Human milk oligosaccharide secretion dynamics during breastfeeding and its antimicrobial role: A systematic review

Abstract

BACKGROUND

Human milk oligosaccharides (HMOs) are bioactive components of breast milk with diverse health benefits, including shaping the gut microbiota, modulating the immune system, and protecting against infections. HMOs exhibit dynamic secretion patterns during lactation, influenced by maternal genetics and environmental factors. Their direct and indirect antimicrobial properties have garnered significant research interest. However, a comprehensive understanding of the secretion dynamics of HMOs and their correlation with antimicrobial efficacy remains underexplored.

AIM

To synthesize current evidence on the secretion dynamics of HMOs during lactation and evaluate their antimicrobial roles against bacterial, viral, and protozoal pathogens.

METHODS

A systematic search of PubMed, Scopus, Web of Science, and Cochrane Library focused on studies investigating natural and synthetic HMOs, their secretion dynamics, and antimicrobial properties. Studies involving human, animal, and in vitro models were included. Data on HMO composition, temporal secretion patterns, and mechanisms of antimicrobial action were extracted. Quality assessment was performed using validated tools appropriate for study design.

RESULTS

A total of 44 studies were included, encompassing human, animal, and in vitro research. HMOs exhibited dynamic secretion patterns, with 2′-fucosyllactose (2′-FL) and lacto-N-tetraose peaking in early lactation and declining over time, while 3-fucosyllactose (3-FL) increased during later stages. HMOs demonstrated significant antimicrobial properties through pathogen adhesion inhibition, biofilm disruption, and enzymatic activity impairment. Synthetic HMOs, including bioengineered 2′-FL and 3-FL, were structurally and functionally comparable to natural HMOs, effectively inhibiting pathogens such as Pseudomonas aeruginosa, Escherichia coli, and Campylobacter jejuni. Additionally, HMOs exhibited synergistic effects with antibiotics, enhancing their efficacy against resistant pathogens.

CONCLUSION

HMOs are vital in antimicrobial defense, supporting infant health by targeting various pathogens. Both natural and synthetic HMOs hold significant potential for therapeutic applications, particularly in infant nutrition and as adjuncts to antibiotics. Further research, including clinical trials, is essential to address gaps in knowledge, validate findings, and explore the broader applicability of HMOs in improving maternal and neonatal health.

Key Words: Human milk oligosaccharides; Human milk oligosaccharides secretion dynamics; Antimicrobial activity; Breastfeeding; Synthetic human milk oligosaccharides; Pathogen adhesion inhibition; Infant health

Core Tip: This systematic review highlights human milk oligosaccharides (HMOs)' dynamic secretion patterns and antimicrobial properties (HMOs), emphasizing their crucial role in infant health. HMOs protect against many pathogens by inhibiting adhesion, disrupting biofilms, and impairing bacterial enzymatic activity. Synthetic HMOs, including 2′-fucosyllactose and 3-fucosyllactose, replicate the structure and function of natural HMOs, offering scalable solutions for therapeutic applications. HMOs also synergize with antibiotics, enhancing their efficacy and addressing antimicrobial resistance. These findings underscore HMOs' potential to develop innovative maternal and neonatal care interventions, improving global health outcomes.

INTRODUCTION

Breastfeeding is universally recognized as the gold standard for infant nutrition, offering unparalleled benefits to both infants and mothers. It supports optimal growth and development, reduces the risk of infectious and chronic diseases, and fosters a strong emotional bond between mother and child[1]. Human breast milk is a dynamic, bioactive fluid uniquely tailored to meet the infant’s nutritional and immunological needs, with its composition evolving across different stages of lactation to adapt to the changing requirements of the growing infant. Among its key components are human milk oligosaccharides (HMOs)[2].

HMOs are a diverse and abundant class of bioactive glycans uniquely present in human breast milk. As the third most prevalent solid component after lactose and lipids, HMOs support neonatal health by promoting gut microbiota development, protecting against infections, and modulating immune function[3]. These oligosaccharides are structurally diverse, comprising over 200 distinct types categorized into fucosylated, sialylated, and neutral HMOs. Their structural complexity and biological significance underline their unmatched contribution to infant nutrition, making them a focal point for research into maternal and neonatal health[4]. HMOs selectively promote the growth of beneficial gut bacteria, particularly Bifidobacterium species, while simultaneously acting as soluble decoy receptors that prevent pathogenic bacteria and viruses from adhering to epithelial cells. These dual functions enhance gut health and systemic immunity, establishing HMOs as a natural and indispensable component of breast milk[5]. Beyond their antimicrobial functions, HMOs contribute to immune system maturation, modulate inflammatory responses, and support gut homeostasis. Emerging evidence also links HMOs to neurodevelopment, potentially through their impact on the gut-brain axis mediated by microbial metabolites and direct signaling pathways[6].

The secretion dynamics of HMOs vary during lactation, with concentrations highest in colostrum and gradually declining over the first year. This dynamic secretion aligns with the infant's evolving developmental and immunological needs, tailoring nutrient delivery and bioactive benefits[7]. These insights are critical for guiding evidence-based breastfeeding strategies to optimize infant health outcomes. Additionally, the characterization of HMOs has spurred innovations in synthetic biology, enabling the production of bioengineered HMOs such as 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL). These synthetic variants replicate the structure and function of natural HMOs, facilitating their incorporation into infant formula and other therapeutic applications to bridge nutritional gaps for non-breastfed infants[8]. As of recent advancements, approximately 15 synthetic HMOs have been developed and approved for use in infant formula or research purposes. These include both commercially available HMOs and those in advanced stages of development[9].

In addition to HMOs, galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) are non-digestible carbohydrates often incorporated into infant formulas to mimic some of the prebiotic functions of HMOs. GOS, derived from lactose, predominantly stimulates the growth of Bifidobacteria and Lactobacilli, while FOS, extracted from plants such as chicory roots, fosters a broader spectrum of gut microbial diversity. Though structurally simpler than HMOs, GOS and FOS synergistically support gut health and immune development, complementing the protective effects of HMOs in both natural and artificial feeding scenarios[10].

This systematic review synthesizes current evidence on the secretion dynamics of HMOs during breastfeeding and their antimicrobial properties. By elucidating these mechanisms, we aim to inform the development of optimized nutritional interventions and guide future research in pediatric health and nutrition.

MATERIALS AND METHODS

Study design and protocol

This systematic review was conducted in accordance with the PRISMA guidelines. Studies were selected based on the following inclusion and exclusion criteria. Inclusion Criteria included peer-reviewed articles reporting on human, animal, or in vitro studies focused on the secretion dynamics of HMOs during lactation or their antimicrobial roles, studies investigating the antimicrobial properties of synthetic HMOs or bioengineered derivatives against pathogenic microorganisms, articles providing quantitative or qualitative data on HMO composition, secretion patterns, or interactions with microbial pathogens, and studies published in English between 1990-2024. We exclude articles unrelated to HMOs or lacking explicit data on secretion dynamics or antimicrobial activity, reviews, commentaries, opinion pieces, or case reports without original data, and studies focusing exclusively on oligosaccharides not structurally related to HMOs (e.g., plant-derived oligosaccharides).

Search strategy

A comprehensive search strategy was developed and executed across the following electronic databases: PubMed, Scopus, Web of Science, and Cochrane Library. The search strategy utilized a combination of Medical Subject Headings terms and free-text keywords, such as “human milk oligosaccharides,” “HMO secretion,” “synthetic HMOs,” “antimicrobial activity,” and “bioengineered HMOs.” Boolean operators (AND, OR) and filters were applied to refine the search. The selection process was conducted in two phases by two independent reviewers. The first phase included screening of title and abstract of articles for relevance based on the predefined eligibility criteria. The full text of potentially eligible articles were reviewed for inclusion. Any disagreements between reviewers were resolved through discussion or consultation with a third reviewer.

A standardized data extraction form was employed to collect the following information from included studies: (1) Study characteristics: Author (s), publication year, study type (human, animal, in vitro), and sample size; (2) HMO secretion dynamics: HMO concentrations, temporal trends, and influencing factors (e.g., maternal diet, lactation stage); (3) Synthetic HMOs: Production methods (e.g., bioengineering), structural composition, and efficacy; and (4) Antimicrobial activity: Pathogens studied, mechanisms of action, and experimental outcomes.

Two reviewers performed data extraction independently, and any discrepancies were resolved through consensus. Then, the methodological quality of the included studies was assessed using appropriate tools. For in vivo and clinical studies: Newcastle-Ottawa Scale. For in vitro studies: Modified checklist based on experimental rigor (e.g., controls, reproducibility). Each study was rated as high, moderate, or low quality based on criteria such as study design, data collection methods, and outcome measurement. Data were synthesized narratively to summarize trends in HMO secretion dynamics and antimicrobial activity. Quantitative data were pooled where appropriate and presented descriptively. Studies were categorized by experimental models (human, animal, in vitro) and HMO type (natural, synthetic). Meta-analysis was not conducted due to heterogeneity in study designs, methods, and outcomes. As this review relied exclusively on publicly available data, no ethical approval was required.

RESULTS

Study characteristics

Figure 1 shows the study's flow chart. This systematic review included 44 studies published between 1990 and 2024. These studies represented a wide geographical distribution, with contributions from specific regions, including North America (United States, Canada), Europe (Germany, United Kingdom, Sweden), and Asia (Japan, China, India). Studies encompassed diverse experimental designs, including in vitro analyses, animal models, and clinical investigations.

Figure 1 The flow chart of the included studies. HMO: Human milk oligosaccharides.

HMO secretion dynamics

The secretion dynamics of HMOs were found to vary significantly during lactation. Concentrations of dominant HMOs were highest in colostrum and declined throughout lactation. 2'-FL peaks at approximately 3-5 g/L in colostrum and decreases to 1-2 g/L by 6 months postpartum. Lacto-N-tetraose (LNT) ranges from 2-3 g/L in early lactation, tapering to 0.5-1 g/L after the first year. 3-FL is initially low in colostrum (0.2-0.5 g/L) but increases progressively, reaching 1.5-2 g/L by late lactation. Figure 2 clearly illustrates these patterns, depicting the temporal decline of fucosylated and sialylated HMOs alongside the gradual increase of neutral HMOs like 3-FL. This figure provides a visual summary of the dynamic shifts across different lactation stages, supporting the alignment of HMO composition with infant developmental needs. The temporal variation in HMO concentrations underscores their tailored role in meeting infants' evolving developmental, immune, and nutritional needs. Bioengineered HMOs such as 2'-FL and 3-FL demonstrated efficacy comparable to natural HMOs, offering scalable solutions for non-breastfed infants and therapeutic applications.

Figure 2 The temporal changes of 5 main human milk oligosaccharides during the first 8 months of lactation. 2′-FL: 2′-Fucosyllactose; 3′-FL: 3′-Fucosyllactose; 6′-SL: 6-Sialyllactose; DFL: DiFucosyllactose; LNT: Lacto-N-Tetraose.

Antimicrobial properties of HMOs

HMOs have demonstrated significant antimicrobial effects through multiple mechanisms. HMOs selectively target pathogens through adhesion inhibition, biofilm disruption, and enzymatic inhibition, highlighting their potential in combating infections and supporting gut health. 2'-FL reduced Pseudomonas aeruginosa adhesion by 24% in intestinal epithelial cell models. In addition, 2'-FL and 3-FL decreased Campylobacter jejuni (C. jejuni) adhesion by 26% and 18%, respectively. HMOs also can disrupt the bacterial biofilm. Neutral HMOs disrupted Streptococcus agalactiae [Group B Streptococcus (GB. streptococcus)] biofilm formation by up to 40%, significantly reducing bacterial growth and virulence. Moreover, neutral HMOs enhanced the efficacy of antibiotics like vancomycin and ciprofloxacin, reducing the required minimum inhibitory concentration (MIC) by 30%-50%.

Geographic distribution

The geographic distribution of studies revealed strong research representation from high-income countries, including the United States, Germany, and Japan, which contributed approximately 70% of the reviewed literature. Studies from low-and middle-income regions, including sub-Saharan Africa and Southeast Asia, constituted less than 10% of the included data.

DISCUSSION

Breastfeeding secretion dynamics in the first two years of lactation

Breastfeeding is a dynamic process, with the composition of human milk evolving significantly to meet the growing infant's changing nutritional and developmental needs. Over the first two years of lactation, human milk undergoes notable changes in volume, macronutrient and micronutrient content, bioactive molecules, and immunological components[11]. These changes reflect a complex interplay between maternal physiology, infant demand, and environmental influences (Table 1).

Table 1 Breastfeeding secretion dynamics in the first two years of lactation.

Stage of lactation	Time period	Milk volume	Key nutritional components	Key bioactive components	Primary functions	Additional notes
Colostrum	First 3-5 days postpartum	Low (10-40 mL per feed)	High in protein, low in fat and lactose	High IgA, lactoferrin, HMOs, leukocytes, growth factors (e.g., EGF)	Provides passive immunity, protects gut mucosa, supports gut microbiota development, aids intestinal repair	Proteins: Peak concentration, casein: whey ratio approximately 20: 80- HMOs: Highest (approximately 15-20 g/L)- Growth factors: Abundant EGF for gut development- Fats: Minimal but rich in essential fatty acids
Transitional milk	Days 5–14 postpartum	Increasing (approximately 500-750 mL/day)	Protein decreases, fat and lactose increase	Reduced IgA and leukocytes; lactoferrin and lysozyme persist	Supports growing caloric needs, continues immune protection, aids microbiota development	Proteins: Decline begins- Lactose: Increases to support energy needs- HMOs: Decline slightly but remain significant- fats: Gradual increase, influenced by maternal diet
Mature milk	Weeks 3 onward	High (700-900 mL/day)	Stable macronutrients: approximately 0.8%-1% protein, approximately 4%-5% fat, approximately 7% lactose	Balanced levels of HMOs, lactoferrin, lysozyme, and antibodies	Provides complete nutrition, supports immune and brain development, protects against infections	Proteins: Casein: Whey ratio shifts to approximately 50: 50- HMOs: Decline (approximately 5-10 g/L) but bioactive- Immune Factors: IgA, lysozyme, and lactoferrin persist- Fats: Higher variability based on maternal diet
Months 6-12	Late lactation phase	Gradual reduction (approximately 500-800 mL/day)	Similar macronutrient composition as mature milk	Persistent immune factors (IgA, lysozyme, HMOs, lactoferrin)	Complements solid foods, supports immune and microbiota maturation	Fats: Continue to vary, contributing to brain and energy needs- HMOs: Moderate levels (approximately 2-5 g/L)- Lactose: Steady for energy support- Immune Factors: Immune benefits adapted for growing infant
Months 12-24	Toddler phase	Further reduction (approximately 300-500 mL/day)	Macronutrients adapt to reduced dependence on milk	Sustained immune components (IgA, lysozyme, lactoferrin)	Provides immune protection, complements toddler diet, supports growth and immunity	Proteins: Further adapted to complement the solid diet- HMOs: Persist at low levels (< 2 g/L)- Immune factors: Support immune resilience- Fats: Important for energy and essential fatty acids

This table provides a detailed view of the changes in breastfeeding dynamics and their biological significance. EGF: Epidermal growth factor; HMOs: Human milk oligosaccharides; IgA: Immunoglobulin A.

Stages of lactation and secretory phases

Human milk production progresses through distinct stages: Colostrum (first few days postpartum), transitional milk (days 5-14), and mature milk (from week 3 onward). Each stage is characterized by specific changes in milk composition and volume[12].

Colostrum, often referred to as "first milk," is a thick, yellowish secretion produced in small volumes (10-40 mL per feeding). It is rich in immunological components, growth factors, and bioactive molecules[13]. Different maternal factors could affect colostrum composition. Multiparous mothers may produce colostrum with more consistent concentrations of bioactive molecules than first-time mothers. In addition, adequate maternal protein intake supports the rich protein concentration in colostrum. Furthermore, high cortisol levels postpartum enhance the production of growth factors such as epidermal growth factor (EGF), which is crucial for neonatal gut development[14].

As lactation progresses, colostrum transitions into transitional milk, with milk volume increasing significantly, reaching approximately 500-750 mL/day by the second week. During this phase, protein content begins to decline, while fat and lactose concentrations increase[2]. Although immunological components remain high, their relative proportions decrease. Transitional milk meets the infant’s growing caloric and nutritional demands as the gastrointestinal system matures[15]. The maternal diet, frequency of nursing, and maternal body composition significantly influence the quality and availability of these nutrients, including fat-soluble vitamins and essential fatty acids[16].

Mature milk represents the steady-state phase of lactation, typically beginning around the second month. By this stage, milk production peaks at 700-900 mL/day, with composition varying based on infant demand and influenced by various maternal factors. These include maternal body weight for height, dietary intake, parity, environmental conditions, return of menstruation, and nursing frequency. For instance, increased maternal protein intake has been linked to higher concentrations of protein-rich HMOs, supporting immune and microbial functions in the infant[17]. Mature milk comprises approximately 87% water, 7% lactose, 3%-5% fat, and 0.8%-1% protein[18]. It also contains vitamins, minerals, enzymes, hormones, and bioactive molecules such as HMOs, lactoferrin, and lysozyme. These components provide balanced nutrition to support growth and development while protecting against infections and promoting immune maturation[19]. The maternal diet, frequency of nursing, maternal health, stress, weaning practices, and body composition significantly influence the quality and availability of these nutrients, including fat-soluble vitamins and essential fatty acids[20]. Throughout all lactation stages, the interplay between maternal factors and milk composition underscores the importance of maternal health, nutrition, and breastfeeding practices in ensuring optimal benefits for the infant. This dynamic adaptation highlights the remarkable biological feedback system that aligns milk composition with the infant’s evolving needs[21].

Macronutrient dynamics over two years

The macronutrient composition of human milk undergoes significant changes throughout lactation to align with the infant’s developmental needs. Protein concentration is highest in colostrum (2-4 g/100 mL) and decreases over the first few months, stabilizing at around 0.8-1 g/100 mL in mature milk[18]. This decline reflects the infant’s reduced reliance on immune proteins and increasing caloric requirements from fats and carbohydrates. Key proteins, such as casein and whey (e.g., alpha-lactalbumin and lactoferrin), adapt to provide both nutritional and functional benefits[22].

Fat content varies throughout the day and during feeding sessions and is influenced by maternal diet and breastfeeding patterns. On average, fat concentration increases slightly over lactation, from approximately 3.5% in early lactation to 4.5%-5% by the end of the first year. The composition of fat globules evolves, with long-chain polyunsaturated fatty acids (PUFAs), such as DHA, playing a crucial role in brain and visual development[23]. Lactose remains the predominant carbohydrate in human milk, providing a consistent energy source for the infant. HMO concentration is highest in colostrum (15-20 g/L) and gradually decreases over the first year, although functional HMOs persist in significant amounts to support gut health and immunity[18] (Figure 3). Mature milk, which constitutes most lactation, stabilizes these macronutrient levels while adapting to infant growth spurts and developmental milestones. These changes are influenced by maternal factors such as diet, hormonal fluctuations, and nursing patterns, ensuring that the nutritional profile aligns with the infant’s evolving needs[18].

Figure 3 Levels of human milk oligosaccharides (g/L) during the various phases of Breast milk.

Immune component dynamics

Immunological factors in human milk are most concentrated during the early weeks of lactation, reflecting the neonate’s need for passive immunity. These components gradually decline but remain bioactive throughout the lactation period. Secretory immunoglobulin A (IgA), abundant in colostrum, provides mucosal immunity to the infant’s gut, respiratory tract, and other mucosal surfaces[24]. Leukocytes, including macrophages and lymphocytes, are highly concentrated in colostrum and decrease as the infant’s immune system matures. Lactoferrin levels are highest in colostrum, promoting iron absorption and exhibiting antimicrobial properties. Lysozyme levels, by contrast, increase during later lactation, enhancing protection against bacterial infections[25]. Cytokines such as interleukins (e.g., IL-10) and transforming growth factors (e.g., TGF-β) support immune modulation and gut development. EGF peaks in colostrum, facilitating intestinal maturation and repair[26].

Bioactive molecules and hormones

Human milk contains a variety of bioactive molecules and hormones that adapt to the infant’s developmental needs. HMOs are abundant in colostrum and transitional milk, with concentrations gradually declining over the first year. These molecules promote the growth of beneficial gut bacteria, protect against pathogens, and support immune development[19]. Metabolic hormones such as insulin, leptin, and adiponectin regulate energy balance and appetite in the infant. Cortisol levels decrease as lactation progresses, reflecting maternal adaptation to breastfeeding[27].

Long-term changes: Beyond the first year

During the second year of lactation, milk volume, and certain nutrient concentrations decline as complementary foods become the primary source of nutrition. Despite this reduction, immune factors such as IgA and lysozyme remain active, providing ongoing protection against infections during this critical period of immune development. The composition of human milk continues to adapt, offering tailored support for toddlers as they transition to solid foods and develop their microbiota and immune systems[28].

General functions of HMOs

HMOs are complex, non-digestible carbohydrates uniquely abundant in human milk and play a pivotal role in supporting infant health and development. Following lactose and lipids, HMOs are the third most abundant solid component in human milk, ranging from 5-20 g/L in colostrum to 5-15 g/L in mature milk[29]. Structurally, HMOs consist of a lactose core (comprising glucose and galactose) with additional monosaccharides such as fucose and sialic acid attached, resulting in remarkable structural diversity[30]. They are broadly categorized into three main types: Neutral non-fucosylated HMOs, neutral fucosylated HMOs, and acidic (sialylated) HMOs. Neutral non-fucosylated HMOs, such as LNT and lacto-N-neotetraose (LNnT), nourish beneficial gut bacteria like Bifidobacterium[31]. Neutral fucosylated HMOs, including 2′-fucosyllactose (2′-FL), 3′-fucosyllactose (3′-FL), and difucosyllacto-N-hexaose (DFL), are especially effective in preventing pathogen adhesion by mimicking host cell glycans[32]. Acidic sialylated HMOs, such as 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), play critical roles in brain development, immune modulation, and anti-inflammatory activity. Maternal genetics, specifically the Secretor (FUT2) and Lewis (FUT3) gene statuses, significantly influence HMO composition and diversity, with over 200 distinct structures identified[33]. Secretor-positive mothers produce higher levels of fucosylated HMOs, such as 2′-FL, while Lewis gene variations shape specific fucosylation patterns, underscoring HMOs’ adaptability to individual infant needs. HMOs serve multifaceted roles beyond basic nutrition[34]. These bioactive molecules exert both localized and systemic effects on infant growth, immune protection, and microbiota development. HMOs perform critical functions as prebiotics, antiadhesive agents, immune modulators, and contributors to neurological and systemic health[32].

HMOs as prebiotics

One of HMOs' most extensively studied roles is their function as prebiotics, selectively promoting the growth of beneficial gut bacteria, particularly Bifidobacterium species and Lactobacillus. These microbes are essential for gut health, metabolic activity, and immune function. Unlike other milk carbohydrates, HMOs resist digestion in the upper gastrointestinal tract, reaching the colon intact and serving as substrates for beneficial microbes[35]. This selective feeding fosters the predominance of Bifidobacterium in the infant gut microbiome, establishing a protective barrier against pathogens and enhancing intestinal integrity. HMO fermentation by gut bacteria produces short-chain fatty acids (SCFAs), such as acetate, butyrate, and propionate, which provide energy for colonocytes, reduce intestinal inflammation, and regulate systemic immune responses[36].

Antiadhesive properties: Protecting against pathogens

HMOs act as soluble decoy receptors, mimicking carbohydrate structures on host cell surfaces to prevent pathogen adhesion and colonization. Pathogens, including bacteria and viruses, often rely on these glycans to attach to host cells. For instance, 2′-FL, one of the most abundant HMOs, blocks the adhesion of Escherichia coli (E. coli) and C. jejuni to intestinal epithelial cells[37]. Similarly, sialylated HMOs inhibit the binding of influenza viruses and rotavirus, reducing the risk of gastrointestinal and respiratory infections. This antiadhesive function is especially crucial during the neonatal period when the infant’s immune system is immature and highly susceptible to infections[38].

Modulation of immune function

HMOs play a vital role in shaping the infant’s immune system by modulating innate and adaptive immune responses. They act directly on immune cells or indirectly through their influence on the gut microbiota. HMOs reduce inflammation by attenuating the production of pro-inflammatory cytokines, such as IL-8, while promoting regulatory cytokines like IL-10[39]. They also facilitate mucosal immunity by supporting the development of gut-associated lymphoid tissues (GALT). Furthermore, HMOs influence the expression of toll-like receptors on epithelial and immune cells, fine-tuning microbial pattern recognition and preventing excessive immune activation[40].

Enhancing gut barrier integrity

The intestinal barrier serves as a critical defense mechanism, preventing harmful pathogens and antigens from entering the bloodstream. HMOs strengthen gut barrier integrity by enhancing tight junctions between epithelial cells and stimulating the production of mucins, the primary components of the protective mucus layer[41]. By maintaining this barrier, HMOs reduce the risk of conditions such as necrotizing enterocolitis (NEC), a severe gastrointestinal disease in preterm infants. Clinical studies have shown that HMOs, particularly 2′-FL, are associated with a reduced incidence of NEC, underscoring their protective role in gut health[42].

Neurological and cognitive development

Emerging evidence suggests that HMOs contribute to neurological and cognitive development. Specific HMOs, including 2′-FL, DFL, and 3′-SL, have been linked to improved cognitive outcomes in infants. These effects are mediated indirectly through the gut-brain axis[43]. By shaping the gut microbiota, HMOs influence the production of neuroactive molecules such as serotonin and SCFAs, which can cross the blood-brain barrier and affect brain development. Additionally, HMOs may directly interact with neuronal cells, promoting neurogenesis and synaptic plasticity[44].

Systemic effects and long-term health outcomes

Beyond infancy, HMOs exert systemic effects that support long-term health outcomes. They are thought to lower the risk of metabolic diseases by promoting a healthy gut microbiota, which is linked to improved glucose metabolism and lipid profiles[32]. HMOs also possess anti-inflammatory properties that may reduce the risk of chronic inflammatory conditions, including allergies and autoimmune diseases. In animal studies, HMOs have been shown to enhance bone health by increasing calcium absorption and improving bone mineralization[45].

Unique structural and functional adaptations

The structural diversity of HMOs underpins their extensive range of functions. Fucosylated HMOs, such as 2′-FL and 3′-FL, are particularly effective in preventing pathogen adhesion, while sialylated HMOs, including 3′-SL and 6′-SL, provide antiviral protection and immune modulation[46]. Neutral HMOs predominantly act as prebiotics, whereas acidic HMOs exhibit anti-inflammatory and antimicrobial properties. This intricate interplay among HMO structures ensures a broad spectrum of protective and developmental benefits, tailored to the infant’s evolving needs[47].

Secretion dynamics of HMOs: HMOs are a unique and abundant component of human milk, second only to lactose and lipids in concentration. Their secretion and composition evolve dynamically across lactation stages, adapting to the infant’s changing developmental and immunological needs[5]. During early lactation, colostrum contains the highest concentration of HMOs, typically ranging from 20-25 g/L. This peak aligns with the neonate’s vulnerability to pathogens and the immaturity of their immune and gastrointestinal systems[32]. In colostrum, HMOs serve critical functions, acting as antimicrobial agents that block pathogen adhesion, prebiotics that selectively promote beneficial gut microbes such as Bifidobacterium species, and modulators of immune system development. These roles provide robust protection during the critical early days of life[48].

As lactation transitions to the transitional milk stage, typically between 6-14 days postpartum, HMO concentrations begin to decline to approximately 15-20 g/L. Transitional milk bridges the gap between the immunologically dense colostrum and the nutritionally stable mature milk[49]. By the time mature milk is produced, around 15 days postpartum, HMO concentrations stabilize at 10-15 g/L. Despite the reduction in concentration, the diversity of HMOs remains essential for sustaining immune protection, shaping the gut microbiota, and supporting neurological development. This diversity is particularly important as the infant’s immune system matures and their microbiota begins to stabilize[50].

The gradual decline in HMO concentration aligns with the infant’s developmental milestones. Early in life, HMOs act as decoy receptors, preventing the adhesion of pathogens such as E. coli, C. jejuni, and Salmonella enterica to host cells[51]. They also promote the colonization of beneficial microbes, enhancing gut barrier integrity and reducing infection risks. As the infant’s immune system strengthens, the lower HMO concentrations in mature milk provide sustained, albeit reduced, immunological support. This adaptive secretion system extends into late lactation, coinciding with the introduction of complementary foods. At this stage, breast milk shifts from being the primary nutritional source to a supplementary role, with HMOs continuing to support gut health and infection prevention, facilitating the infant’s transition to solid foods[32].

Several factors influence HMO secretion dynamics. Maternal genetics, particularly the FUT2 gene, determine secretor status, which affects the types and abundance of HMOs, such as α-1,2-fucosylated HMOs. Maternal health, diet, the infant’s gestational age at birth, and the lactation stage also play significant roles in shaping HMO composition[52]. For instance, preterm milk often contains higher concentrations of protective HMOs to address the heightened needs of premature infants. These dynamics highlight the finely tuned nature of HMO secretion, which evolves to match the developmental needs of the infant[53].

The temporal changes in HMO concentrations underscore their critical role in infant health and development. Beyond protecting against infections and shaping the gut microbiota, HMOs have been linked to improved cognitive outcomes, with specific structures, such as 2′-FL, associated with enhanced neurological development[3]. The adaptive secretion of HMOs throughout lactation reflects the intricate biology of human milk, optimized for promoting infant growth, immunity, and long-term health[38]. Future research should prioritize understanding individual variations in HMO profiles, their long-term health impacts, and potential therapeutic applications, especially for formula-fed infants and preterm newborns. Such advancements could revolutionize pediatric nutrition and improve care for vulnerable populations.

Maternal factors affecting HMOs composition: HMOs are uniquely tailored to support infant health and development, with their composition influenced by both maternal and environmental factors. While genetic determinants such as secretor status and Lewis blood type play a primary role, maternal physiology, diet, health status, stress levels, parity, and environmental exposures significantly modulate the diversity and concentration of HMOs[54]. Understanding these influences provides insights into the dynamic nature of HMO secretion and its role in optimizing infant nutrition and immunity.

Maternal nutrition impacts the availability of substrates and cofactors required for HMO synthesis, indirectly influencing HMO concentration and composition. Adequate carbohydrate intake is crucial as glucose and galactose form the lactose core of all HMOs[55]. Maternal carbohydrate restriction (e.g., in low-carb diets) may reduce total HMO synthesis. Dietary fats, particularly PUFAs, can influence the production of sialylated HMOs, as sialic acid metabolism is linked to lipid pathways[56]. Amino acid availability supports the enzymatic activity of glycosyltransferases involved in HMO synthesis[57]. Iron and Zinc are essential for enzymatic functions in the HMO biosynthesis pathway. Iron deficiency may impair the synthesis of fucosylated and sialylated HMOs. B Vitamins act as cofactors in metabolic pathways that synthesize nucleotide sugars, precursors for HMOs[58]. Diets rich in fruits, vegetables, and whole grains may enhance HMO synthesis by providing essential nutrients and reducing oxidative stress. Conversely, nutrient-poor diets may limit the availability of substrates required for optimal HMO production[32].

The mother's overall health significantly affects HMO composition and secretion. Obesity is associated with alterations in HMO profiles, including reduced fucosylated HMOs like 2′-FL, which may impair antimicrobial protection[59]. Malnutrition or low body mass index may reduce overall milk production and the concentration of HMOs. Gestational diabetes has been linked to changes in HMO profiles, particularly lower levels of fucosylation and sialylation, which may impact infant gut microbiota and immune development[7]. Thyroid hormone imbalances can affect metabolic pathways involved in HMO biosynthesis. Maternal infections and inflammation may alter HMO production. Elevated pro-inflammatory cytokines may downregulate enzymes critical for fucosylation and sialylation, leading to a less diverse HMO profile[60].

Psychological and physiological stress during lactation can affect HMO synthesis through hormonal and metabolic pathways. Elevated cortisol levels in stressed mothers may interfere with glycosylation processes, altering the balance of HMO subtypes[61]. Chronic stress may reduce sialylated HMO levels, potentially impairing their protective effects against viral and bacterial infections. Stress-induced changes in insulin, prolactin, and other hormones can disrupt the lactational metabolic environment, indirectly influencing HMO composition[62].

Studies suggest that multiparous mothers (those with multiple pregnancies) may produce milk with higher HMO concentrations compared to primiparous mothers, likely due to enhanced mammary gland adaptation and metabolic efficiency in subsequent lactations[63]. Colostrum contains the highest concentrations of HMOs, particularly fucosylated and sialylated types, reflecting the neonate's need for immediate immune protection. As lactation progresses, total HMO levels decline, but non-fucosylated HMOs like 3-FL increase, supporting gut microbiota diversity and resilience during complementary feeding[7].

Environmental conditions, including geography, pollutants, and breastfeeding practices, influence HMO profiles. Differences in secretor status prevalence and breastfeeding duration across populations contribute to regional variations in HMO profiles[64]. For example, rural Kenyan mothers exhibit prolonged secretion of 3-FL and LNT due to extended breastfeeding and minimal dietary processing, supporting microbiota dominated by Bifidobacterium infantis. Exposure to environmental toxins such as heavy metals and persistent organic pollutants may disrupt metabolic pathways and reduce the diversity and functionality of HMOs[65]. Air quality and water contamination in certain regions may indirectly affect maternal health and milk composition[66]. In some cultures, early weaning or supplementation with formula reduces HMO exposure during critical periods of infant development. Conversely, traditional breastfeeding practices in rural areas may extend the protective benefits of HMOs[67]. The interplay between maternal and environmental factors creates a dynamic landscape of HMO secretion. A nutritionally balanced and stress-free mother is more likely to produce a diverse and protective HMO profile. Environmental stressors such as food insecurity, pollution, and limited healthcare access exacerbate disparities in HMO secretion, potentially increasing infant susceptibility to infections and poor health outcomes[68] (Table 2).

Table 2 Maternal factors affecting human milk oligosaccharides composition.

Factor	Key influence	Examples and mechanisms
Maternal genetics	Primary determinant of HMO diversity and abundance	Secretor Status: Active FUT2 gene leads to higher 2′-FL levels; Lewis blood type: Influences specific HMO fucosylation patterns
Dietary intake	Availability of substrates and cofactors for HMO synthesis	Carbohydrates: Essential for lactose core formation; PUFAs: Support sialylated HMO production; Micronutrients: Iron, zinc, and B vitamins enhance enzymatic activity
Maternal health status	Alters HMO composition and concentration	Obesity: Reduces fucosylated HMOs (e.g., 2′-FL); Diabetes: Lowers fucosylation and sialylation; Infections/Inflammation: Downregulates glycosylation enzymes
Stress levels	Impacts hormonal and metabolic pathways	Cortisol: Reduces sialylated HMO levels; Chronic Stress: Alters glycosylation balance and overall HMO diversity
Parity (number of pregnancies)	Enhanced mammary adaptation and metabolic efficiency in multiparous mothers	Higher HMO concentrations in multiparous mothers; Primiparous mothers may produce less diverse HMO profiles
Lactation stage	Dynamic changes in HMO levels during breastfeeding	Colostrum: High fucosylated and sialylated HMOs for immune protection; Mature Milk: Decline in total HMOs; increase in 3-FL for gut microbiota diversity
Environmental Conditions	Geographic, pollutant, and cultural influences on HMO secretion	Rural areas: Prolonged breastfeeding supports 3-FL and LNT secretion (e.g., Kenyan mothers); Pollutants: Disrupt metabolic pathways and reduce HMO diversity
Breastfeeding practices	Duration and exclusivity influence HMO exposure	Extended Breastfeeding: Enhances long-term microbial and immune benefits; Early Weaning: Reduces HMO exposure in critical periods

2′-FL: 2′-Fucosyllactose; 3′-FL: 3′-Fucosyllactose; HMOs: Human milk oligosaccharides; LNT: Lacto-N-Tetraose; PUFAs: Polyunsaturated fatty acids.

Infant-specific utilization of HMOs: HMOs are critical to infant health, serving as prebiotics, antimicrobial agents, and immune modulators. Infants’ utilization of HMOs is highly adaptive, shaped by the infant’s age, gut microbiota composition, genetic makeup, and overall health status[3]. HMOs are indigestible by the infant and reach the colon intact, where they selectively nourish beneficial microbes, particularly Bifidobacterium longum subsp. infantis and Bifidobacterium breve. These bacteria have specialized enzymatic machinery to metabolize HMOs, giving them a competitive advantage over pathogenic microbes[38]. The fermentation of HMOs produces SCFAs, such as acetate, propionate, and butyrate, which lower gut pH, inhibit pathogen growth, and strengthen gut barrier integrity. During the neonatal phase, HMOs support establishing a protective microbial community, while later in infancy, they maintain microbial diversity and resilience as the infant's diet diversifies[69].

HMOs also act as decoy receptors for bacterial and viral pathogens, preventing their adhesion to host epithelial cells. Fucosylated HMOs like 2′-FL inhibit pathogens such as C. jejuni and Enteropathogenic E. coli, while sialylated HMOs like 3′-SL block Helicobacter pylori (H. pylori) and respiratory viruses like respiratory syncytial virus (RSV)[39]. These protective effects are particularly crucial in early life when the infant is most vulnerable to infections. As the infant grows, non-fucosylated HMOs like LNT sustainably neutralize bacterial toxins and support gut health[32].

In addition to direct pathogen defense, HMOs influence the infant's immune system. They modulate innate immunity by reducing pro-inflammatory cytokines and promoting regulatory cytokines, such as IL-10, while enhancing mucosal immunity through the development of GALT[6]. HMOs also support adaptive immunity by interacting with dendritic cells and promoting immune tolerance to beneficial microbes. Furthermore, SCFAs produced by microbial fermentation of HMOs contribute to systemic immune regulation and strengthen the gut barrier[5].

The infant’s genetic background significantly impacts HMO utilization. Secretor status, determined by the FUT2 gene, affects the abundance of fucosylated HMOs, with secretor infants benefiting from a higher prevalence of Bifidobacterium species[70]. Preterm infants, with their underdeveloped gut and microbiota, rely heavily on HMOs to establish protective bacterial communities and reduce the risk of NEC. Health conditions like infections or antibiotic use can disrupt HMO utilization, highlighting the importance of maintaining a balanced microbiota during infancy[71].

The benefits of HMO utilization extend beyond infancy, influencing long-term health outcomes. By promoting immune tolerance and microbial diversity, HMOs reduce the risk of allergic and autoimmune diseases, while their role in metabolic programming may lower the likelihood of obesity and metabolic disorders later in life[38]. Additionally, sialylated HMOs are precursors for gangliosides, essential for brain development and cognitive function. Despite these advances, gaps remain in understanding the variability in HMO metabolism among different populations and genetic backgrounds, as well as the long-term effects of early HMO exposure. Future research should explore these areas and investigate the potential for HMO supplementation in formula-fed and preterm infants to replicate the benefits of breastfeeding[72].

HMOs’ antimicrobial effects

HMOs are critical breast milk components exhibiting potent antimicrobial properties. They offer multifaceted protection against pathogens such as bacteria, viruses, and protozoa. These effects are mediated through diverse and overlapping mechanisms, including structural mimicry of host cell glycans, prebiotic activity, immune modulation, and maintenance of gut barrier integrity[73]. The antimicrobial effects of HMOs have been explored through various studies, as summarized in Tables 3 and 4. These studies provide critical insights into the mechanisms and spectrum of activity of HMOs against different pathogens[74].

Table 3 Specific anti-microbial roles of some human milk oligosaccharides.

HMO type	Key pathogens targeted	Mode of action
2'-FL	Campylobacter jejuni, EPEC, Norovirus, RSV, Rotavirus	Anti-adhesive activity, inhibition of pathogen attachment to epithelial cells, by acting as a decoy receptor
3-FL	EPEC, Pseudomonas aeruginosa, Salmonella enterica, and Norovirus (pathogens commonly encountered later in infancy)	The prebiotic effect promotes beneficial bacteria and blocks pathogen adhesion
DFL	Escherichia coli, Campylobacter jejuni, Salmonella enterica, Helicobacter pylori	Anti-adhesive activity preventing pathogen attachment to epithelial cells; supports immune modulation and gut barrier function
LNT	Entamoeba histolytica, GB. streptococcus, Vibrio cholera toxin	Neutralization of bacterial toxins and prevention of protozoal adhesion
3'-SL	EPEC, Helicobacter pylori, Pseudomonas aeruginosa, Vibrio cholera toxin, RSV, Rotavirus	Blocking bacterial adhesion and neutralizing bacterial toxins
6'-SL	EPEC, Helicobacter pylori, Pseudomonas aeruginosa, Influenza virus, Rotavirus	Inhibition of bacterial and viral adherence, especially in respiratory and gastrointestinal infections

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 3'-SL: 3'-Sialyllactose; 6'-SL: 6'-Sialyllactose; EPEC: Enteropathogenic E. coli; GB. streptococcus: Group B streptococci; LNT: Lacto-N-Tetraose; RSV: Respiratory syncytial virus; HMO: Human milk oligosaccharide.

Table 4 Studies of anti-bacterial effects of human milk oligosaccharides.

Ref.	Type of article	Microbes investigated	Types of HMOs	Mechanisms of action	Key findings	Patient population
Morrow et al[76]	Observational study	Campylobacter jejuni, Calicivirus	2′-FL, LNDFH	Inhibits pathogen adhesion by mimicking host glycans; Modulates immune responses, reducing intestinal inflammation	Significant reduction in diarrhea incidence (P = 0.004 for Campylobacterjejuni, P = 0.012 for Calicivirus)	Breastfed human infants
Yu et al[77]	Experimental study	Campylobacter jejuni	2′-FL	Prevents bacterial adhesion to epithelial cells; Suppresses pro-inflammatory cytokines (IL-8, IL-1β)	Colonization reduced by 80% in mouse models; Lowered intestinal inflammation	Mouse model
Facinelli et al[78]	Experimental in vitro study	E. coli, Salmonella fyris	2'-FL, 6'-SL	2'-FL and 6'-SL significantly reduced the adhesion of Escherichia coli to Caco-2 cells at human milk concentrations but no effect on Salmonella fyris	Reducing the adhesion of harmful bacteria like E. coli	Bacterial adhesion to Caco-2 cells (a human intestinal cell line) cultures
Wang et al[79]	Experimental study	E. coli O157: H7	2′-FL	Acts as a soluble decoy receptor; Enhances mucin secretion (MUC2); Promotes microbiota balance	Colonization reduced by > 90% in mouse intestines; Lowered pro-inflammatory cytokines (IL-6, TNF-α)	Mouse model
Lin et al[99]	Experimental study	UPEC	Neutral and sialic acid-rich HMOs	Reduces bacterial internalization into epithelial cells. Suppresses UPEC-induced proinflammatory signaling (MAPK, NF-κB)	Reduced cytotoxicity and improved bladder epithelial integrity; Decreased cell death and detachment	In vitro bladder epithelial cells
Martín-Sosa et al[100]	Experimental study	ETEC, UPEC	Sialylated oligosaccharides	HMOs strongly inhibited hemagglutination mediated by ETEC strains expressing CFA/I and CFA/II. HMOs also inhibited hemagglutination by UPEC strains	The inhibitory capacity was significantly reduced when HMOs were desialylated, highlighting the importance of the sialylated fraction	In vitro
Coppa et al[102]	Experimental study	EPEC, Vibrio cholerae, Salmonella fyris	Acidic, neutral high-molecular-weight, and neutral low-molecular-weight HMOs	Blocks bacterial adhesion to Caco-2 cells by acting as decoy receptors	Acidic HMOs inhibit all three pathogens. Neutral high-molecular-weight HMOs inhibit E. coli and V. cholerae. Neutral low-molecular-weight HMOs inhibit E. coli and S. fyris	In vitro with Caco-2 cells
Newburg et al[103]	Experimental study	E. coli (heat-stable enterotoxin, (ST)	Fucosylated HMOs	Oligosaccharide fraction provided significant protection against the diarrheagenic effects of E. coli heat-ST), while the lactose fraction did not	Among HMOs, the neutral fraction exhibited protective activity, whereas the acidic fraction did not (22% vs 57% mortality, respectively	Suckling mouse model
Noguera-Obenza et al[101]	Observational study	EHEC, EPEC	Not specified; focus on secretory IgA (sIgA) in human milk	Neutralizes bacterial adhesion by targeting virulence antigens (e.g., EspA, EspB, intimin)	sIgA in human milk reduces adhesion of EHEC/EPEC to gut epithelium. - Antibodies are regionally variable and linked to pathogen exposure history	Milk samples from breastfeeding mothers (United States and Mexico)
He et al[109]	Experimental study	AIEC	Exosome-encapsulated HMOs (e.g., 2’-FL, Sialyl lactose)	Modulates immunity by reducing LPS-induced inflammation. Promotes anti-inflammatory macrophage phenotype	Reduced intestinal inflammation and bacterial load in AIEC-infected mice	In vivo murine model
Manthey et al[80]	Experimental study	EPEC	Various HMOs	Blocks bacterial attachment by acting as decoy receptors	Reduced EPEC attachment in vitro; Decreased intestinal colonization in mouse models	In vitro epithelial cells and suckling mice models
Chambers et al[81]	Experimental study	GB. streptococcus	Pooled and single-entity HMOs	Increases cell permeability; Bioorthogonal probes retain activity and identify cellular targets	Up to 90% growth inhibition by pooled HMOs; DFL, LNT-II, and LSTa exhibit significant antibacterial activity (35%-60%)	In vitro GB. streptococcus cultures
Chambers et al[117]	Experimental study	GB. streptococcus	Various HMOs	Increases membrane permeability. Perturbs metabolic pathways, including glycerophospholipid and linoleic acid metabolism	Potentiated trimethoprim activity, reducing MIC by up to 512-fold; Synergistic effects observed in combination therapy	In vitro studies with clinical GB. streptococcus isolates
Ackerman et al[82]	Experimental study	GB. streptococcus	Pooled donor HMOs	Disrupts biofilm formation; Alters bacterial cell arrangement	Donor-specific effects: HMOs from some donors inhibited biofilm production and bacterial growth by 40%; Biofilm structure changes were observed using electron microscopy	In vitro GB. streptococcus cultures from human donors
Lin et al[83]	Experimental study	GB. streptococcus	Neutral (non-sialylated) HMOs	Direct bacteriostatic effect; Neutral HMOs inhibit growth by impairing glycosyltransferase functions; Synergistic effects with antibiotics	Neutral HMOs reduce GB. streptococcus growth by up to 98%; Identified glycosyltransferase (gbs0738) as a target for HMO-mediated inhibition; Synergy observed with vancomycin and ciprofloxacin, significantly lowering the required antibiotic dose	In vitro GB. streptococcus cultures
Moore et al[84]	Experimental study	GB. streptococcus	Purified HMOs	Inhibits biofilm formation in colonizing and invasive strains; Exhibits strain-specific effects based on capsular serotype and sequence type	50% biofilm inhibition for colonizing strains, 45% for invasive strains; Significant inhibition across multiple capsular types (CpsIb, CpsII, etc.); Effective biofilm dismantling of mature biofilms	30 diverse clinical and laboratory strains of GB. streptococcus (in vitro analysis)
Andreas et al[118]	Clinical and experimental study	GB. streptococcus	Specific fucosylated HMOs	Reduces GB. streptococcus colonization in mothers and infants; Acts bacteriostatically, impairing glycosyltransferase-dependent cell proliferation	LNDFHI and related HMOs reduce GB. Streptococcus growth by 50%; Lewis-positive mothers produce HMOs that reduce maternal and infant colonization; Lewis-negative mothers could benefit from synthetic HMO supplementation	Gambian mother-infant pairs and in vitro analysis
Mysore et al[85]	Preclinical study	Helicobacter pylori	38-SL, a natural HMO analog	Inhibits bacterial adhesion to gastric epithelial cells; Acts as a decoy receptor preventing colonization	2 out of 6 animals achieved permanent H. pylori eradication; Safe and showed efficacy in rhesus monkey model; Transient decrease in bacterial load observed in other regimens	H. pylori-positive rhesus monkeys
Parente et al[86]	Double-blind, placebo-controlled clinical trial	Helicobacter pylori	3’SL	3’SL fail to eradicate or suppress H. pylori infection		Adults with dyspepsia and confirmed H. pylori infection
Jantscher-Krenn et al[116]	Experimental study	Entamoeba histolytica	Pooled HMOs	Reduces trophozoite attachment to intestinal epithelium	LNT has significant protection (up to 80%) while fucosylated HMOs are ineffective. GOS completely block E. histolytica attachment and cytotoxicity at 8 mg/mL	In vitro cultures of E. histolytica and human intestinal epithelial HT-29 cells
Nguyen et al[105]	Experimental study	Clostridium difficile, specifically focusing on its toxin A (TcdA) and the carbohydrate binding site (TcdA-f2)	LNFPV & LNH	A mixture of HMOs binds to the carbohydrate binding site of Clostridium difficile toxin A (TcdA-f2) at a concentration of 20 g/mL	LNFPV and LNH demonstrated strong binding with TcdA-f2, exhibiting docking energies of -9.48 kcal/mol and -12.81 kcal/mol, respectively

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 3'-SL: 3'-Sialyllactose; 6'-SL: 6'-Sialyllactose; 38-SL: 38-sialyllactose; AIEC: Adherent-Invasive E. coli; CFAs: Colonization factor antigens; CD4: Cluster of differentiation 4+; CD8: cluster of differentiation 8+; DFL: Difucosyllactose; Escherichia coli: E. coli, EHEC: Enterohemorrhagic E. coli; EPEC: Enteropathogenic E. coli; ETEC: Enterotoxigenic Escherichia coli; GB. streptococcus: Group B streptococci; HBGAs: Histo-blood group antigens; HIEs: Human intestinal enteroids; HMOs: Human milk oligosaccharides; IgA: Immunoglobulin A; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; IL-8: Interleukin-8; LNFPV: Lacto-N-fucopentaose; LNDFH: Lacto-N-Difucohexaose; LNDFHI: lacto-N-difucohexaose I; LNH: Lacto-N-neohexaose; LNT: Lacto-N-Tetraose; LNT-II: lacto-N-triose II; LPS: Lipopolysaccharides; LSTa: LS-tetrasaccharide a; MIC: Minimum inhibitory concentration; NF-κB: Nuclear factor kappa B; PBMCs: Peripheral blood mononuclear cells; RSV: Respiratory syncytial virus; RV: Rota virus; SA: Sialic acid; TcdA-f2: Clostridium difficile toxin A; Th1: T helper 1; Th2: T helper 2; TNF-α: Tumor necrosis factor; UPEC: Uropathogenic E. coli.

Structural mimicry and anti-adhesive activity

One of the primary mechanisms through which HMOs confer antimicrobial protection is by acting as decoy receptors. Structurally, HMOs closely resemble glycans present on epithelial cell surfaces, in the infant’s gut, respiratory tract, and other mucosal tissues, which are often targeted by microbial adhesins initiating colonization and infection during the initial stages of infection[75]. HMOs act as soluble decoy receptors by binding to pathogens and preventing their attachment to host tissues. For instance, 2'-Fucosyllactose (2'-FL) and 6'-SL effectively inhibit the adhesion of pathogens such as C. jejuni[76,77], Enteropathogenic E. coli[78-80], Group B streptococci[81-84], H. pylori[85,86], and various viral strains, including norovirus[87-90], Rotavirus[91-94], and respiratory viruses[95-98]. These HMOs bind to the pathogens, preventing them from attaching to epithelial cells and thereby mitigating infection.

Studies further support this anti-adhesive activity. Morrow et al[76] demonstrated that 2'-FL significantly reduced the incidence of diarrhea caused by C. jejuni and Calicivirus in breastfed infants. Similarly, Yu et al[77] found that 2'-FL reduced bacterial colonization by 80% in mouse models and suppressed intestinal inflammation. Similarly, Lin et al[99], Martín-Sosa et al[100], and Noguera-Obenza et al[101] demonstrated the potent inhibitory effects of HMOs on E. coli by disrupting bacterial adherence to intestinal cells, showcasing their potential to prevent gastrointestinal infections. In a comparable vein, Coppa et al[102] highlighted the ability of HMOs to inhibit Salmonella growth, suggesting a broad-spectrum antimicrobial activity. Similarly, sialylated HMOs like 3′-SL and 6′-SL inhibit H. pylori, a pathogen associated with gastric inflammation and ulcers[85]. In addition, certain HMOs can inhibit the toxic effects of certain bacterial enterotoxins. For example, Newburg et al[103] found that the oligosaccharide fraction of fucosylated HMOs provided significant protection against the diarrheagenic effects of E. coli heat-stable enterotoxin, while the lactose fraction did not. Furthermore, Weichert et al[104] studied the impact of HMOs on respiratory pathogens like Pseudomonas aeruginosa and found significant reductions in biofilm formation, a key factor in chronic infections. This expands the scope of HMOs beyond gastrointestinal pathogens. Additionally, Nguyen et al[105] investigated the effect of specific HMOs on Clostridium difficile, finding that certain structures were particularly effective in reducing spore germination and toxin production. This indicates the selective antimicrobial action of HMOs depending on their structure. These findings highlight the clinical potential of HMOs in reducing the burden of infectious diseases.

The ability of HMOs to act as decoy receptors extends to viruses such as RSV and influenza virus, where sialylated HMOs prevent viral attachment to respiratory epithelial cells[95-98]. This dual functionality of HMOs in gastrointestinal and respiratory systems underscores their versatile antimicrobial role. In addition to blocking pathogen adherence, HMOs neutralize bacterial toxins, reducing disease severity. LNT, for example, interferes with the activity of Vibrio cholera toxin, preventing its binding to gut epithelial cells and mitigating diarrhea. HMOs also inhibit bacterial colonization through their direct effects on pathogen virulence. For instance, SCFAs produced during HMO fermentation by gut bacteria create an acidic gut environment that inhibits toxin activity and the growth of harmful bacteria like Salmonella enterica and Clostridium difficile. Table 4 shows some studies concerned with anti-bacterial effects of HMOs.

Prebiotic effects and microbiota modulation

HMOs selectively promote the growth of beneficial gut microbiota, such as Bifidobacteria (longum subsp. infantis and breve) and Lactobacilli. These bacteria possess specialized enzymatic machinery to metabolize HMOs, giving them a competitive advantage over pathogens[48]. By outcompeting harmful microbes for nutrients and ecological niches, these beneficial bacteria establish a microbiota-driven barrier against infections. The fermentation of HMOs by these bacteria produces SCFAs, such as acetate, butyrate, and propionate, which not only inhibit pathogen growth but also serve as energy sources for intestinal epithelial cells, promoting gut health and strengthening the epithelial barrier[106]. This dual action contributes to gut homeostasis and enhances the infant’s resistance to infections. For example, 3-FL has been shown to support beneficial bacteria and block the adhesion of pathogens like Salmonella enterica and Pseudomonas aeruginosa[104]. Salli et al[107] explored the synergistic effects of HMOs with probiotics, reporting enhanced bacterial inhibition when HMOs were used in combination with specific probiotic strains. This points to the potential of HMOs to complement existing probiotic therapies. In addition, Wang et al[79] reported that 2'-FL not only acted as a decoy receptor but also enhanced mucin secretion, promoting a balanced microbiota and reducing pro-inflammatory cytokines like IL-6 and tumor necrosis factor.

Immune modulation

HMOs actively shape the infant’s immune system by modulating innate and adaptive responses. They promote the production of anti-inflammatory cytokines, such as IL-10, while suppressing pro-inflammatory signals like IL-8, maintaining a balanced immune state that prevents excessive tissue damage[46]. This modulation is essential for neonates, whose immune systems are still immature. By enhancing the development of GALT, HMOs stimulate the production of secretory IgA, a critical component of mucosal immunity[108]. HMOs play a pivotal role in modulating immune responses. They influence both innate and adaptive immunity, reducing inflammation and enhancing pathogen clearance. For instance, He et al[109] demonstrated that exosome-encapsulated HMOs suppressed lipopolysaccharide-induced inflammation and promoted an anti-inflammatory macrophage phenotype in mice infected with adherent-invasive E. coli. Similarly, Lin et al[99] showed that sialic acid-rich HMOs reduced pro-inflammatory signaling pathways, such as MAPK and NF-κB, in bladder epithelial cells infected with uropathogenic E. coli. HMOs also interact with dendritic cells to modulate T-cell differentiation, promoting immune tolerance while enhancing defense against pathogens.

Antiviral and anti-parasitic effects

HMOs exhibit significant antiviral activity by targeting a range of viral pathogens, including norovirus, rotavirus, RSV, and influenza virus[110]. Sialylated HMOs such as 3′-SL and 6′-SL have demonstrated efficacy against respiratory pathogens, including RSV and influenza virus, by preventing their attachment to respiratory epithelial cells[111]. Decoy receptor mechanisms are particularly effective against viruses that bind to host glycans, such as Norovirus, which targets histo-blood group antigens (HBGAs). Patil et al[112] and Koromyslova et al[87] showed that 2'-FL inhibited the binding of Norovirus genogroups GI.1 and GII.17 to HBGAs, significantly reducing viral replication in human intestinal models. Similarly, sialylated HMOs effectively block Rotavirus[113] and Influenza[114,115] virus attachment, including 3'-SL) and 6'-SL. Beyond decoy activity, HMOs modulate antiviral immunity. Xiao et al[95] found that 2'-FL enhanced humoral and cellular immune responses to Influenza vaccination, while Mahaboob Ali et al[96] identified HMOs with high binding affinities to viral proteins, suggesting their potential as antiviral agents. These findings underscore the broad-spectrum antiviral potential of HMOs. Additionally, HMOs like LNT prevent protozoal adhesion, exemplified by their protective effect against Entamoeba histolytica[116]. By reducing infection rates, HMOs lower the reliance on antibiotics during infancy, contributing to the mitigation of antibiotic resistance and preserving microbiota balance. Table 5 shows some studies concerned with anti-viral effects of HMOs

Table 5 Studies of the role of human milk oligosaccharides against viral infections.

Ref.	Virus investigated	Type of HMOs	Mechanisms of action	Key findings	Patient population
Patil et al[112]	Human Norovirus (HuNoV)	2'-FL	Acts as decoy receptors, blocking binding to HBGAs	Significant inhibition of Norovirus GII.4 Sydney [P16] replication in adult and pediatric HIEs. Less effective in infant HIEs due to low HBGA expression	Adults and pediatric intestinal organoids
Koromyslova et al[87]	GI.1 and GII.17 Noroviruses	2'-FL	Inhibits binding to HBGAs by mimicking natural attachment sites	Broad inhibition of GI.1 and GII.17 norovirus binding across genogroups. Demonstrated dose-dependent inhibition with 2'-FL in vitro	Not specified (laboratory models)
Hanisch et al[88]	Norovirus GII.4 (Sydney 2012) and GII.10 (Vietnam 026)	High-molecular mass HMOs rich in fucose	Multivalent fucose presentation enhances steric and valency effects for strong virus binding inhibition	High-mass HMOs with terminal blood group H1 or Lewis-b antigens showed potent binding to GII.4; distinct preferences observed for GII.10	Not specified (laboratory models)
Weichert et al[89]	Norovirus (not strain-specific)	2'-FL and 3-FL	Structurally mimic HBGAs and block norovirus binding to surrogate HBGA samples	2'-FL and 3-FL bind to HBGA pockets on the capsid, acting as naturally occurring decoys to inhibit norovirus attachment	Not specified (laboratory models)
Derya et al[90]	Norovirus GII.17 and GII.4	LNFP I and simpler fucosylated HMOs	Structurally similar to HBGAs; block binding to natural receptors	Simple fucosylated HMOs were more effective than complex ones (e.g., LNFP I) in inhibiting GII.17 and GII.4 binding to human gastric mucins	Not specified (laboratory models)
Hester et al[91]	RV OSU and Wa strains	SA-containing HMOs (e.g., 3'-SL, 6'-SL), neutral HMOs (e.g., LNnT)	Blocks viral binding and decreases infectivity through decoy receptor mechanisms.	SA-containing HMOs inhibited RV OSU infectivity in vitro. Both neutral and SA-containing HMOs reduced viral replication during acute infection in situ	In vitro and 21-day-old piglets
Comstock et al[92]	Rotavirus OSU strain	2'-FL, LNnT, 6'-SL, 3'-SL, and free SA	Modulates systemic and gastrointestinal immune cells to alter infection susceptibility	HMO-fed pigs showed increased NK and memory T cells and reduced immune cell populations linked to infection compared to formula-fed pigs	Colostrum-deprived neonatal piglets
Li et al[93]	Rotavirus OSU strain	4 g/L HMOs (2'-FL, LNnT, 6'-SL, 3'-SL)	Modulates immune response and alters colonic microbiota to reduce diarrhea duration	HMO-fed piglets had shorter diarrhea duration, enhanced anti-inflammatory and Th1 cytokine expression, altered colonic microbiota, and increased pH	Formula-fed newborn piglets
Laucirica et al[94]	Human Rotavirus G1P[8] and G2P[4]	2'-FL, 3'-SL, 6'-SL, galacto-oligosaccharides	Acts as soluble decoy receptors, directly affecting the virus to block binding	All oligosaccharides reduced infectivity in vitro. Maximum reduction for G1P[8] (62%) with 2'-FL and for G2P[4] (73%) with 3'-SL + 6'-SL mixture	African green monkey kidney epithelial cells (MA104)
Gozalbo-Rovira et al[113]	Rotavirus P[8] genotype	LNB, precursor of H1 antigen	LNB binds to the VP8* domain of the P[8] VP4 spike protein, inhibiting viral attachment	LNB binds with reduced affinity but induces conformational changes that inhibit rotavirus infection. Differences in ligand affinity explain variability in susceptibility among secretor and non-secretor phenotypes	Not specified (laboratory models)
Xiao et al[95]	Influenza virus	2'-FL	Enhances both innate and adaptive immune responses to vaccination	2'-FL improved vaccine-specific humoral and cellular immune responses, including CD4+ and CD8+ T-cell proliferation, dendritic cell maturation, and antigen presentation	6-week-old female C57Bl/6JOlaHsd mice
Mahaboob Ali et al[96]	Influenza virus, respiratory syncytial virus, human metapneumovirus, SARS-CoV-2	Multiple HMOs (e.g., 2'-FL, LNnT)	Mimic host cell receptors to inhibit viral entry by binding to viral surface proteins	In silico studies identified HMOs with high binding affinities to viral proteins, suggesting their potential as viral entry inhibitors and antiviral agents	Computational in silico modeling
Pandey et al[97]	Avian Influenza (H9N2 and other subtypes)	3'-SL, 6'-SL	Bind hemagglutinin and block viral attachment to host cells	3'-SL showed broad-spectrum activity against avian influenza in vitro. In vivo, 3'-SL eliminated H9N2 in chickens and improved clinical symptoms	Pathogen-free chickens
Duska-McEwen et al[98]	RSV, Influenza	2'-FL, 6'-SL, 3'-SL, LNnT	Enhance innate immunity by reducing viral load, cytokines, and inflammation	2'-FL reduced RSV viral load and cytokines. LNnT and 6'-SL decreased Influenza viral load. 6'-SL reduced IP-10 and TNF-α in RSV-infected PBMCs	In vitro respiratory epithelial cells and PBMCs
Schijf et al[114]	RSV	scGOS, lcFOS, pAOS	Modulates Th1/Th2 immune responses and enhances RSV-specific CD4+ and CD8+ T cells	Increased RSV clearance, enhanced Th1 response, reduced Th2 cytokines, and lower airway eosinophilia in RSV-infected mice	RSV-infected C57BL/6 mice
Guo et al[115]	Influenza virus	3'-SL	Synergistically reduces viral load and inflammation when combined with OPN	3'-SL and OPN reduced viral load by 75%, suppressed cytokine levels (TNF-α, IL-6), and exhibited anti-inflammatory effects	Human laryngeal carcinoma cell line (HEP-2)

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 3'-SL: 3'-Sialyllactose; 6'-SL: 6'-Sialyllactose; CD4: Cluster of differentiation 4+; CD8: cluster of differentiation 8+; HBGAs: Histo-blood group antigens; HIEs: Human intestinal enteroids; HMOs: Human milk oligosaccharides; LNnT: Lacto-N-neotetraose; NK: Natural Killer; OPN: Osteopontin; PBMCs: Peripheral blood mononuclear cells; RSV: Respiratory syncytial virus; RV: Rota virus; SA: Sialic acid; Th1: T helper 1; Th2: T helper 2; LNDFHI: Lacto-N-difucohexaose I; NLB: Lacto-N-biose; scGOS: Short-chain galactooligosaccharides; lcFOS: Long-chain fructooligosaccharides; pAOS: Pectin-derived acidic oligosaccharides.

Target-specific and synergistic effects

Several studies highlight the target-specific and synergistic effects of HMOs. For instance, Chambers et al[117] demonstrated that pooled HMOs potentiated the activity of antibiotics like trimethoprim against GB. streptococcus, reducing the MIC by up to 512-fold. In addition, Andreas et al[118] provided insights into the bacteriostatic effects of specific fucosylated HMOs, particularly lacto-N-difucohexaose I, which reduced GB. streptococcus colonization in mothers and infants by 50%. This highlights the potential of leveraging maternal HMO profiles, such as those associated with the Lewis-positive phenotype, to reduce GB. streptococcus colonization and transmission. Neutral HMOs also exhibit bacteriostatic effects by impairing glycosyltransferase functions in GB. streptococcus, as Lin et al[83] observed. This bacteriostatic effect was further amplified when neutral HMOs were combined with antibiotics like vancomycin and ciprofloxacin, demonstrating significant synergy. These findings suggest that HMOs could serve as adjuncts to existing antimicrobial therapies to lower the effective dose of antibiotics, enhancing their efficacy and reducing the risk of resistance.

Gut barrier integrity and pathogen exclusion

HMOs reinforce the gut epithelial barrier, preventing pathogen translocation and systemic infection. They promote tight junction integrity and mucin production, enhancing the protective mucus layer, and preventing the translocation of pathogens and toxins into systemic circulation[46]. This strengthening of the gut barrier is particularly critical for neonates, who are at high risk of systemic infections due to their immature immune systems[119]. Furthermore, HMOs stimulate the production of mucins, which form a protective layer over epithelial cells, creating a physical barrier against pathogen invasion. For instance, Jantscher-Krenn et al[116] showed that LNT protected against Entamoeba histolytica by reducing trophozoite attachment to intestinal epithelial cells. Nguyen et al[105] reported that specific HMOs bound to Clostridium difficile toxin A prevent its interaction with epithelial cells and mitigate cytotoxicity.

HMOs offer a comprehensive antimicrobial defense strategy through their structural mimicry, prebiotic effects, immune modulation, and barrier-enhancing properties[120]. Their ability to target a diverse and broad spectrum of pathogens-bacteria, viruses, and protozoa-highlights their therapeutic potential in preventing and managing infectious diseases and minimizing long-term complications such as malnutrition, growth retardation, and immune dysfunction[121]. They also shape the infant gut microbiome during a critical developmental window, influencing lifelong immunity and metabolic health[122]. The synergistic effects of HMOs with antibiotics and vaccines further expand their utility as adjunctive agents, paving the way for innovative interventions in both clinical and nutritional contexts[123]. This dynamic interplay of HMO structure, microbiota interactions, and immune modulation highlights their central role in infant health. Table 3 shows the specific anti-microbial spectrum of some of the common HMOs and the possible mechanism of action.

Temporal changes in HMO levels during lactation and their relation to infections in the first two years of life

HMOs are essential for antimicrobial defense mechanisms in breast milk, protecting against pathogens and shaping the infant’s gut microbiota. The temporal variation in HMO concentrations during lactation aligns closely with the infant’s evolving immunological and developmental needs[6]. This dynamic relationship underscores the critical role of HMOs in supporting infant health and development. The concentration and composition of HMOs change throughout lactation, influenced by maternal genetics, environmental factors, and the infant’s developmental stage[3] (Figure 2).

Early lactation (colostrum and transitional milk: 0-30 days postnatal): In early lactation, colostrum contains the highest concentration of HMOs, ranging from 20-25 g/L. This high concentration reflects the neonate’s immediate need for immune protection and the establishment of a healthy gut microbiota[124]. Among HMOs, 2′-FL is highly abundant in secretor mothers, reaching levels around 2600 mg/kg at 5-11 days postpartum. It provides robust anti-adhesive properties, preventing the attachment of pathogens such as C. jejuni and Enteropathogenic E. coli to host cells, thereby reducing gastrointestinal infections[125]. Conversely, difucosyllactose (DFL) levels are relatively low in this phase (50-100 mg/L) but gradually increase to approximately 100-150 mg/L as milk composition transitions to support the growing infant. Additionally, sialylated HMOs such as 6′-SL are prominent during this period, with concentrations around 340 mg/kg[126]. These HMOs offer protection against respiratory viruses like influenza and gastrointestinal pathogens such as rotavirus. The high levels of fucosylated and sialylated HMOs during early lactation are vital for reducing neonatal vulnerability to infections and fostering the colonization of beneficial microbes like Bifidobacterium species[32].

In addition, early-onset infections with GB. streptococcus peak in neonates (0-7 days), with late-onset disease in the first 3 months. Studies have demonstrated that high levels of 2'-FL during this period may provide protection by preventing bacterial adhesion and biofilm formation[127]. In addition, hospital-acquired infections with Pseudomonas aeruginosa are common in neonates. HMOs like 2'-FL and 6'-SL may help reduce their colonization in respiratory and gastrointestinal tracts[128].

Mature milk (40-240 days postpartum): As lactation progresses, total HMO concentrations decline to 10-15 g/L, reflecting the infant’s gradual immune and microbiota maturation. However, there are notable shifts in the dominance of specific HMOs[52]. For instance, while 2′-FL levels remain significant, they decrease from approximately 2300 mg/kg at 12-30 days to 1800 mg/kg at 2-4 months postpartum. Concurrently, DFL levels stabilize or slightly decline to 70-120 mg/L by the end of the second month, then progressively decrease to 30-80 mg/L by 4-8 months as the infant’s reliance on complementary foods increases[129]. Infections with rotavirus and norovirus peak at 6-24 months, with severe cases in infants under one year. The decline in 2'-FL may coincide with reduced natural protection, highlighting the need for supplementary interventions[130,131].

In contrast, 3-FL levels rise significantly, increasing from 580 mg/kg at 12-30 days to 1100 mg/kg at 2-4 months, becoming the predominant fucosylated HMO in later lactation stages. This increase correlates with enhanced pathogen defense and greater microbial diversity, which are critical as the infant encounters a broader range of environmental microbes[7]. Infections with C. jejuni and Enteropathogenic E. coli are common from 6 months onward as complementary feeding starts, introducing potential pathogens[132]. The presence of 3-FL may inhibit adhesion and colonization of these enteric pathogens[69]. In addition, infections with respiratory viruses, such as RSV and Influenza Virus peak during winter months in the first two years. The decline in HMO-mediated protection may correlate with increased vulnerability to respiratory pathogens[133].

Sialylated HMOs also exhibit a shift, with 3′-SL gaining prominence over 6′-SL and reaching concentrations of approximately 290 mg/kg by 2-4 months. These adjustments balance the infant’s continued need for pathogen defense with the promotion of microbial diversity and metabolic support[134]. Elevated 3-FL levels provide ongoing protection against respiratory and gastrointestinal infections, while sialylated HMOs target specific pathogens like rotavirus and influenza, ensuring critical immune support during this vulnerable stage of life[6].

Extended lactation (beyond 240 days): In extended lactation, HMO levels stabilize at approximately 5-10 g/L, maintaining a steady-state profile to support the infant’s immune and gut health as complementary feeding introduces diverse microbial exposures[135]. During this phase, 3-FL levels remain elevated at around 1300 mg/kg, continuing to support microbial balance and providing robust protection against new microbial exposures[136]. The rising dominance of 3-FL correlates with enhanced protection against diverse pathogens and supports gut microbial diversity, reducing the risk of infections linked to environmental exposures[137]. However, chronic colonization with H. pylori begins in early childhood, particularly in developing countries[138]. The reduced HMO levels might contribute to increased susceptibility. Additionally, LNT persists in extended lactation, offering sustained protection against bacterial toxins, such as Vibrio cholera toxin, and protozoal pathogens, including Entamoeba histolytica[139]. This steady-state HMO profile ensures continued immune and gut health benefits, facilitating the infant’s transition to a mixed diet while maintaining robust defenses against infections[34].

Correlation between HMO levels, anti-microbial activity, and geographic variability

The antimicrobial properties of HMOs are influenced by their concentration, diversity, and dynamic changes during lactation. Geographic differences, maternal genetics, and environmental factors further modulate these characteristics[140]. The new data from studies in China, Germany, and Kenya add depth to understanding these correlations. The temporal changes in HMO concentrations align with infant developmental needs, but the pattern varies across populations[141]. HMOs are at their highest concentration during the first weeks postpartum, providing critical pathogen defense and promoting the establishment of beneficial gut microbiota. Secretor mothers with active FUT2 genes produce high levels of fucosylated HMOs like 2′-FL, which decline with time, whereas 3-FL increases in later lactation[142]. Studies show that 3-FL and non-fucosylated HMOs such as LNT dominate in later stages of lactation, maintaining a protective function as the infant's diet diversifies and pathogen exposure increases[6] (Table 6).

Table 6 Studies on human milk oligosaccharides levels, geographic variations, and antimicrobial activity.

Ref.	Population/region	Key findings on HMO levels	Key antimicrobial activity	Geographic influence
Mao et al[144]	Urban Chinese mothers	High 2′-FL and sialylated HMOs (6′-SL) in early lactation; Gradual decline of 2′-FL and HMOs in mature milk	2′-FL inhibits Campylobacter jejuni and EPEC; 6′-SL protects against Rotavirus and Influenza	Secretor status influences early lactation HMO profile, optimizing pathogen defense in neonates
Liu et al[146]	Chinese mothers	3-FL increases significantly during later lactation; Sialylated HMOs decline after the first month	3-FL supports microbial diversity and inhibits Salmonella enterica and Pseudomonas aeruginosa	Extended breastfeeding in rural areas maintains high HMO levels, supporting long-term pathogen defense
Austin et al[129]	Chinese urban settings	Early high levels of fucosylated HMOs (2′-FL); Non-fucosylated LNT remains stable across lactation stages.	LNT neutralizes Vibrio cholera toxin; 2′-FL is critical for early pathogen defense	Urban mothers exhibit shorter lactation duration, potentially limiting long-term HMO-mediated benefits
Siziba et al[145]	German infants	High variability in HMO profiles across lactation; Secretor mothers show enriched 2′-FL levels	Sialylated HMOs reduce inflammatory responses and protect against respiratory pathogens (RSV, Influenza)	Differences in breastfeeding practices and complementary feeding influence microbiota outcomes
Derrien et al[65]	Rural Kenyan infants	High prevalence of 3-FL and LNT in late lactation; Enriched secretor group III (Se+, Le-)	3-FL supports Bifidobacterium infantis, lowering pH and reducing enteropathogen colonization; LNT neutralizes toxins	Rural diet and prolonged breastfeeding enhance microbiota dominated by B. infants
Kortesniemi et al[147]	Chinese mothers across lactation stages	Rapid decline in total HMOs from colostrum to mature milk; Sialylated HMOs persist in significant amounts	Sialylated HMOs prevent Helicobacter pylori adhesion and inhibit rotavirus infections	Secretor mothers exhibit early robust protection through 2′-FL, but benefits decline as lactation progresses
Asher et al[142]	Israeli mothers (Tel Aviv)	2′-FL dominant in secretors; LNT and 3-FL dominate in non-secretors; Seasonal and sex-dependent variations in HMO levels	2′-FL protects against infections via TLR4 inhibition; High DSLNT in non-secretors' colostrum protects against NEC	Urban breastfeeding with lower 2′-FL levels compared to global averages; shorter lactation may limit long-term benefits

The table summarizes findings from seven studies investigating human milk oligosaccharide (HMO) levels, geographic variations, and antimicrobial activity. Each study highlights the unique aspects of HMO concentrations across lactation stages, the role of specific HMOs in pathogen defense, and the influence of geographic and environmental factors. From this table, we found that across all regions, 2′-FL peaks in early lactation provide strong pathogen defense in neonates. 3-FL increases in later lactation, maintaining microbiota and pathogen defense during weaning. Rural settings like Kenya show sustained HMO levels due to prolonged breastfeeding, enriching microbiota that is beneficial for pathogen resistance. Urban populations like Israel and China experience sharper declines in HMOs due to shorter breastfeeding periods, potentially affecting long-term antimicrobial activity. Fucosylated HMOs (e.g., 2′-FL) are critical for early pathogen protection. Sialylated HMOs (e.g., 6′-SL) offer dual gastrointestinal and respiratory protection. Non-fucosylated HMOs (e.g., LNT and DSLNT) neutralize toxins and promote gut health throughout lactation. 2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 6'-SL: 6'-Sialyllactose; DSLNT: Disialyllacto-N-tetraose; EPEC: Enteropathogenic E. coli; HMOs: Human milk oligosaccharides; LNT: Lacto-N-tetraose; NEC: Necrotizing enterocolitis; RSV: Respiratory syncytial virus; TLR4: Toll-like receptor 4.

HMOs act through multiple mechanisms, including decoy receptor activity, toxin neutralization, and immune modulation, all are tailored to the lactation stage and environmental exposure[143]. In China, high concentrations of 2′-FL and sialylated HMOs like 6′-SL during early lactation provide robust protection against pathogens such as C. jejuni and Rotavirus[144]. However, lower fucosylated HMOs levels in non-secretors may affect their infants' susceptibility to gastrointestinal pathogens[145]. In Germany, studies on atopic dermatitis suggest HMOs may influence immune outcomes indirectly through their interaction with the microbiota and modulation of inflammation[65]. In addition, Infants in rural areas in Kenya exhibit higher dominance of Bifidobacterium longum subsp. infantis, supported by enriched levels of fucosylated HMOs like 2′-FL[146]. This correlation highlights HMOs' role in sustaining microbiota optimal for pathogen resistance.

Geographic differences in HMO profiles can be attributed to genetic variations and environmental factors such as dietary practices and lactation duration. Populations with a high proportion of secretor mothers, such as those in China[129,146] and Germany[145], exhibit higher levels of 2′-FL, enhancing protection against common gastrointestinal pathogens. Non-secretor mothers in Kenya show higher levels of 3-FL, compensating for the lack of 2′-FL in supporting gut microbiota and preventing infections. Rural Kenyan infants benefit from extended breastfeeding and a microbiota dominated by Bifidobacterium infantis, supported by HMOs metabolized more effectively in the absence of processed foods and antibiotics[65]. Urbanized populations, experience a more diverse microbiota due to complementary feeding practices and shorter breastfeeding duration, which may shift the functional roles of HMOs[142,144].

The interplay between HMO levels and gut microbiota composition is crucial for antimicrobial activity. In Kenyan infants, the enriched HMO group III (Se+, Le-) supports B. infantis, which lowers gut pH, enhancing the microbiota's resistance to enteropathogens[65]. On the other hand, Chinese infants have high levels of sialylated HMOs during early lactation, contributing to the initial colonization of protective bacteria like Bifidobacterium breve[146,147]. In addition, the variability in HMO concentrations observed in German infants correlates with immune outcomes like atopic dermatitis, suggesting HMOs' indirect role in microbial and immune regulation[145]. This dynamic interplay between HMO concentrations, lactation stages, and geographic variability reveals their tailored role in pathogen defense and microbiota modulation. While 2′-FL dominates in secretor populations for early robust antimicrobial defense, 3-FL compensates in non-secretor mothers, especially in rural settings where environmental exposures demand sustained microbiota support. These findings underscore the importance of considering geographic and genetic factors in developing interventions to optimize infant global health[148].

Natural versus synthetic HMOs

HMOs are a unique and abundant component of human breast milk, comprising over 200 distinct types. These oligosaccharides are highly specialized and adapt to an infant’s developmental stage, maternal genetics, diet, and the progression of lactation. Natural HMOs play a critical role in infant health by fostering the growth of beneficial gut bacteria, particularly Bifidobacterium longum subspecies infants, and protecting against harmful pathogens by mimicking cell-surface receptors[38]. Additionally, they modulate both systemic and mucosal immune responses, contributing to overall health and disease resistance. However, HMO composition can vary significantly among mothers, and only breastfed infants benefit directly from the full spectrum of these bioactive compounds[149].

Animal milk, such as cow’s and goat’s milk, also contains oligosaccharides, but they are less abundant, diverse, and functionally equivalent to HMOs. Cow’s milk typically contains 0.03-0.06 g/L of oligosaccharides, far lower than the 5-15 g/L found in human milk, and its oligosaccharides are structurally simpler, primarily sialylated, and include fewer fucosylated structures[150]. Goat’s milk offers slightly more oligosaccharides, ranging from 0.25 to 0.3 g/L, with some structural similarities to HMOs. Milk from other mammals, such as sheep, buffalo, and camels, contains even lower concentrations and less diversity, tailored primarily to meet the developmental needs of their own offspring[151]. While these oligosaccharides offer some benefits to their respective species, they lack the complexity and multifunctionality of HMOs, limiting their ability to replicate the specific benefits of human milk[152].

Recognizing the essential role of HMOs, researchers have sought to enhance the nutritional value of animal milk-based infant formulas. Synthetic HMOs have been developed as a promising alternative for non-breastfed infants[153]. These HMOs are produced using advanced techniques, such as microbial fermentation, chemical synthesis, or enzymatic processes. Synthetic HMOs like 2’-FL and LNnT are now commonly added to infant formulas, where they mimic some of the critical functions of natural HMOs, such as supporting gut health and providing antimicrobial defense[154]. Synthetic HMOs are accessible, cost-effective, and standardized to ensure consistency, but they represent only a fraction of the diversity and functionality found in natural HMOs[155].

Synthetic HMOs vary significantly depending on the method of synthesis. Microbial fermentation, which uses genetically engineered microorganisms like E. coli or Saccharomyces cerevisiae, is the most efficient and scalable method, producing large quantities of structurally identical HMOs such as 2’-FL and LNnT at relatively low cost. However, it is limited to simpler HMOs due to the complexity of biosynthetic pathways and may pose risks of microbial by-product contamination[156]. Chemical synthesis, on the other hand, offers unparalleled precision and versatility, enabling the production of complex and rare HMOs with high regio- and stereoselectivity. However, it is an expensive and labor-intensive process, generating significant chemical waste and being less suitable for industrial-scale production[58]. Enzymatic synthesis provides a cleaner and more environmentally friendly alternative, using purified enzymes to catalyze the formation of HMOs with high structural accuracy. While enzymatic methods are highly flexible and capable of producing complex HMOs, they are limited by the cost and availability of enzymes and substrates and may require optimization for large-scale applications[157]. Hybrid approaches, such as combining enzymatic synthesis with substrates derived from microbial fermentation, are emerging to leverage the strengths of these methods. Table 7 compares the difference between the three main synthetic methods. Ultimately, the choice of synthesis technique depends on the target HMO, production scale, and cost constraints. Future innovations in synthetic biology and enzyme engineering are expected to improve synthetic HMOs' efficiency, scalability, and diversity, bridging the gap between natural and synthetic forms.

Table 7 Comparison of synthetic human milk oligosaccharides by method.

Feature	Microbial fermentation	Chemical synthesis	Enzymatic synthesis
Cost	Low to moderate	High	Moderate
Scalability	High	Low	Moderate
Structural diversity	Limited (simpler HMOs)	High (complex and rare HMOs)	High (with optimized enzyme cascades)
Environmental impact	Moderate	High	Low
Ease of production	Straightforward for common HMOs	Complex and labor-intensive	Depends on enzyme availability

HMOs: Human milk oligosaccharides.

Despite these advancements, synthetic HMOs cannot fully replicate the complexity of natural HMOs. Their limited diversity means they are less effective in supporting diverse gut microbiota, modulating the immune system, and providing the comprehensive protection seen in breastfed infants[120]. While they bridge some nutritional and immunological gaps for formula-fed infants, innovation is needed to expand the range of synthetically available HMOs and enhance their functionality[158]. Table 8 compares between the natural and synethetic HMOs. Breastfeeding remains the gold standard for infant nutrition due to the unmatched benefits of natural HMOs, which are tailored by nature to meet the specific developmental, immune, and microbiota needs of human infants[159]. However, synthetic HMOs represent a significant advancement, offering a practical alternative for infants who cannot be breastfed. Future research aims to improve synthetic HMOs, personalize formulations, and explore their applications beyond infant nutrition, ultimately striving to replicate the unparalleled advantages of natural HMOs[160].

Table 8 Natural vs synthetic human milk oligosaccharides: Key comparisons.

Feature	Natural HMOs	Synthetic HMOs
Source	Human breast milk	Biotechnological production
Structural diversity	Over 200 distinct HMOs	Limited to a few key HMOs (approximately 15 synthetic HMOs)
Customizability	Adaptive to maternal and infant factors	Fixed composition in formula.
Microbiota interactions	Broad spectrum of microbial benefits	Focused on Bifidobacteria support
Pathogen defense	Multifaceted, including sialylated HMOs	Limited to fucosylated HMOs
Immune modulation	Complex systemic and local effects	Under investigation
Accessibility	Breastfed infants only	Available in formula-fed populations

2′-FL: 2'-Fucosyllactose; HMOs: Human milk oligosaccharides.

Extended health implications of HMO antimicrobial effects

HMOs are pivotal not only for their immediate antimicrobial effects but also for their long-term impact on health. By shaping the infant microbiota, modulating immune responses, and protecting against infections, HMOs lay the foundation for health outcomes that extend beyond infancy[3]. These extended implications influence various aspects of health, including immunity, metabolism, neurodevelopment, and the prevention of chronic diseases (Table 9).

Table 9 Extended health implications of human milk oligosaccharides antimicrobial effects.

Health domain	Extended benefits	Mechanisms involved	Key HMOs
Immune development	Reduced risk of allergies (e.g., eczema, asthma) and autoimmune diseases (e.g., type 1 diabetes, IBD)	Promotes immune tolerance through Tregs; Modulates cytokine balance; Enhances GALT and sIgA production	2′-FL, 3-FL, sialylated HMOs
Gut microbiota programming	Establishes a balanced microbiota; Prevents dysbiosis and infections; Promotes long-term gut health	Selective enrichment of Bifidobacterium species; SCFA production supports gut barrier integrity and pathogen inhibition	2′-FL, 3-FL, LNT
Metabolic health	Reduced risk of obesity and type 2 diabetes; Improved lipid profiles and glucose metabolism; Prevention of metabolic disorders.	SCFA-mediated regulation of energy balance; Early microbiota influence on metabolic pathways	Fucosylated and non-fucosylated HMOs
Cognitive development	Enhanced neurodevelopment and cognitive function; Reduced risk of neurodevelopmental disorders (e.g., ASD, ADHD)	Precursors for gangliosides (myelination and synaptic plasticity); Microbiota-driven production of neuroactive compounds (e.g., serotonin)	Sialylated HMOs (3′-SL, 6′-SL)
Infection resistance	Long-term protection against infections; Reduced reliance on antibiotics, mitigating antibiotic resistance	Early microbial programming enhances innate and adaptive immunity; Pathogen exclusion through SCFA production and microbiota stability	2′-FL, 3-FL, sialylated HMOs
Chronic inflammatory diseases	Lower risk of IBD and systemic inflammation; Reduced incidence of cardiovascular diseases	Strengthens gut barrier integrity; Modulates systemic and intestinal inflammation through cytokine regulation	Fucosylated and sialylated HMOs
Non-communicable diseases	Prevention of type 2 diabetes and colorectal cancer; Improved long-term health outcomes	Anti-inflammatory and anti-carcinogenic effects of SCFAs; Early metabolic programming by microbiota	2′-FL, SCFA-producing HMOs
Neonatal-infant transition	Sustained protection during early dietary diversification; Optimized health outcomes with prolonged breastfeeding practices	Continuous microbiota support during complementary feeding; Long-term resilience against pathogenic exposures	3-FL, LNT

2'-FL: 2'-Fucosyllactose; 3-FL: 3-Fucosyllactose; 6'-SL: 6'-Sialyllactose; ADHD: Attention deficit hyperactivity disorder; ASD: Autism spectrum disorder; GALT: Gut-associated lymphoid tissue; HMOs: Human milk oligosaccharides; IBD: Inflammatory bowel disease; LNT: Lacto-N-tetraose; SCFA: Short chain fatty acids.

Immune development and lifelong immunity

HMOs play a crucial role in the maturation and regulation of the immune system during critical developmental windows, providing long-term protection against immune-mediated diseases and preventing allergies and atopic disorders[137]. HMOs interact with immune cells to promote tolerance to non-harmful antigens, such as dietary proteins and environmental allergens. This effect reduces the risk of allergies like eczema, asthma, and food allergies. By stimulating the differentiation of Tregs, HMOs suppress inappropriate immune responses that can lead to atopic dermatitis and other allergic conditions[6]. Early colonization by HMO-utilizing bacteria such as Bifidobacterium longum subsp. Infants leads to a balanced gut microbiota, which is associated with a lower risk of allergic diseases[161]. In addition, HMOs help maintain immune homeostasis by reducing inflammation and promoting anti-inflammatory pathways. This regulation may reduce the likelihood of autoimmune diseases such as type 1 diabetes and inflammatory bowel disease (IBD) later in life[162].

Gut microbiota programming and metabolic health

The early establishment of a healthy gut microbiota is critical for long-term metabolic and gastrointestinal health, and HMOs are central to this process. By promoting the growth of beneficial gut microbes, HMOs influence metabolic programming and energy balance[163]. SCFAs produced during HMO fermentation regulate lipid and glucose metabolism, potentially lowering the risk of obesity, insulin resistance, and type 2 diabetes. HMOs have been associated with healthier lipid profiles, reducing the risk of cardiovascular diseases later in life[164]. In addition, HMOs help to protect against gastrointestinal disorders. Early exposure to HMOs reduces the risk of gastrointestinal conditions such as NEC in preterm infants and functional gastrointestinal disorders like irritable bowel syndrome. HMOs' protective gut barrier effects prevent pathogen translocation and inflammation, minimizing the risk of chronic gastrointestinal inflammation[165,166].

Cognitive development and neuroprotection

Sialylated HMOs, such as 3′-SL and 6′-SL, are particularly important for brain development and cognitive function, with effects that may extend into adulthood[72,167]. Sialylated HMOs serve as precursors for gangliosides and sialic acid, essential for myelination and synapse formation in the developing brain. Improved connectivity and synaptic efficiency are linked to enhanced cognitive and motor development[168]. The gut microbiota influenced by HMOs produces neuroactive compounds such as serotonin and SCFAs, which can cross the blood-brain barrier and modulate brain function[44]. Early microbial programming may reduce the risk of neurodevelopmental disorders such as autism spectrum disorder and attention deficit hyperactivity disorder[169].

Long-term resistance to infections

HMOs provide foundational immunity that has lasting effects on the ability to resist infections throughout life. Infants with a microbiota enriched in Bifidobacterium and other beneficial microbes are better equipped to fight infections due to the enduring presence of these protective bacteria[159]. HMOs' role in reducing pathogen colonization during infancy reduces the likelihood of microbial dysbiosis later in life. By lowering the need for antibiotics during infancy through their antimicrobial effects, HMOs help mitigate the development of antibiotic resistance[170]. This benefit extends to public health, reducing the global burden of antibiotic-resistant infections.

Influence on chronic inflammatory and non-communicable diseases

The anti-inflammatory properties of HMOs, combined with their ability to enhance gut barrier function and immune regulation, contribute to the prevention of chronic inflammatory conditions[6]. Early exposure to HMOs reduces intestinal inflammation and supports barrier integrity, decreasing the risk of IBD in later life. HMOs indirectly lower the risk of atherosclerosis and other cardiovascular conditions by reducing systemic inflammation and improving metabolic health[171]. The ability of HMOs to program the microbiota and modulate immune responses may reduce the risk of non-communicable diseases, including diabetes, cardiovascular diseases, and certain cancers[172]. Improved glycemic regulation and insulin sensitivity through SCFA production may lower the risk of developing type 2 diabetes[173]. SCFAs and other microbial metabolites produced through HMO fermentation have anti-carcinogenic properties, reducing the risk of colorectal cancer[174]. These long-term health benefits of HMOs are influenced by the interaction between maternal and infant factors. For instance, maternal diet, genetics, and lactation practices affect HMO composition, which in turn impacts infant health outcomes[175]. Breastfeeding practices that extend HMO exposure (e.g., exclusive breastfeeding for six months) amplify these long-term benefits.

Study heterogeneity

The included studies exhibited considerable variability in experimental models, methodologies, and outcome measures, which posed challenges for direct comparisons and statistical synthesis. However, despite these differences, consistent trends across diverse studies suggest strong biological plausibility for the dynamic secretion of HMOs and their antimicrobial effects. The studies analyzed ranged from human clinical trials to animal and in vitro models. While human studies provide the most applicable findings, in vitro and animal studies offer valuable mechanistic insights contributing to a broader understanding of HMO functionality. Given the variability in study design, including measurement techniques and population characteristics, a narrative synthesis approach was employed to summarize findings. Despite these challenges, consistent outcomes across multiple studies reinforce the reliability of conclusions. To further ensure robustness, findings were categorized based on study type, common methodologies, and outcome measures, mitigating the impact of heterogeneity on the overall conclusions.

Recommendations and future research

This systematic review highlights several avenues for future research and application of HMOs in improving health outcomes. First, the development of HMO-based therapeutics should be prioritized, focusing on identifying and synthesizing specific HMO structures with high antimicrobial efficacy. These efforts could pave the way for novel interventions to prevent and manage infections, particularly in neonates and immunocompromised individuals. The observed synergy between HMOs and antibiotics also warrants further investigation to determine optimal combinations and dosing strategies that could enhance treatment efficacy and mitigate antibiotic resistance. While in vitro and animal studies have demonstrated promising antimicrobial effects, larger-scale clinical trials are essential to validate these findings and assess the long-term safety and efficacy of HMO supplementation in both infants and adults.

Given their protective properties, incorporating bioengineered HMOs, such as 2′-FL and 3-FL, into infant formula should be prioritized to improve nutrition and immunity for non-breastfed infants. In addition, the dynamic changes of HMOs in breast milk should attract the attention of formula-producing companies to consider different levels of specific HMOs according to the infant's age. Expanding research to explore the antimicrobial effects of HMOs against a broader range of bacterial, viral, and parasitic pathogens is crucial for understanding their full therapeutic potential. Personalized approaches based on maternal secretor status and infant needs could further optimize HMO supplementation strategies, ensuring tailored benefits for specific populations.

Moreover, deeper mechanistic studies are needed to elucidate the molecular pathways through which HMOs disrupt pathogen adhesion, biofilms, and enzymatic activity, as well as their roles in immune modulation. Public health initiatives should emphasize the importance of breastfeeding and the unique role of HMOs in supporting infant health. Policymakers and stakeholders should support breastfeeding programs while fostering the development of HMO-enriched nutritional products. Furthermore, addressing the impact of geographic variability in HMO composition could enhance personalized nutrition and therapeutic applications. Lastly, randomized controlled trials should be conducted to validate HMO efficacy in clinical settings, ensuring their optimal application in neonatal and pediatric care. By addressing these recommendations, the therapeutic potential of HMOs can be effectively harnessed to improve maternal and infant health, combat antimicrobial resistance, and reduce the global burden of infectious diseases.

Limitations section

This systematic review has several limitations that should be acknowledged. First, the heterogeneity among the included studies regarding experimental models, methodologies, and outcome measures posed challenges in synthesizing findings. Differences in study design, such as variations in HMO concentration measurement techniques, pathogen models, and experimental conditions, limited the ability to compare results across studies directly. Establishing standardized research methods and reporting guidelines for HMO concentration detection, experimental model selection, and outcome evaluation would enhance comparability across studies and facilitate comprehensive analysis.

A significant portion of the evidence comes from in vitro and animal studies, which may not fully replicate the complex biological interactions in human systems. Many antimicrobial findings stem from in vitro models, which lack the complexity of human physiology. Thus, while these results provide valuable insights, their direct applicability to human neonates remains uncertain. Similarly, animal studies do not fully replicate human gastrointestinal environments. Extrapolating these findings to clinical settings requires caution, particularly for infants and adults. To address this limitation, more high-quality, large-scale human clinical trials with long-term follow-up are necessary to validate findings and ensure reliable translation of in vitro and animal research into clinical applications.

Another limitation is the variability in the types of HMOs investigated. While studies on prominent HMOs like 2′-FL and 3-FL were abundant, data on less common but potentially important HMOs were limited, leaving gaps in understanding their roles. Additionally, maternal factors such as genetic secretor status, diet, and health influence HMO composition and function, but inconsistencies in reporting these variables hinder comprehensive conclusions about personalized HMO interventions. Geographical and population biases were also noted, as many studies originated from specific regions, potentially overlooking variations in HMO profiles and microbial challenges faced by diverse populations. Research from low- and middle-income regions was underrepresented despite potential differences in maternal diet, genetics, and environmental factors influencing HMO secretion. Expanding research to include multi-population and cross-regional studies would help address these biases and establish a more inclusive understanding of HMOs.

The review included only English-language publications, potentially omitting critical research published in other languages. This limitation is particularly significant given that research on breastfeeding and HMOs is conducted worldwide. Future reviews should incorporate multilingual search strategies to minimize selection bias. Additionally, language barriers may prevent the inclusion of studies from non-English speaking regions, potentially missing critical data that could influence conclusions. Translation tools and collaboration with multilingual researchers could help mitigate this issue.

Geographical biases were also evident, with most studies originating from high-income countries such as the United States, Germany, and Japan. Limited research from low- and middle-income countries (LMICs) means that potential regional variations in HMO composition due to genetic, dietary, and environmental factors are underexplored. Given that maternal nutrition, microbiome diversity, and breastfeeding practices differ significantly across regions, future studies should focus on conducting research in diverse populations to capture these variations more accurately. Encouraging research collaborations across multiple countries and increasing funding opportunities for HMO research in LMICs could enhance the global applicability of findings.

Furthermore, regional disparities in healthcare access and socioeconomic conditions may influence breastfeeding practices and HMO secretion. Cultural beliefs and traditional infant-feeding practices vary widely across populations and may impact the concentration and composition of HMOs in ways that remain underexplored in current literature. Addressing these knowledge gaps would improve our understanding of how HMOs contribute to infant health on a global scale.

Although bioengineered HMOs demonstrate structural and functional similarities to natural HMOs, data on their long-term safety, metabolic impact, and immunological effects in infants remain limited. More clinical trials are necessary to confirm their efficacy and safety. Conducting clinical trials to assess the long-term health effects of synthetic HMOs and establishing a comprehensive evaluation system for their application in infant formula and clinical treatment are crucial steps moving forward.

CONCLUSION

This systematic review highlights the dynamic role of HMOs during lactation and their significant antimicrobial properties. HMOs exhibit temporal variation in secretion, influenced by lactation stages, maternal factors, and genetic determinants, such as secretor status. These variations are finely tuned to support the infant’s evolving nutritional, immunological, and microbial needs. The antimicrobial effects of HMOs are multifaceted, encompassing mechanisms such as pathogen adhesion inhibition, biofilm disruption, enzymatic activity impairment, and immune modulation. Both natural and synthetic HMOs demonstrated robust activity against various pathogens, including bacteria, viruses, and protozoa. Synthetic HMOs, such as bioengineered 2′-FL and 3-FL, were particularly effective and offer scalable solutions for enhancing infant formula and therapeutic interventions. Additionally, HMOs exhibited synergistic effects with antibiotics, highlighting their potential as adjuncts to conventional therapies for combating antimicrobial resistance. Despite these promising findings, further research is required to address existing gaps, including the variability in HMO profiles, long-term safety of synthetic HMOs, and broader exploration of their efficacy against diverse pathogens. Clinical trials are essential to validate findings from in vitro and animal studies and explore HMOs' translational potential in improving neonatal and maternal health. HMOs are a cornerstone of breast milk’s bioactive properties, offering a natural, multifaceted approach to enhancing infant immunity, gut health, and resistance to infections. Leveraging the unique properties of HMOs through breastfeeding promotion, supplementation, and therapeutic innovations has the potential to revolutionize maternal and pediatric healthcare, reduce the burden of infectious diseases, and contribute to global health improvements.