined from human milk and blood samples) varies from 55 to 73%, Se−Le+ varies from 20 to 31%, Se+Le− varies from 6 to 11% and Se−Le− varies from 3 to 5% [
25,
27,
50,
51]. In African population from Burkina Faso, the
SeLe distribution (from saliva samples) was reported to be 54% (Se+Le+), 14% (Se−Le+), 25% (Se+Le−) and 7% (Se−Le−) [
52]. A study in a semi-isolated Black community in Northern Brazil reported a very similar
SeLe phenotypes (from saliva and blood samples) distribution to Burkina Faso [
53]. A study conducted in the state of São Paulo, Brazil [
54] with 827 participants regardless of ethnicity revealed a prevalence of Se+Le+ phenotype (from blood samples) in 78% of the population, similarly with the Se+Le+ prevalence in our cohort (76%; from human milk samples), which was also composed by different ethnicities. Our cohort presented a
SeLe distribution closer to the Caucasian/Asian than the semi-isolated Black from Northern Brazil. In our cohort, only one lactating mother was assigned to Group 4 (Se−Le−), which agrees with the rare occurrence of this phenotype in Caucasians and Asians [
22,
27].
Our study demonstrated a difference in HMOs profile and concentrations among
SeLe groups, supporting previously reported results [
23,
25,
26,
27,
55]. Furthermore, our results showed that the different HMOs composition of the 3
SeLe groups had no influence on infant’s weight gain and anthropometric parameters, which reinforces that human milk is nutritionally adequate, apart from the different HMOs composition. Similarly, Sprenger et al. (2017) did not observe differences in anthropometric parameters between infants who consumed breast milk with low or high 2’-FL content (Se− and Se+ milk, respectively) [
56]. However, as in the study of Sprenger et al. (2017), our cohort was composed of healthy term-born infants. Charbonneau et al. (2016) reported a difference in HMOs composition from Se− mothers having healthy or severely stunted infants, which was not observed in Se+ mothers [
36]. Therefore, the influence of
SeLe milk groups on other populations, such as preterm or malnourished infants remains to be studied.
In a systematic review of 21 studies on HMOs concentrations, Thurl et al. (2017) reported TF-LNH, 2’-FL, DF-LNH II, and LNFP I to be the most abundant neutral HMOs and 6’-SL the most abundant acidic HMO in Se+ term human milk. However, the authors warned about a possible bias on the high concentrations of TF-LNH and DF-LNH II, which needs to be confirmed [
47]. The systematic review reported concentrations of 33 HMOs and the results corroborate that the 16 HMOs analyzed in our study are among the most representative in human milk, except for DFLNHc and DFpLNnH (in term milk), which were not reported in the systematic review [
47]. Our results largely agree with the concentrations reported in the above-mentioned systematic review and with Kunz et al. (2017), not included in the review [
26]. The most abundant HMO in Se+Le+ e Se+Le− was 2’-FL, followed by LNDFH I in Se+Le+ and by LNFP I in Se+Le−. Among the acidic HMOs, we also observed the highest concentration of 6’-SL not only in Se+ but also in all
SeLe groups. However, Kunz et al. (2017) reported a higher LNT concentration in Se−Le+ than in Se+Le+ and Se+Le−, which was not observed in our cohort.
Although our samples were collected in a single time point, we could observe some of the dynamics in HMOs concentrations during lactation reported in longitudinal studies, investigating correlations of HMOs concentrations and time postpartum. A significant increase in 3’-FL concentrations during the first months of lactation, as well as a significant decrease in LNFP I and LSTc have been previously reported [
23,
29,
47,
57,
58,
59,
60]. We observed a significant positive correlation between 3’-FL and time postpartum and significant negative correlations between several HMOs—including LNFP I and LSTc—and time postpartum in Se+Le+ milk (
Table 5). As in the study of McGuire et al. (2017) [
28], 6’-SL, LSTc and LNH negative correlations with time postpartum were also observed. Total acidic HMOs presented the highest negative Spearman rank correlation coefficient (r = −0.79) with time postpartum in Se+Le+, which is in accordance with a significant decrease of acidic HMOs in the first three months of lactation reported in the systematic review of HMOs concentrations [
47]. However, although associated with the variability in the concentrations of some HMOs, time postpartum does not explain variability alone. For example, the HMO with the highest coefficient of variation in Se+Le+ milk was LNDFH II (CV 293%,
Table 4), but the correlation of LNDFH II concentrations with time postpartum had a low correlation coefficient (r = 0.27,
Table 5), although significant. This is even more evident with DFpLNnH in Se+Le+, with a CV of 154% (
Table 4), but without significant correlation with time postpartum (r = 0.01,
Table 5).
There are some hypotheses about the role of HMOs on infant allergy development, related to the establishment of the gut microbiota, but the influence of maternal allergic diseases on HMOs composition and concentrations have not been substantially studied [
30,
61,
62,
63]. In our study, we investigated whether maternal allergic disease (asthma, rhinitis or eczema) could explain, at least in part, the great variability in the concentrations of HMOs observed in each
SeLe milk group. We observed significantly higher DFpLNnH concentrations in the milk from Se+Le+ and Se+Le− women with allergic disease than in those without allergic disease. When we considered secretor status alone, there were no differences in DFpLNnH concentrations or another HMO between the groups. Sjögren et al. (2007) [
62] performed—in a smaller cohort—the only study previously published that investigated associations between maternal allergic diseases and HMOs concentrations and found no differences in the concentrations of nine neutral HMOs from colostrum of allergic and non-allergic mothers. However, DFpLNnH was not included in the analysis [
62]. Furthermore, in our cohort, Se+ mothers presented a higher prevalence of allergic disease than Se− mothers, which agrees with a higher prevalence of asthma and a higher susceptibility to asthma exacerbation observed in Se+, particularly in blood group O/Se+ individuals [
64,
65]. On the other hand, a 1968 study reported no differences in the incidence of allergic diseases between Se+ and Se− individuals [
66]. Similarly, Sprenger et al. (2017) did not observe differences in allergy prevalence on mothers presenting or not 2’-FL in the milk (Se+ and Se−, respectively) [
30].
We observed differences in 2’-FL and 3’-FL concentrations between overweight and eutrophic Se+ mothers, as well as a positive correlation between 2’-FL concentrations in Se+ milk and maternal BMI. However, the difference in 2’-FL and 3’-FL concentrations from mothers with distinct nutritional statuses was no longer significant when considering the Lewis phenotype together. Positive correlations between maternal BMI or weight and total HMOs or 2’-FL concentrations have been previously reported [
27,
28]. It has been hypothesized that maternal diet may influence HMOs concentrations, yet Azad et al. (2018) found no associations between the overall diet quality and HMOs concentrations [
29]. More studies are needed to verify dietary and nutritional effects on HMOs composition and concentrations.
To date, few studies have investigated associations between parity and HMOs concentrations, without agreement. While Elwakiel et al. (2018) found no associations [
27], Azad et al. (2018) observed higher LNT and LNnT and lower 3’-FL concentrations in multiparous mothers [
29]. Interestingly, we observed a significant positive correlation between parity and some HMOs—including LNT+LNnT—and a negative correlation between parity and 3’-FL, similarly to Azad et al. (2018), but only in Se+Le− mothers (
Table A2). However, Se+Le− was a small group, which may limit conclusions. We found no correlations between HMOs concentrations and parity in other
SeLe groups.
We observed unprecedented significant differences (
p < 0.04) in some HMOs concentrations between Se+Le− milk from mothers of boys and girls, even with a small number of mother-infant pairs in Se+Le− group. As previously reported, we found no associations between HMOs concentrations and socioeconomic status as well as with the type of delivery in any
SeLe group [
27,
29,
59].
Exclusively breastfed infants present lower weight gain—although adequate—during the first year of life than formula-fed infants [
67,
68]. There is a possible effect of breastfeeding on obesity prevention, related to the lower weight gain in the first year of life, but the mechanisms involved in this protection are not yet understood [
69]. Importantly, the intricate composition and high amounts of HMOs are responsible for crucial differences between human milk and infant formula, which may contribute to the differences in health outcomes between breastfed and formula-fed infants [
4,
70]. HMOs may influence infant growth and the development of obesity directly since they are absorbed into the circulation and can induce systemic effects [
71,
72]. A study demonstrated that the HMO lacto-N-fucopentaose III modulates metabolic functions by improving glucose tolerance, insulin sensitivity and suppressing lipogenesis in the liver of diet-induced obese mice [
73]. Additionally, HMOs may also modulate infant growth and obesity development indirectly by altering the gut microbiome [
36,
74]. Inverse associations between concentrations of individual HMOs and infant body composition have been previously reported. Alderete et al. (2015) observed that each 1 µg/mL increase in LNFP I concentration was associated with a 0.4 g lower infant weight at 1 month postpartum. At 6 months, inverse associations were also observed between LNFP I and body weight, lean and fat mass, as well as between LNnT and fat mass [
35]. A higher proportion of LSTc in milk was associated with a lower infant’s weight-for-age (WAZ) score in the study conducted by Davis et al. (2016), who also observed that a higher proportion of 3’-SL, LNFP I + III and DFLNHa contributed positively to WAZ and height-for-age scores [
75]. In our study, although the infants—all exclusively breastfed—presented adequate weight gain, we demonstrated inverse associations between concentrations of specific HMOs in breast milk—including LNFP I, LNT+LNnT and LSTc —and infant weight and weight gain at 1 month after birth (
Table 5). The observed inverse associations between HMOs concentrations and infant weight gain suggest a potential role of HMOs on infant growth and metabolism, which deserves future investigations.
Among the major strengths of our study are the accurate, extensively validated method utilized for HMOs quantification and the standardized human milk collection procedure, which occurred in short time intervals to minimize natural diurnal variations and biases caused by milk sampling. Another important strength of our study is that our population was composed by exclusively breastfed infants, since the consumption of solid foods and water besides breastfeeding can impact infant growth, adding some bias on the results about the influence of human milk components on infant’s health. However, our study has some limitations. Although we selected 16 representative HMOs from the ~150 different structures occurring in human milk, it is possible that maternal and infant factors are associated with other HMOs not included in our analyses. The utilization of the ISAAC questionnaire for the screening of maternal allergy is also considered a limitation, however, there is no validated instrument available to be applied in the adult population. Furthermore, the small number of Se−Le+, Se+Le−, and Se−Le− mothers impaired more robust conclusions regarding these groups.