Effects of Infant Formula With Human Milk
Oligosaccharides on Growth and Morb idity:
A Randomized Multicenter Trial
Giuseppe Puccio,
y
Philippe Alliet,
Cinzia Cajozzo,
y
Elke Janssens,
Giovanni Corsello,
jj
Norbert Sprenger,
z
Susan Wernimont,
§
Delphine Egli,
jj
Laura Gosoniu, and
ô
Philippe Steenhout
ABSTRACT
Objectives: The aim of the study was to evaluate the effects of infant
formula supplemented with 2 human milk oligosaccharides (HMOs) on
infant growth, tolerance, and morbidity.
Methods: Healthy infants, 0 to 14 days old, were randomized to an intact-
protein, cow’s milk–based infant formula (control, n ¼ 87) or the same
formula with 1.0 g/L 2
0
fucosyllactose (2
0
FL) and 0.5 g/L lacto-N-neotetraose
(LNnT) (test, n ¼ 88) from enrollment to 6 months; all infants received
standard follow-up formula without HMOs from 6 to 12 months. Primary
endpoint was weight gain through 4 months. Secondary endpoints included
additional anthropometric measures, gastrointestinal tolerance, behavioral
patterns, and morbidity through age 12 months.
Results: Weight gain was similar in both groups (mean difference [95%
confidence interval] test vs control: 0.30 [1.94, 1.34] g/day; lower bound of
95% confidence interval was above noninferiority margin [3 g/day]).
Digestive symptoms and behavioral patterns were similar between groups;
exceptions included softer stool (P ¼ 0.021) and fewer nighttime wake-ups
(P ¼ 0.036) in the test group at 2 months. Infants receiving test (vs control) had
significantly fewer parental reports (P ¼ 0.004–0.047) of bronchitis through 4
(2.3% vs 12.6%), 6 (6.8% vs 21.8%), and 12 months (10.2% vs 27.6%); lower
respiratory tract infection (adverse event cluster) through 12 months (19.3% vs
34.5%); antipyretics use through 4 months (15.9% vs 29.9%); and antibiotics
use through 6 (34.1% vs 49.4%) and 12 months (42.0% vs 60.9%).
Conclusions: Infant formula with 2
0
FL and LNnT is safe, well-tolerated, and
supports age-appropriate growth. Secondary outcome findings showing
associations between consuming HMO-supplemented formula and lower
parent-reported morbidity (particularly bronchitis) and medication use
(antipyretics and antibiotics) warrant confirmation in future studies.
Key Words: 2
0
fucosyllactose, bronchitis, lacto-N- neote traose , safety, tolerance
(JPGN 2017;64: 624–631)
H
uman milk contains an abundance of structurally diverse
oligosaccharides, known collectively as human milk oligo-
saccharides (HMOs), which represent the third largest solid com-
ponent of human milk after lactose and lipids (1,2). Three classes of
oligosaccharides can be distinguished in human milk: neutral
What Is Known
Human milk contains structurally diverse oligosac-
charides, which may support gastrointestinal and
immune functions in breast-fed infants.
Numerous studies have reported a lower incidence of
infections, including respiratory tract infections, in breast-
fed infants compared with those fed infant formula.
What Is New
Infant formula supplemented with 2
0
fucosyllactose
and lacto-N-neotetraose, 2 oligosaccharides com-
monly found in human milk, is safe, well-tolerated
and supports age-appropriate growth.
Secondary outcome findings of lower morbidity
(particularly bronchitis) and medication use (anti-
pyretics and antibiotics) were reported in infants
fed supplemented formula, associations that warrant
confirmation in future studies.
Received July 29, 2016; accepted January 13, 2017.
From the
Dipartimento Materno Infantile, AOUP ‘‘Paolo Giaccone,’’
Universita` di Palermo, Palermo, Italy, the
y
Department of Paediatrics,
Jessa Hospital, Hasselt, Belgium, the
z
Nestle´ Nutrition R&D, King of
Prussia, PA, the
§
Nestle´ Nutrition, Vevey, the
jj
Nestle´ Research Center,
Nestec Ltd, Lausanne, and the
ô
Nestle´ Health Science, Epalinges,
Switzerland.
Address correspondence and reprint requests to Giuseppe Puccio, MD,
Dipartimento Materno Infantile, AOUP ‘‘Paolo Giaccone,’’ Universita`
di Palermo, Via Alfonso Giordano 3, 90127 Palermo, Italy
(e-mail: gipuccio@gmail.com).
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text, and links to the digital files are provided in the
HTML text of this article on the journal’s Web site (www.jpgn.org).
www.clinicaltrials.gov registration number: NCT01715246.
This study was sponsored by Nestec Ltd. The infant formula evaluated
in this study was prepared for the purpose of the clinical trial by
Nestle
´
Product Technology Center Konolfingen and was provided free
of charge.
Portions of this study were presented in abstract form at the 3rd International
Conference on Nutrition and Growth, Vienna, Austria, March 17–19, 2016.
Nestle
´
provided grants and nonfinancial support to AOUP ‘‘Paolo Giaccone,’’
Palermo, Italy, and to the Department of Paediatrics, Jessa Hospital, Hasselt,
Belgium. G.P. and C.C. have also worked on other studies sponsored by
Nestle
´
. N.S. and L.G. are current employees of Nestle
´
Research Center,
Nestec Ltd. S.W. is a former employee of Nestle
´
Nutrition. D.E. is a current
employee of Nestle
´
Nutrition. P.S. is a current employee of Nestle
´
Health
Sciences. The remaining authors report no conflicts of interest.
Copyright
#
2017 The Author(s). Published by Wolters Kluwer Health, Inc.
on behalf of the European Society for Pediatric Gastroenterology,
Hepatology, and Nutrition and the North American Society for Pediatric
Gastroenterology, Hepatology, and Nutrition. This is an open-access
article distributed under the terms of the Creative Commons Attribution-
Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it
is permissible to download and share the work provided it is properly
cited. The work cannot be changed in any way or used commercially
without permission from the journal.
DOI: 10.1097/MPG.0000000000001520
ORIGINAL ARTICLE:NUTRITION
624 JPGN
Volume 64, Number 4, April 2017
fucosylated (including 2
0
fucosyllactose, 2
0
FL), neutral nonfucosy-
lated (including lacto-N-neotetraose, LNnT), and acidic (including
3
0
sialyllactose, 3
0
SL, and 6
0
sialyllactose, 6
0
SL) (3–5). In contrast,
cow’s milk contains relatively low oligosaccharide content with
limited structural diversity (6). Although the types and concen-
trations of HMOs vary considerably among lactating women and
over time, 2 that are commonly found in abundance in human milk
are 2
0
FL and LNnT. Levels of 2
0
FL typically range from 1.1 to 4.3 g/L
in mature milk based on pooled data from secretors and nonsecretors
(3,5,7–17). Levels of LNnT in mature milk typically range from 0.1
to 0.6 g/L (5,9,10,12,15,18–20), with higher levels more commonly
observed within the first month of lactation (12).
Emerging research has suggested the importance of HMOs in
enhancing the development of the intestinal microbiota and sup-
porting immune protection in breast-fed infants (21,22). Both 2
0
FL
and LNnT promote the growth of Bifidobacterium species in
preclinical models (23,24). In addition to this, in mice infected
with adherent-invasive Escherichia coli (AIEC), 2
0
FL was effective
in reducing colonization by AIEC, modulating AIEC-induced
CD14 expression, and attenuating inflammation associated with
E. coli invasion (25). In healthy infants, the relative absorption of
2
0
FL was similar among breast-fed infants and those fed formula
supplemented with 2
0
FL (26), suggesting that 2
0
FL and possibly
other HMOs may exert effects beyond the gastrointestinal (GI) tract
(27,28). These findings, in conjunction with the documented differ-
ences in oligosaccharide composition between human and bovine
milk, support the rationale for supplementing cow’s milk–based
infant formulas with 2
0
FL and LNnT. Although the safety of HMOs
has been previously established in preclinical studies (29,30),
clinical data regarding the effects of HMO supplementation on
infant growth and tolerability are extremely limited (26). Therefore,
the primary objective of this study was to evaluate the growth of
infants fed a new cow’s milk–based formula supplemented with
2
0
FL and LNnT. Secondary objectives included the evaluation of
anthropometric measures, GI tolerance, and behavioral patterns, as
well as morbidity through age 12 months.
METHODS
This was a multicenter, randomized, double-blind trial of 2
parallel groups of formula-fed infants who were enrolled at 14
days of age. Formula-fed infants who met the eligibility criteria
were randomized equally to receive either control or test formula
through 6 months of age. Randomization was carried out using a
permuted block algorithm with Medidata Balance (New York, NY)
and was stratified by infant sex and delivery method (vaginal or
cesarean) to ensure balance of infant sex and delivery mode between
groups. Parents/caregivers (hereafter ‘‘parents’’), investigators and
study support staff were blinded to the study formulas; formulas
were coded by the manufacturer (Nestle
´
Product Technology
Center, Konolfingen, Switzerland) using a single nonspeaking code
per formula group.
The control formula was an intact protein, cow’s milk–
based, whey-predominant infant formula with long-chain polyun-
saturated fatty acids (67 kcal/100 mL reconstituted formula; 1.8 g
protein/100 kcal with a whey:casein ratio of 70%:30%). The test
formula was identical to control except for the addition of 2 HMOs
(Glycom A/S, Kongens Lyngby, Denmark) providing 1.0 g 2
0
FL and
0.5 g LNnT per liter of reconstituted formula. Although not directly
measured, the osmolarities of the 2 study formulas were likely
similar (or even slightly lower in the test formula) because HMOs,
which replaced the same amount of lactose in the test formula, have
higher molecular weight than lactose. The test and control formulas
were distributed at study visits until age 6 months, when infants in
both formula groups were switched to an intact protein, cow’s
milk–based, follow-up formula without HMOs for feedings through
age 12 months. Parents were advised to feed the study formulas to
their infants as they deemed appropriate, based on the infant’s
appetite, age, and weight. Complementary foods were allowed
beginning at age 4 months.
The study was conducted between October 2012 and July
2015 in the Dipartimento Materno Infantile AOUP ‘‘Paolo
Giaccone,’’ Universita
`
di Palermo, Palermo, Italy, and in the
Department of Paediatrics at Jessa Hospital in Hasselt, Belgium.
The study was approved by the ethical committees of both hospitals.
Trial conduct complied with the Declaration of Helsinki and the
International Conference on Harmonization guidelines for Good
Clinical Practice. Informed consent was obtained from the parent or
legal guardian of each infant before enrollment.
Participants
We enrolled healthy infants using the following inclusion
criteria: 14 days of age; gestation 37 to 42 weeks; birth weight
2500 to 4500 g; mothers had independently elected, before enroll-
ment, not to breast-feed; exclusive formula-feeding at time of
enrollment; and parent/legal guardian informed consent. Exclusion
criteria were congenital illness or malformation that could affect
growth; significant prenatal and/or serious postnatal disease before
enrollment; minor parent(s); parents not expected to comply with
study procedures; and current or previous participation in another
clinical trial.
Study Visits
Infants completed a baseline visit at 14 days of age,
followed by visits at 1, 2, 3, 4, 6, and 12 months of age. At the
baseline visit, demographic information and participant character-
istics were collected, a clinical examination was performed and
anthropometrics (weight, length, and head circumference) were
obtained. At each subsequent visit, a clinical examination was
performed, anthropometrics were obtained, and digestive tolerance,
behavioral patterns, and formula intake were assessed using a paper
diary completed by the parent reflecting 3 consecutive days before
the visit. Parents also provided information on illness symptoms and
medication use in the diary, which was reviewed and confirmed by
the physician at each study visit.
Outcome Measures
The primary outcome was weight gain (g/day) between enroll-
ment and age 4 months (calculated as the difference in infant weight
between the baseline visit and the visit at age 4 months, divided by the
number of days between these 2 visits), as recommended in guidelines
from the American Academy of Pediatrics Task Force on Clinical
Testing of Infant Formulas (31). The period from 0 to 4 months
represents the timeframe when infant formula is used as the sole
source of nutrition. Secondary outcomes included other anthropo-
metric measures (weight, length, body mass index [BMI], head
circumference, and corresponding z scores), digestive tolerance
(flatulence, spitting-up, and vomiting), stool characteristics (stool
consistency and frequency), behavior patterns (restlessness, colic,
and nighttime awakenings), formula intake, and morbidity (parent-
reported adverse events [AEs] and concomitant medications).
Detailed information on outcome measurement procedures is
provided in Supplemental Digital Content 1 (http://links.lww.com/
MPG/A886). Briefly, anthropometrics were measured by trained
study personnel using standard procedures; z scores were calculated
using the WHO 2006 Child Growth Standards (32). Diaries
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625
completed by parents in the 3-day period before each postbaseline
study visit provided information on number of stools per day and
stool consistency (1 ¼ hard, 7 ¼ watery; stool images and descrip-
tions provided to parents). Parents were also asked to respond to a
series of structured questions in the diary designed to assess the
frequency with which their child exhibited symptoms related to
digestive tolerance and behavioral patterns (ie, flatulence, spitting-
up, vomiting, colic, restless and irritable, and waking up during the
night) by selecting one of the response options (ie, never, some-
times, or often). During the initial baseline visit, parents were
instructed on how and when to fill out the diary, and at each of
the subsequent visits, the investigators reviewed the diary with the
parents to verify the completeness and accuracy of the diary entries.
The diary also included a grid for the parent to record any
illness symptoms including fever. Although parents were not
expected to diagnose specific illnesses, they were asked to convey
in the diary any illness diagnoses provided to them by either the
child’s primary care physician or the study physician when the child
was ill. Parents also could record in the grid any hospitalizations
(reason, duration) and intake of medications since the prior visit,
and the associated start and stop dates. The information recorded in
the diary by the parent was reviewed and verified by the study
physician at each study visit before being recorded in the study
database as reported AEs or reported medication use. AEs were then
coded and categorized by a single physician who was not involved
in study conduct using the Medical Dictionary for Regulatory
Activities (MedDRA) System, Organ, and Class (SOC) categories
as well as the Preferred Terms (PTs) within each SOC category.
Several PTs within the ‘‘Infections and Infestations’’ SOC category
were identified a priori as being of particular interest, including
upper respiratory tract infection, pyrexia, rhinitis, bronchitis,
bronchiolitis, otitis media, pharyngitis and gastroenteritis. In
addition to this, 3 AE clusters were identified: upper respiratory
tract infection, lower respiratory tract infection, and otitis/ear
infection (see Supplemental Digital Content 1, http://links.lww.
com/MPG/A886, for PTs included in each AE cluster). Similarly,
recorded medications were coded and categorized into groups,
including antibiotics, antipyretics, and gastroesophageal reflux
disease medications. Data related to AEs and medication use were
expressed as the proportion of infants in each group with at least 1
episode of the specific AE or medication use.
Statistical Analysis
The sample size was determined according to guidelines
from the American Academy of Pediatrics Task Force on Clinical
Testing of Infant Formulas (31). Specifically, power calculations
were performed using the software package R version 2.13.2 (2011;
The R Foundation for Statistical Computing, Vienna, Austria).
Assuming 80% power, a ¼ 0.025 for the 1-sided weight gain
noninferiority comparison between groups, a noninferiority margin
of 3 g/day (31), and a standard deviation of 6.1 g/day (33), a total
of 66 infants were needed in each formula-fed group. Enrollment of
approximately 88 infants per group was planned to account for a
25% dropout rate.
The primary outcome (weight gain, g/day from baseline to 4
months) was analyzed using an analysis of covariance model
adjusted for multiple covariates/factors including baseline weight,
treatment, sex, and center. Missing postbaseline weight gain was
imputed using the respective treatment visit mean by sex. Adjusted
mean and standard error (SE) of weight gain was reported. Weight
gain in the test group was considered noninferior to control if the
lower bound of the 2-sided 95% confidence interval (CI) for
the difference between groups (test minus control) was above the
noninferiority margin of 3 g/day (31). The primary outcome was
analyzed in both the per-protocol (PP) and intention-to-treat (ITT)
populations. The PP population included infants with none of the
following major protocol violations before age 4 months: being
hospitalized for >3 consecutive days 1 week before age 4 months,
being off study formula for 3 consecutive days, and taking 4
teaspoons of complementary food per day. The ITT population
included all infants randomized to study formula.
Secondary outcomes were evaluated in the ITT population,
without adjustment for multiple testing. Anthropometrics were
analyzed using a mixed-effect model repeated measures approach
adjusted for multiple covariates/factors and their interactions
including baseline anthropometric assessments, sex, center, visit,
treatment, sex visit, and sex treatment. The Cochran-Mantel-
Haenszel test was used to evaluate the linear association between
study formula and digestive tolerance, behavioral patterns, and stool
consistency. Stool consistency was also compared between groups
using a Student t test at each visit. Stool frequency was analyzed
using the negative binomial generalized linear model. Morbidity
outcomes were analyzed using the Fisher exact test and reported as
odds ratio (OR) and 95% CI (See Supplemental Digital Content 2,
http://links.lww.com/MPG/A887, for additional details on statistical
analyses).
RESULTS
A total of 175 infants were enrolled (95 from the site in
Belgium and 80 from the site in Italy), with 87 and 88 randomized to
receive the control and test formulas, respectively (Fig. 1). Forty-
four (25.1%) infants (20 in control; 24 in test) withdrew before the
primary outcome assessment (4-month follow-up). The dropout rate
was comparable between groups and was accounted for in the
sample size calculation. The most common reason for discontinu-
ation was an AE (n ¼ 11 in control; n ¼ 12 in test). Other reasons for
discontinuation before 4 months included parent/guardian request
(n ¼ 3 in control; n ¼ 6 in test); lost to follow-up/missing (n ¼ 5in
control; n ¼ 6 in test); and other (n ¼ 1 in control; n ¼ 0 in test).
Infant baseline characteristics and anthropometrics were compar-
able between groups (Table 1).
Growth
In the PP population, adjusted mean (SE) weight gain
through age 4 months was 29.84 (0.60) g/day for infants fed the
test formula and 30.15 (0.58) g/day for those fed control (Table 2).
Mean difference (95% CI) in weight gain between groups was
0.30 (1.94, 1.34) g/day, with the lower limit of the 95% CI
above the predefined noninferiority margin of 3 g/day, indicating
similar weight gain in the 2 groups. Results were similar in the ITT
population. Mean weight, length, head circumference, and BMI
were not significantly different between groups at any study visit
(see Supplemental Digital Content 3, http://links.lww.com/MPG/
A888). Likewise, mean weight-for-age, length-for-age, head cir-
cumference-for-age, and BMI-for-age z scores (Fig. 2) did not differ
significantly between the groups at any study visit and tracked
closely with the WHO growth standards.
Formula Intake and Tolerance
Mean daily formula intake was similar between groups at all
study visits. At age 4 months, mean intake was 908 mL in the test
group and 929 mL in the control group (P ¼ 0.54). GI symptoms,
including flatulence, spitting-up and vomiting, were not significantly
different at any study visit between the groups, indicating no associ-
ation between type of formula and digestive tolerance. No differences
were observed between the groups in the proportion of stools in each
Puccio et al JPGN
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626 www.jpgn.org
consistency category. Comparison of mean SE stool consistency
ratings showed that the test group had a tendency toward softer stools
at 1 month (5.9 0.2 vs 5.5 0.2, P ¼ 0.064) and had significantly
softer stools at 2 months (6.1 0.1 vs 5.7 0.2, P ¼ 0.021); no
significant differences were observed at other visits. No significant
differences in stool frequency were reported at any visit.
Behavioral Patterns
Parent-reported infant behavioral patterns including restless-
ness/irritability and colic were similar in the test and control groups
at each study visit, although among the subgroup of infants deliv-
ered by cesarean section, colic at 4 months was reported less
frequently in the test group (P ¼ 0.035). Specifically, among cesar-
ean-born infants, colic was reported as ‘‘never’’ and ‘‘sometimes’’
in 96% and 4%, respectively, in the test group compared to 74% and
26% in the control group. In addition to this, at 2 months, nighttime
awakenings were reported less frequently in the test group
(P ¼ 0.036), with parents reporting ‘‘never,’’ ‘‘sometimes,’’ and
‘‘often’’ in 27%, 69%, and 4% of infants in the test group, compared
with 25%, 57%, and 18% in the control group. This difference,
however, did not persist after age 2 months.
Morbidity
The overall percentage of infants who had at least 1 reported
AE was similar in both groups during the 4-month exclusive feeding
period (test: 84.1%; control 90.8%). At 4 months, at least 1 serious
AE was reported in 6 infants (6.8%) in the test group and 10 infants
(11.5%) in the control group. Only 1 infant experienced an AE
(cow’s milk-protein allergy) considered to be related to the study
formula (infant was assigned to test). There were no statistically
significant differences between groups in the cumulative incidence
of reported AEs when analyzed by SOC categories. The percentage
of infants who reported at least 1 AE in the SOC category of
‘‘Infections and Infestations’’ from 0 to 12 months was, however,
numerically lower in the test group, a difference that approached
statistical significance (69.3% vs 82.8%, OR 0.47, 95% CI 0.21–
1.02, P ¼ 0.051). No statistically significant differences were
observed between groups in other SOC categories.
Regarding the a priori-identified AEs of interest within the
‘‘Infections and Infestations’’ SOC category, as shown in Figure 3,
infants receiving test had significantly fewer reports of bronchitis
through 4, 6, and 12 months, and the AE cluster of lower respiratory
tract infection through 12 months. Similar statistically significant
differences between the test and control groups were observed in the
subgroup of infants born by cesarean section for bronchitis through
12 months (3.1% vs 34.4%, OR 0.06, 95% CI 0.00–0.50, P ¼ 0.003)
and lower respiratory tract infection through 6 (6.3% vs 28.1%, OR
0.17, 95% CI 0.02–0.96, P ¼ 0.043) and 12 months (12.5% vs
40.6%, OR 0.21, 95% CI 0.04–0.83, P ¼ 0.022). Fewer reports of
the AE cluster of otitis/ear infection were observed in the test group
through 12 months, but the difference between groups did not reach
statistical significance (6.8% vs 12.6%, P ¼ 0.213). There were no
TABLE 1. Baseline characteristics of study participants
,y
Infant characteristics Control (n ¼ 87) Test (n ¼ 88)
Age, days 7.7 3.3 8.6 3.3
Sex (n, % male) 44 (50.6) 44 (50.0)
Gestational age, wk 39.2 1.0 39.2 1.1
Mode of delivery (n, % cesarean) 32 (36.8) 32 (36.4)
APGAR scores at birth
5 min 9.0 0.7 8.8 1.0
10 min 9.7 0.6 9.7 0.8
Weight, kg 3.4 0.4 3.4 0.4
Length, cm 50.9 1.9 50.7 1.7
Head circumference, cm 35.4 1.3 35.3 1.2
Data on baseline characteristics were collected at randomization unless
otherwise noted.
y
Values are mean standard deviation unless otherwise noted.
Randomized (n =175)
Control formula (n = 87)
Received study feeding (n = 87)
Test formula with 2 HMOs (n = 88)
Received study feeding (n = 88)
Allocation
Discontinued or
withdrawn (n = 20)
Included in ITT analysis (n = 87)
Included in PP analysis (n = 75)
Completed 6-month intervention (n = 64)
Completed 12-month study (n = 64)
Discontinued or
withdrawn (n = 24)
Included in ITT analysis (n = 88)
Included in PP analysis (n = 71)
Completed 6-month intervention (n = 58)
Completed 12-month study (n=57)
4-month follow-up
for primary analysis
6-month follow-up
12-month follow-up
FIGURE 1. Flow of study participants. Ninety-five infants (47 control, 48 test) were enrolled at the site in Belgium; 80 infants (40 control, 40 test)
were enrolled at the site in Italy. HMOs ¼ human milk oligosaccharides; ITT ¼ intention-to-treat; PP ¼ per-protocol.
JPGN
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627
other statistically significant findings for the reported incidences of
AE PTs or AE clusters. Infants receiving test had significantly fewer
reports of the use of antipyretics through 4 months; however, this
difference was not statistically significant at 6 or 12 months (Fig. 3).
Infants receiving test also had significantly fewer reports of anti-
biotic use through 6 and 12 months.
DISCUSSION
To our knowledge, this is the first randomized, controlled
trial of infant formula supplemented with both 2
0
FL (1 g/L) and
LNnT (0.5 g/L), 2 abundant oligosaccharides in breast milk that
collectively represent 37% of total HMOs (34). We found that
compared with infants fed the control formula without HMOs, those
fed the test formula: manifested similar age-appropriate growth, had
indications of improved GI comfort in the first few months of age,
and had lower rates of parent-reported morbidities related to lower
respiratory tract infections as well as antipyretic and antibiotic use.
As the study was powered for the primary outcome (weight gain
from 0 to 4 months of age), the findings related to secondary
outcomes warrant confirmation in future studies.
Growth and Tolerance
The study demonstrated comparable weight gain in the test
and control groups and growth tracked closely with the WHO
growth standards through 12 months of age. The study also showed
that test formula supplemented with 1 g/L 2
0
FL and 0.5 g/L LNnT is
well tolerated. Interestingly, infants fed the test formula tended to
have softer stools at 1 month of age and had significantly softer
stools at age 2 months. Although these differences did not persist at
later time points, the findings highlight the beneficial effect of
HMOs on stool characteristics during the first few months of life
when the neonatal GI tract undergoes profound growth and func-
tional maturation (35). Moreover, the proportion of parents report-
ing that their infants woke up ‘‘often’’ at night was lower in the test
group at 2 months of age, although this finding did not persist at
later time points. The lack of statistical significance for this end-
point after 2 months may be attributable to inadequate power. In
addition to this, among cesarean-born infants, parents reported less
colic in the test group at age 4 months. Collectively, these findings
suggest that infant formula supplemented with 1 g/L 2
0
FL and 0.5 g/L
TABLE 2. Comparison of weight gain from enrollment to 4 months of age between groups
Population Groups Weight gain, g/day LS Mean (SE)
Difference between groups
(test control)
Estimate 95% CI
PP Test (n ¼ 71) 29.84 (0.60) 0.30 1.94, 1.34
Control (n ¼ 75) 30.15 (0.58)
ITT Test (n ¼ 88) 29.39 (0.54) 0.13 1.63, 1.37
Control (n ¼ 87) 29.52 (0.54)
CI ¼ confidence interval; ITT ¼ intention-to-treat; LS ¼ least squares; PP ¼ per-protocol; SE ¼ standard error.
From an analysis of covariance model with treatment, sex, and center as fixed effects and baseline weight as a covariate.
2
AB
CD
1
0
−1
Weight-for-age z score
Head circumference-
for-age-z score
−2
≤14 days
12
Age, mo
A
g
e, mo
34612
2
1
0
−1
−2
≤14 days
1234612
2
1
0
−1
Length-for-age-z scoreBMI-for-age-z score
−2
≤14 days
12
Age, mo
A
g
e, mo
34612
2
1
0
−1
−2
≤14 days
1234612
FIGURE 2. Anthropometric z scores for weight-for-age (A), length-for-age (B), head circumference-for-age (C) and BMI-for-age (D) from
enrollment to 12 months of age based on the 2006 World Health Organization Child Growth Standards. Triangles/dashed line ¼ control; Circles/
solid line ¼ test. BMI ¼ body mass index.
Puccio et al JPGN
Volume 64, Number 4, April 2017
628 www.jpgn.org