Real-world evidence study on tolerance and growth in infants fed an infant formula with two human milk oligosaccharides vs mixed fed and exclusively breastfed infants

Human milk oligosaccharides (HMOs) are found in abundance in human milk and make up the largest solid component after lactose and lipids [1‐4]. HMOs can become characterized as complex group of more than 200 different, nondigestible, and non-nutritional carbohydrates, providing an energy source for beneficial intestinal bacteria. There is evidence that HMOs improve the host defense by strengthening the gut barrier and immune-modulating effects and other mechanisms. Bovine milk, in contrast to human milk, contains relatively low levels of oligosaccharides, and the prevalence of fucosylated oligosaccharides, in particular, is quite low [5]. 2′-fucosyllactose (2′FL) is a trisaccharide composed of glucose, galactose, and fucose and is one of the most abundant HMOs. Levels of 2′FL vary depending on the secretor blood group status of an individual woman as well as ethnicity and stage of lactation, with 2′FL levels from about 0.9 to above 4 g/L in mature milk among secretors [6‐14]. Another predominant HMO in human milk is lacto-N-neotetraose (LNnT) at levels ranging from 0.1 to 0.6 g/L with higher levels within the first month of lactation [7‐10, 15‐17].

Advancements in manufacturing technology now enable the synthesis of HMOs, and preclinical studies have established their safety for the purposes of supplementation of infant formulas [18, 19]. Safety, tolerance, and adequate growth as well as potential clinical benefits have been demonstrated in randomized controlled trials (RCTs) of term infant formulas supplemented with 2′FL alone and in combination with LNnT [20‐22]. An RCT in the United States of America (USA) found that infants receiving formula supplemented with either galacto-oligosaccharides (GOS) or GOS + 2′FL demonstrated adequate growth and good tolerance [21]. Another RCT conducted in Belgium and Italy examined a study formula containing 1.0 g/L 2′FL and 0.5 g/L of LNnT in the test arms, while the control arm received standard formula without HMOs [20]. The HMO-supplemented formula was again well-tolerated and supported age-appropriate growth. A third study in the USA compared tolerance in infants receiving a 100% whey, partially hydrolyzed infant formula with the probiotic Bifidobacterium lactis with and without the further addition of 2’FL and found that the HMOs-supplemented formula was well-tolerated [22].

Evidence is emerging that HMOs play an important role in the development of a balanced intestinal microbiota and in supporting immune protection in breastfed infants [23‐25]. Preclinical models have found that both 2’FL and LNnT promote the growth of Bifidobacterium species [26, 27]. Additionally, in a RCT of a term infant formula supplemented with 2’FL and LNnT, lower rates of parent-reported morbidity (particularly lower respiratory tract illnesses such as bronchitis) and lower use of antipyretics and antibiotics in the group receiving HMOs-supplemented formula were reported compared to the control infants [20]. Stool samples collected for microbiota assessment and metabolic signature at 3 months showed that the addition of 2’FL and LNnT shifted the stool microbiota closer to that observed in breastfed infants both in composition and function [28]. Collectively, these findings, in conjunction with the documented differences in HMOs composition between human and bovine milk, have provided a solid rationale for the benefits of bovine milk-based infant formulas with HMOs.

While the evidence provided to date in RCTs is supportive of the safety and tolerance of HMOs-supplemented infant formula, studies are needed in a real-world setting because results from a highly controlled RCT do not always translate outside of the trial setting [29]. Additionally, a relatively large proportion of infants in real-world settings are fed with both human milk and formula [30‐32], a mixed feeding regimen not studied in RCTs. The current study was thus designed to complement and enhance existing RCTs by assessing the growth, safety, and tolerance of healthy term infants, consuming an infant formula supplemented with HMOs either exclusively or mixed with human milk in a real-world setting.

The results indicate that formula-fed infants, either exclusively or mixed fed, receiving the formula supplemented with 2′FL and LNnT, had age-appropriate growth in line with the WHO standards and comparable to BF infants. Growth was also comparable to that seen in previous studies with West and South European infant populations [35]. By week 8, GI tolerance as indicated by low IGSQ scores was comparable in the formula-fed infants with that in BF infants indicating the formula is well tolerated. The incidence of adverse events in all groups was low. As shown at Table 4, 7.8% (n = 4) of the AEs were potentially formula related. Despite the season of the year (fall-winter), cases of bronchitis were lower than expected from the literature [36]. Therefore, the results were not added as secondary outcomes.

The results of this RWE study are comparable to those from previous RCTs that have examined anthropometry and GI tolerance for term infant formulas supplemented with HMOs. One RCT was a multicenter, double-blind trial that enrolled 175 healthy term infants in Italy and Belgium at less than 14 days of age who were fed study formula for 6 months [20]. The HMO-supplemented formula (2′FL + LNnT) demonstrated age-appropriate growth as well as good tolerance as measured by parents. Another RCT included 189 term infants in the USA who were exclusively formula fed until 4 months of age [21]. Formulas with 2′FL (at two different dosages) and GOS were well-tolerated based on parental reports, and no significant differences were observed for growth compared to a control group. Notably, neither of those trials utilized a validated tool to assess tolerance, and thus, tolerance outcomes cannot easily be compared across studies. A recent RCT used the same validated IGSQ tool as in the current study to assess tolerance [22]. The HMO-supplemented formula was well tolerated as evidenced by similar IGSQ scores at week 6 between the groups with (mean [SD] = 20.9 [4.8]) and without (mean [SD] = 20.7 [4.3]) the addition of 2′FL. These scores are similar to those observed in FF in the current study at week 8 (mean [SD] = 19.1[4.5]). Additionally, a single-arm study of a formula supplemented with 2′FL fed to fussy infants showed significant improvement in IGSQ scores after 3 weeks of feeding (baseline mean [SD] = 34.1 [10.0]; week 3 mean [SD] = 21.4 [7.0]; p < 0.001) [37]. Although we did not limit our study to fussy infants, we also saw an improvement in IGSQ in FF in our study from baseline to week 8 (mean difference [95% confidence interval] =  − 6.639 [− 9.497, − 3.782], p < 0.0001). The improvement in GI tolerance in our study might be partially related to the natural evolvement of GI tolerance which improves with increasing age but could potentially also be attributed to the composition of the study formula including the two HMOs. Almost all infants switched to the study formula at the beginning of the study, i.e., they were receiving a different formula prior to enrollment (44 of 46 FF infants and 21 of 22 MF infants), suggesting that the HMO-containing study formula has better GI tolerance than the formulas without HMOs consumed prior to enrollment. Only one other real-world study has been conducted to our knowledge; a study with similar design to the current study was conducted in Spain and had very similar results for both growth and IGSQ scores [38]. The agreements between the previous RCTs, the Spanish real-world study, and the current real-world study are reassuring that growth, safety, and tolerance of HMO-supplemented formula are consistent and robust across different geographical populations.

This study has several strengths. First, GI burden was measured using a validated instrument, the 13-item IGSQ based on five separate domains of feeding tolerance. The use of a validated instrument provides information that is interpretable and meaningful to practicing clinicians. Second, this was an RWE study, a design distinct from the RCT, simpler, less restrictive, but still in line with current clinical practices, enhancing the generalizability of the results and providing complementary data to RCTs. The published prevalence of infants who are mixed fed indicates that at age 1 month, 30% of infants receive mixed feedings [30, 31], similar to that observed in this study. Thus, the demonstration of appropriate growth and good tolerance in the mixed feeding group of infants in this study provides important evidence not found in the RCTs conducted to date. Some limitations of the study should also be noted. An open-label, non-randomized design increases the risk for bias, in particular for response bias (even for validated questionnaires), and higher attrition rates and missing data. In a study with specifically defined feeding regimens such as ours, randomization is however not possible. The main aim of randomization is to have study groups with comparable characteristics. We therefore compared the baseline characteristics in our three groups, and there are no substantial differences except for infant age at enrollment and parents’ smoking status. Infant age at enrollment was slightly lower in MF compared with both BF and FF. As it may take longer for mothers to establish exclusive breastfeeding or formula-fed patterns, the younger age in MF at enrollment can be expected. To account for the slight difference in baseline age between the groups, we included baseline age in the statistical models to reduce potential risk of bias arising by the non-randomized nature of our trial. Smoking was higher among the parents in FF compared with BF which for the fathers could be linked to the slightly lower education level in FF compared with BF. Alternately, the parents in BF may have underreported their smoking habits to make a positive impression, a form of social-desirability bias. The study formula was supplemented with just a single level of 2′FL and LNnT, and thus, this study cannot assess whether the observed growth and tolerance effects might differ over a wider range of levels of these HMOs. Additionally, this study, while multicenter, took place within only two countries (Germany and Austria), and its results may not be generalizable outside of Northwest Europe. Furthermore, the authors want to point out that even if supplemented with HMOs, infant formula is not comparable with the gold standard “human breast milk” concerning multiple aspects. The aim of research in infant formula is of cause not to replace human breast milk — but to have the best possible substitute available in case breastfeeding fails or breast milk is not available.

In conclusion, this is one of the first studies to use real-world evidence to examine the supplementation of infant formula with HMOs. The results obtained were similar to those found in more tightly controlled RCTs, indicating robust effects for growth, safety, and tolerance in association with HMO-supplemented infant formulas.