Important Insights into Human Milk Oligosaccharides

• Human milk oligosaccharides (HMOs) are non-digestible complex carbohydrates that are highly abundant in human milk. More than 200 different HMOs have been identified, a reflection of the diversity across this biologically active component of human milk. _^1,2

• A recent study led by dsm-firmenich scientists brings to light a unique view into the dynamic and variable nature of HMO composition.³
• dsm-firmenich’s research team utilized an innovative approach to determine the most abundant HMOs observed globally in term human milk throughout the course of lactation. The learnings gained offer an important perspective on HMO innovation in infant nutrition. 

Human milk is the gold standard for early life nutrition, and human milk oligosaccharides (HMOs) are the third largest solid component of human milk. HMOs are increasingly being recognized for their importance for infant health and development.^4,5

With clinical evidence for the ability of HMOs to support a balanced microbiome and the immune system,^6,7 these oligosaccharides have recently been added to infant formulas, offering some of the benefits of human milk to formula-fed infants. As this innovation in infant nutrition unfolds, scientists continue to research HMOs to more fully understand why they are such a prominent component of human milk and their role in early human development.

As human milk composition is the inspiration for infant formulas, it is essential that infant-targeted HMO innovation is informed by HMO levels and concentrations in human milk. To this end, a group of dsm-firmenich scientists recently completed research to assess and rank HMO concentration from healthy mothers throughout lactation at a global level. The team was led by Buket Soyyilmaz, MSc, Associate Scientist, and included contributions from Marta Hanna Miks, PhD, DSc, Senior Regulatory and Scientific Affairs Manager, and Christoph Rohrig, PhD, Head of HMO Regulatory Affairs, among other research assistants.

The team aimed to illustrate the considerable variability and the dynamic nature of HMOs across lactation. Dr. Rohrig explains, “HMOs are not static ingredients; their concentrations change throughout the course of lactation. Further, since there are upwards of 200 HMOs in human milk, as individual HMOs change, the collective HMO composition flexes and varies continuously – it is quite dynamic.”

 Soyyilmaz provides an example: “The concentration of total and most individual HMOs will peak shortly after birth, but then decline as lactation continues. For instance, HMOs like 2-fucosyllactose (2'FL) are most abundant in colostrum, but then exhibit a steady decrease as time goes on.^3,8,9 Conversely, we see 3-fucosyllactose (3-FL), an HMO with a similar structure, steadily increase over time – reaching concentrations at 24 months that are ten times higher than those at one month.” ^8,9 Even after the declines described above, 2'FL likely remains the most abundant HMO in human milk at later stages of lactation for most mothers.^3,8 This insight suggests a key role for 2'FL in infant growth and development.¹⁰

In addition to the effects of time, individual genetics plays an important role in the composition and variability of a mother’s HMO profile. According to Dr. Miks, “Differences in HMO composition between mothers is driven largely by genetics, and specifically, by the expression of two important enzymes called fucosyltransferases – referred to as FUT2 and FUT3. The activity of FUT2 and FUT3 is influenced by a woman’s Secretor (Se) and Lewis (Le) blood group status.^4,11,12 Of note, polymorphisms exist in the general population for expression of these enzymes, which means that some mothers possess active genes for one or two of these enzymes, while others do not.” This results in four different milk phenotypes, which gives us four different categories of typical HMO composition.^4,12

Table 1: Milk Groups According to Blood Group and Secretor Status ^3,4,12,13

The four different milk phenotypes are observed in different frequencies throughout the population. It is well-established that approximately 80% of the global population carries the secretor gene – this group is called “secretors” – while approximately 20% do not and are referred to as “non-secretors.”^3,4

Each of the four different milk phenotypes or groups have a somewhat characteristic HMO composition. Given the frequency of women who have the secretor gene, HMO profiles that are associated with secretor phenotypes are the most abundant.

Additional factors influence HMO composition as well, but Dr. Miks explains that the influence of other variables is not yet well-understood. Some of the other elements involved include a mother’s age, health status and diet, an infant’s gestational age at birth, and to some degree, geography.^3,14

Beyond the desire to understand how HMOs change with time and across geography, other research has sought to understand if different HMO profiles result in different health outcomes in infants. While this question remains to be definitively answered, several observational studies suggest there may indeed be links between maternal HMO composition and infant health outcomes.

Lewis et al., reported that infants of secretor mothers had a more rapid colonization of bifidobacteria and suggested this may be due to the HMO profile associated with the secretor phenotype.¹⁵ This finding is consistent with those of the study done by Smith-Brown et al., which also observed a positive association between secretor status and abundance of bifidobacteria.¹⁶ In contrast, another study found the Bifidobacterium genus to be highly enriched in infants of non-secretor mothers.¹⁷

Similarly, infant diarrhea incidence has been linked to mothers’ secretor status.^18,19 Additionally, maternal obesity has been linked to HMO concentration, which in turn has been associated with infant growth during the first six months of life.²⁰ Collectively, these studies suggest an interplay of several variables that ultimately drive HMO composition and an infant’s microbiome and health outcomes. 

Soyyilmaz provides some context for these areas of clinical investigation: “While the data emerging from these and other observational studies suggest intriguing associations between the genetic factors that influence HMO composition and infant health outcomes, inconsistent findings illustrate that the science is not well-established on the impact of a mother’s secretor status on infant health.”

Several studies already describe the concentrations and changing patterns of HMOs, but these assessments are at least partly centered around mothers’ secretor status. ^9,21-24 Taking a different approach, dsm-firmenich’s research team aimed to determine the most abundant HMOs and describe their corresponding concentrations observed globally throughout lactation in term human milk, regardless of variations caused by genetic and non-genetic factors. In this sense, the authors utilized a pooled methodology, combining data on milk samples from all relevant and reliable HMO concentration records they identified.

Although a matter of debate within the scientific community, Soyyilmaz explains, “The advantage of evaluating mean HMO levels in pooled milk is to provide a general proxy that fits the entire population on average. We opted for a mean approach with the aim to see the most abundant HMOs observed on a global scale.” Further, she states “Given the complexity of HMO data, we decided a more pragmatic approach for understanding the most abundant HMOs overall would bring value to the body of literature on HMO levels in human breast milk.”

The general trends identified from dsm-firmenich’s study are in alignment with other studies, i.e., there is an overall decline in total HMO concentration over the course of lactation, with the exceptions of a few individual HMOs increasing over this same time frame.

Their analysis aimed to illustrate the relative proportions of individual HMOs in mature human milk – defined in the study as milk produced during days 14-90 of lactation – in the context of total HMO amounts overall. The results of this analysis are described by Soyyilmaz: “Although there are a large number of individual HMOs, roughly 200, a relatively low number of HMOs make up the majority of the total HMO fraction in human milk. Specifically, it appears that the top six HMOs make up over 50% and the top fifteen make up over 75% of the total HMO component in human milk. These data also tell us that a high number of HMOs must be found in relatively low amounts.”³

The data resulting from dsm-firmenich’s analysis provides meaningful target levels for studying the addition of HMOs to infant formula. Dr. Rohrig tells us, “The pooled approach provides universal addition targets for testing these important oligosaccharides in infant formulas. The secretor or non-secretor status of a mother is more likely the result of an evolutionary biological effect that balances any related benefits between the mother and the infant. Thus, the pooled data show us meaningful levels across the entire population. These levels, then, serve as the guide for testing beneficial outcomes when HMOs are added to infant formula. Additionally, understanding the variability of HMOs helps us set safe levels for the general population.”

Geography is commonly discussed as a factor that influences HMO composition. Previous studies have shed light on the potential influence of geography on HMO profile. For example, McGuire et al., reported that 3-FL levels in Swedish mothers were three times higher than those of mothers in rural Gambia.²⁵ Zhou and colleagues have compiled data that describe the dynamic changes in HMOs specific to the Chinese population.²⁶

dsm-firmenich’s scientists provide some additional context for the role of geography in HMO composition. “Observed regional differences in HMOs are primarily a function of local differences in genetic prevalence, and thus, geography could be thought of as a genetic influence on HMO composition,” explains Dr. Rohrig.  Soyyilmaz agrees, stating “According to available data, differences across geographies do appear, but the significance of them is not fully understood. Certainly, HMO profiles are not identical across various countries, but overall, the most abundant structures appear to be the same.”³ The group agrees that more regional HMO quantitation studies with larger sample sizes are needed. These would be necessary to understand if there are true geographical differences in HMO profiles.

While the body of evidence in support of the role of HMOs in early life nutrition is robust, the scientific community is interested in further research that might drive a more tailored approach to the application of HMOs. The dsm-firmenich researchers shared their perspectives on other questions they hope are explored through clinical studies: “The global levels in pooled human milk provide a good basis for how HMO innovation in infant formula should be approached. The knowledge gained from our study serves as a practical guide for those conducting clinical trials to assess the benefits of HMO supplementation. Human milk is always the standard for infant nutrition, yet we are collectively also responsible for demonstrating that the addition of novel ingredients to infant formula results in meaningful improvements to infant health.” 

The studies completed on HMO composition so far provide ample evidence of the variability, variety, and complexity of HMOs in human milk. dsm-firmenich scientists will continue to gather data on HMO levels to seek answers to some of the questions that persist around the dynamics of HMO levels and concentrations. Infant formula innovation is continuously driven by the goal of achieving a product that is compositionally and functionally closer to human milk. Persistence in uncovering the finer details of the HMO profile that is most representative of healthy mothers all over the globe is a worthy goal.  

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