CLINICAL TRIAL: NUTRITION
Effect of Differential Enteral Protein on Growth and
Neurodevelopment in Infants <1500 g: A Randomized
Controlled Trial
Shivani Dogra, Anup Thakur, Pankaj Garg, and Neelam Kler
ABSTRACT
Objective: The aim of the study was to determine whether higher enteral
protein intake leads to improved head growth at 40 weeks postmenstrual age
(PMA) in preterm infants <32 weeks or 1500 g.
Methods: Randomized controlled trial in which 120 infants were assigned to
either group A with higher enteral protein intake achieved by fortification
with higher protein containing fortifier (1 g/100 mL expressed breast milk)
or to group B with lower enteral protein intake where fortification was done
with standard available protein fortifier (0.4 g /100 mL expressed breast
milk).
Results: The mean (standard deviation) protein intake was higher in group A
as compared to group B; 4.2 (0.47) compared with 3.6 (0.37) g  kg1  day1,
P < 0.001. At 40 weeks PMA, the mean (standard deviation) weekly
occipitofrontal circumference gain was significantly higher in group A as
compared to group B; 0.66 (0.16) compared with 0.60 (0.15) cm/week (mean
difference 0.064, 95% confidence interval [0.004–0.123], [P ¼ 0.04]).
Weight growth velocity in group A was 11.95 (2.2) g  kg1  day1 as
compared to 10.78 (2.6) g  kg1  day1 in group B (mean difference
1.10, 95% confidence interval [0.25–2.07], [P ¼ 0.01]). No difference
was observed in the length between the 2 groups. There was no
difference in growth indices and neurodevelopmental outcomes at 12 to
18 months corrected age in the 2 groups.
Conclusions: Fortification of expressed human milk with fortifier
containing higher protein results in better head growth and weight gain
at 40 weeks PMA in preterm infants <32 weeks or 1500 g without any
benefits on long-term growth and neurodevelopment at 12 to 18 months
corrected age (CTRI/2014/06/004661).
Key Words: expressed breast milk, human milk fortifier, nutrition
(JPGN 2017;64: e126–e132)
O ptimizing nutrition in premature infants is a challenging
task. The nutritional regimen for these infants should be
able to support postnatal growth similar to intrauterine growth.
Most preterm infants are, however, not fed as much protein as they
Received March 18, 2016; accepted October 19, 2016.
From the Department of Neonatology, Institute of Child Health, Sir Ganga
Ram Hospital, Rajinder Nagar, New Delhi, India.
Address correspondence and reprint requests to Prof (Dr) Neelam Kler,
MD, Department of Neonatology, Institute of Child Health, Sir Ganga
Ram Hospital, New Delhi 110060, India
(e-mail: drneelamkler@gmail.com).
www.ctri.nic.in registration number: CTRI/2014/06/004661.
FM 85 human milk fortifier (HMF) was donated by Nestec, Switzerland.
The authors report no conflicts of interest.
Copyright # 2016 by European Society for Pediatric Gastroenterology,
Hepatology, and Nutrition and North American Society for Pediatric
Gastroenterology, Hepatology, and Nutrition
DOI: 10.1097/MPG.0000000000001451
e126
Copyright © ESPGHAL and NASPGHAN. All rights reserved.
What Is Known
 Higher enteral protein improves short-term growth in
preterm very-low-birth-weight infants.
What Is New
 Higher enteral protein intake improves head growth
and weight at 40 weeks postmenstrual age in pre-
term very-low-birth-weight infants.
 Short-term gain may not translate into long-term
benefits of improved growth and neurodevelopmen-
tal outcome.
would have received in utero by active placental transport, which
could possibly result in extrauterine growth restriction. There is
accumulating evidence that postnatal growth restriction of these
babies during the window for brain development can lead to serious
consequences (1).
Human milk is the preferred form of nutrition for all neo-
nates. Premature infants who are predominantly fed human milk
receive significant benefits with respect to host protection and
improved developmental outcomes compared with formula-fed
infants (2). There are, however, concerns about the nutritional
inadequacy of unfortified human milk usage in preterm neonates.
Fortification is a step to overcome this problem. There is evidence
that use of multicomponent fortifier leads to short-term increase in
weight gain, linear growth, head growth, and bone mineralization in
very-low-birth-weight (VLBW) infants. The long-term benefits of
fortification in improving growth and neurodevelopmental out-
comes remain unproven (3).
Most recent guidelines by ESPGHAN Committee on Nutri-
tion recommends aiming at 4.0 to 4.5 g  kg1  day1 protein intake
for infants weighing <1000 g and 3.5 to 4.0 g  kg1  day1 for
infants weighing from1000 to 1800 g (4). Although fortification of
human milk in preterm neonates is a standard global practice to
achieve these intakes of protein, the protein content of human milk
fortifiers (HMFs) varies throughout the world. World Health
Organization guidelines on feeding of VLBW infants does not
support routine multicomponent fortification except in infants
who fail to gain weight despite adequate breast milk feeding (5).
The National Neonatology Forum of India recommends fortifica-
tion in VLBW infants or infants <32 weeks in similar circum-
stances (6). These guidelines present a paradox because it may take
2 to 3 weeks to identify infants failing to gain weight. Subsequent
introduction of HMF therefore translates into significant delays in
JPGN  Volume 64, Number 5, May 2017
JPGN  Volume 64, Number 5, May 2017
maximizing nutrient intake in VLBW infants and its associated
long-term consequences.
The only multicomponent fortifier available in India has a
protein content of 0.4 g/100 mL expressed breast milk (EBM), which
is lower than most of the internationally available fortifiers (0.7–1 g/
100 mL EBM) (7). Moreover it was observed in our subset of VLBW
infants that fortification with this fortifier led to poor gain in head
growth and length during neonatal intensive care unit stay (8). It is
plausible that a higher protein intake would lead to better growth
indices and neurodevelopmental outcomes in these infants. With this
background, we conducted a randomized controlled trial to evaluate
the effect of higher protein intake on the growth of VLBW infants or
infants <32 weeks’ gestation at 40 weeks’ postmenstrual age (PMA).
We also planned to assess the long-term growth and neurodevelop-
mental outcomes of these infants at 12 to 18 months of corrected age.
METHODS
This randomized controlled trial enrolled infants in the
neonatal intensive care unit of a tertiary care center in Northern
India from October 2012 to April 2014. All preterm infants
weighing <1500 g or <32 weeks at birth were enrolled when they
reached a feed volume of 100 mL  kg1  day1. Infants with lethal
congenital malformations were excluded from the study. Consent
for participation was obtained from the parents before enrollment.
The trial was approved by hospital ethics committee and registered
with Clinical trial registry of India (CTRI/2014/06/004661).
Randomization and Blinding
The random sequence numbers with a block size of 4 were
generated by an independent researcher. The sequence was kept in
serially numbered sealed opaque envelopes. Multiple births were
assigned to the same group. Fortifiers were kept in 2 boxes labeled
as A or B. Fortification was done by the care-giving nurse who was
aware of the type of fortifier used in a particular infant. Treating
neonatologists, research personnel, and the families of the enrolled
infants were, however, unaware of the group allocation.
Intervention
The eligible neonates were randomly assigned to either group
A with higher enteral protein intake or to group B with standard
enteral protein intake. Higher protein intake was defined as intake of
human milk containing a multinutrient fortifier (FM 85; Nestle,
Vevey, Switzerland), which increased the protein intake by 1 g/100
mL. Standard protein intake was defined as intake of human milk
fortified with currently available fortifier (Lactodex HMF; Rapta-
kos Brett and Co. Ltd, Mumbai, India), which increased the protein
intake by 0.4 g/100 mL. For calculations we assumed the protein
content of human milk as 1.5 g/100 mL (9). The composition of the
2 fortifiers to be added per 100 mL of EBM is mentioned in Table 1.
We used only mother’s own milk in the study because of
nonavailability of donor human milk. Every possible effort was
made to procure EBM for each infant. When the quantity of EBM
was insufficient then preterm formula milk containing 2.1 g protein/
100 mL was provided to the infants in both the groups. Daily intake
of EBM and formula was recorded. Fortification was done till
discharge or till infants were totally on direct breast-feeds, which-
ever was earlier.
Feeding Protocol
The eligible neonates were assessed daily for feed initiation.
Once infants were hemodynamically stable with soft abdomen and
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Effect of Differential Enteral Protein in Infants
audible bowel sounds, gavage feeds were initiated as intermittent
boluses at 2-hour intervals. Infants who were not on total enteral
feeds were initiated on parenteral nutrition as per unit protocol to
achieve a total protein intake of 3 to 3.5 g  kg1  day1 and calorie
intake of 80 to 90 kcal  kg1  day1. Feeds were advanced by 20 to
30 mL  kg1  day1 as decided by the treating clinician. Infants
were enrolled in the study once they reached a feed volume of
100 mL  kg1  day1, out of which at least 25 mL was EBM. Full
fortification was then started as per allocated group and parenteral
nutrition was discontinued. Advancement of feeds was done till
infants reached a feed volume of 180 mL  kg1  day1. Once
infants were transitioned from gavage feeds to direct feeds, volume
was not controlled but was offered ad lib. Daily feed tolerance data
including abdominal circumference, gastric aspirate volumes if
indicated, and vomiting were recorded. Feed intolerance was
defined if any of the following was observed: abdominal girth
increment >2 cm between feeds, presence of bilious or hemorrhagic
gastric residuals, and presence of abdominal signs such as abdomi-
nal wall discoloration, erythema, or tenderness. Nil per oral hours
(days) were calculated in both the groups.
Outcome Assessment
The primary outcome of our study was difference in head
growth at 40-week PMA in the 2 groups. The secondary growth
outcomes were weight and length at 40 weeks PMA, head growth,
length, and weight at discharge and at 12 to 18 months corrected
age. Other secondary outcomes were culture-positive sepsis; necro-
tizing enterocolitis (NEC); biochemical parameters such as blood
urea nitrogen (BUN), calcium, phosphorus, alkaline phosphatase,
and prealbumin at discharge; and neurodevelopmental outcomes at
12 to 18 months corrected age.
Weight was recorded daily. Length and occipitofrontal cir-
cumference (OFC) were recorded weekly till the time of discharge.
All these measurements were repeated at 40 weeks (3 days) PMA.
Infants were weighed naked at approximately the same time of the
day using electronic balance scales that were accurate up to 5 g. The
weighing scale was calibrated every 6 months and a log book was
maintained. OFC and length were measured to nearest 1 mm. OFC
was measured weekly by using a paper tape placed across the frontal
bones above the eyebrows and over the occipital prominence at the
back of the head. Length was measured weekly using a recumbent
length board. The mean of the 2 measurements of these parameters
was taken. All measurements were taken by the principal investigator.
Weight growth velocity (GV) was calculated by 2-Point
Average Weight model from time to regain birth weight till dis-
charge and 40 weeks PMA. This was calculated by dividing the total
weight difference at 2 points by number of days and average weight
(10). Length increment per week was calculated from length
difference at birth till discharge and 40 weeks PMA. OFC gain
per week was calculated from head circumference difference at
birth till discharge and 40 weeks PMA.
Biochemical analysis including BUN, serum calcium, phos-
phorus, alkaline phosphatase, and prealbumin were done at dis-
charge. Clinical data pertaining to feed intolerance, NEC as defined
by Bell’s stage 2 or beyond (11), culture-proven sepsis, any
intraventricular hemorrhage on cranial ultrasound, and oxygen
requirement at 36 weeks PMA (defined as bronchopulmonary
dysplasia) were recorded.
Long-term Growth and Neurodevelopmental
Outcomes
Follow-up of infants was done at 12 to 18 months corrected
age. Anthropometric measurements were taken by the principal
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Dogra et al
TABLE 1. Composition of human milk fortifiers to be added to 100 mL
expressed breast milk
Component Group A (FM 85) Group B (Lactodex)
Amount of fortifier, g 5 4
Energy, kcal 17.2 13
Protein, g 1.0 0.4
Carbohydrates, g 3.6 2.4
Fat, g 0.01 0.2
Calcium, mg 52 100
Phosphorus, mg 36 50
investigator. Neurodevelopmental outcome was assessed by Devel-
opmental Assessment Scale for Indian Infants (DASII) (12). DASII
consists of 67 items for mental and 163 items for motor develop-
ment and can be used from 1 to 30 months of age. Mental
Developmental Quotient and Motor Developmental Quotient were
calculated by the developmental pediatrician of the institute, who
was blinded to the group allocation.
Sample Size Calculation and Statistical Analysis
We calculated the sample size based on a previous study (13).
To detect a difference of OFC gain of 0.2 cm/week, length differ-
ence of 0.14 cm/week, and weight gain difference of 5.3
g  kg1  day1 with a power of 80% and 2-sided significance of
5%, sample size were estimated. We chose the largest sample size of
60 infants in each group.
Statistical analysis was done by using SPSS software version
17. Quantitative data with normal distribution were compared using
Student t test and those with skewed distribution were analyzed
using Mann-Whitney U test. Nonquantitative data were compared
using Chi square or Fischer exact test as applicable. A P value of
<0.05 was considered significant. Time to event was analyzed using
the Kaplan-Meier survival analysis.
RESULTS
A total of 158 eligible neonates were screened from October
2012 to April 2014. Among these neonates, 38 were excluded due to
various reasons (Fig. 1). A total of 120 neonates were randomized,
60 in each group. At 40 weeks PMA, 57 neonates in group A and 55
neonates in group B were analyzed. At 12 to 18months corrected
age, 44 neonates in group A and 48 neonates in group B were
assessed for long-term growth and neurodevelopment.
The baseline characteristics in both the groups were com-
parable. Infants in group A, however, received higher proportion of
EBM. The protein intake differed by design (Table 2). At 40 weeks
PMA, OFC gain was greater in group A as compared to group B.
Similarly, infants in group A achieved a significantly higher weight
GV. No difference was, however, observed in length increment
between the 2 groups. Similar findings were observed when these
growth parameters were assessed at discharge (Table 3).The
duration of hospital stay (Fig. 2), time to reach full feeds, and time
to regain birth weight were comparable in the 2 groups. No
significant difference was observed in proportion of neonates
who developed bronchopulmonary dysplasia, sepsis, intraventricu-
lar hemorrhage, and NEC (Table 4). On analyzing the biochemical
parameters, it was found that BUN was significantly higher in group
A as compared to group B. All other values including prealbumin,
calcium, phosphorus, and alkaline phosphatase were comparable in
the 2 groups. Long-term growth and neurodevelopmental outcomes
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JPGN  Volume 64, Number 5, May 2017
as assessed by DASII scale at 12 to 18 months were similar in both
the groups (Table 3).
On subgroup analysis in infants who received >50% EBM,
weight GV and OFC gain at discharge were higher in group A as
compared to group B (Table 5). At 40 weeks PMA, weight GV
remained higher in group A; however, there was no significant
difference in other growth parameters. Long-term growth and
neurodevelopmental outcomes as assessed by DASII scale at
12 to 18 months were similar in both the groups (Table 5).
DISCUSSION
Routine fortification of human milk in preterm VLBW
infants is not a standard practice in developing countries. Two
randomized controlled trials from India evaluated the effect of
currently available fortifier (0.4 g/100 mL) on growth and bio-
chemical parameters. Better growth parameters and biochemical
indices (14,15) were achieved in the fortification group as compared
to infants who received unfortified human milk. This fortifier being
low in protein content, however, fails to meet the recommended
protein needs of VLBW infants and extrauterine growth retardation
is common (8). Therefore we conducted a randomized controlled
trial to evaluate the effect of higher protein fortification versus
available standard fortification on growth of preterm VLBW
infants. The 2 fortifiers differed in protein, fat, and calorie content;
however, the estimated protein intake was significantly higher in
the high protein fortification group (4.2 vs 3.6 g  kg1  day1),
whereas estimated intakes of the other nutrients did not differ
between groups.
We found a significantly better head growth in the higher
protein group as compared to standard protein group at 40 weeks
PMA. This could possibly be due to the positive effect of high
protein on head growth. There are various other studies which have
shown that providing higher protein can lead to a better increment in
head circumference (13,16). In a multicentric randomized con-
trolled trial in 150 preterm infants (GA <30þ3 weeks and birth
weight <1250 g), Moya et al (16) compared lower protein contain-
ing HMF powder (control) with liquid HMF containing 20% higher
protein. At 28 days on per protocol analysis, infants in higher
protein group achieved a significantly better head circumference
growth than the control HMF group. Arslanoglu et al (13) in a
clinical trial randomized 32 preterm infants, with gestational age
between 26 and 34 weeks and birth weights of 600 to 1750 g in
standard fortification and adjusted fortification groups. After 4
weeks of enrollment, a greater gain in head circumference
(1.4  0.3 vs 1.0  0.3 cm; P < 0.05) was achieved in infants on
adjusted fortification regimen as compared to standard regimen. In
a randomized controlled trial in preterm infants <31 weeks’
gestation, Miller et al (17), however, did not find any significant
difference in OFC gain in higher fortification (1.4 g protein/100
mL) group as compared to standard fortification (1.0 g protein/100
mL) group.
The weight GV at discharge and 40 weeks PMA was also
significantly higher in the group which received higher protein. The
effect of multicomponent fortification on weight gain is well
established. Several randomized controlled trials have reported
positive effect of higher protein intake on weight gain as found
in our study (16,17). We observed that the weight GV at discharge
was better in higher protein group as compared to standard protein
group but the difference in the 2 groups decreased at 40 weeks
PMA. This could be because fortification was discontinued once
babies were on direct breast-feeds or after discharge and the
beneficial effect of higher protein intake became less obvious.
Protein intake is also known to correlate with linear growth.
Various studies have shown better z scores of length in infants
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JPGN  Volume 64, Number 5, May 2017 Effect of Differential Enteral Protein in Infants

FIGURE 1. Study flow chart.

TABLE 2. Baseline characteristics of enrolled subjects

Baseline characters Group A (n ¼ 60) Group B (n ¼ 60) P

Gestation, wk 29.9 (2.1) 30.4 (2.3) 0.22

Male, n % 36 (60) 37 (61) 0.85
Birth weight, g 1197 (29) 1241 (24) 0.38
SGA, n (%) 14 (23.3) 15 (25%) 0.84
Age at enrolment, days 6.7 (4.7) 6.49 (4.6) 0.80
Enrolment weight, g 1191.5 (264) 1235.5 (207) 0.31
Estimated volume of milk intake, mL  kg1  day1 181 (21) 184 (18) 0.43
Estimated Protein intake, g  kg1  day1 4.2 (0.47) 3.6 (0.37) <0.001

Estimated calorie intake, kcal  kg1  day1 147 (22.8) 147 (15) 0.78
Duration of fortification, days 29.4 (17) 27.9 (18) 0.64
EBM proportion, % 57.8% 47.1% 0.039

EBM ¼ expressed breast milk; SGA ¼ small for gestational age.

All values expressed as mean (standard deviation) unless specified.

For calculation—calorie in 100 mL of EBM ¼ 67 kcal, calorie in 100 mL of formula ¼ 80 kcal.

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Dogra et al JPGN  Volume 64, Number 5, May 2017

TABLE 3. Growth parameters at various time points and neurodevelopmental outcomes

Variables Group A (n ¼ 57) Group B (n ¼ 55) Mean difference, 95% CI P

(a) At discharge
Weight growth velocity, g  kg1  day1 13.66 (3.8) 11.98 (3.3) 1.68 (0.43–2.90) <0.001
OFC gain, cm/wk 0.72 (0.2) 0.64 (0.16) 0.08 (0.01–0.15) 0.02
Length gain, cm/wk 0.94 (0.8) 0.78 (0.26) 0.16 (0.07–0.40) 0.16
(b) At 40 wk
Weight growth velocity, g  kg1  day1 11.95 (2.2) 10.78 (2.6) 1.10 (0.25–2.07) 0.01
OFC gain, cm/wk 0.66 (0.16) 0.60 (0.15) 0.064 (0.004–0.123) 0.04
Length gain, cm/wk 0.77 (0.22) 0.70 (0.24) 0.08 (0.007–0.169) 0.09
(c) At 12–18 mo
Variables Group A (n ¼ 44) Group B (n ¼ 48)
Mean age of assessment, mo 14.15 (1.5) 14.25 (1.5) — 0.78
Weight, g 9998 (115) 9592 (1211) 406 (86–898) 0.10
OFC, cm 46.44 (1.39) 46.20 (1.19) 0.24 (0.29–0.77) 0.38
Length, cm 76.04 (1.73) 76.12 (1.50) 0.075 (0.74–0.59) 0.82
(d) Neurodevelopmental outcome at 12–18 m
Variables Group A (n ¼ 44) Group B (n ¼ 48)
MoDQ 88.2 (12.4) 84.9 (12.9) 3.23 (2.03–8.48) 0.22
MeDQ 88.2 (13.7) 87.5 (12.1) 0.74 (4.6–6.09) 0.74

Variables expressed as mean (standard deviation).

CI ¼ confidence interval; MeDQ ¼ Mental Development Quotient; MoDQ ¼ Motor Development Quotient; OFC ¼ occipitofrontal circumference.

receiving higher protein. In our study, although the length increase
was higher in the high-protein group but it could not reach statistical
significance. It is possible that the quantum of difference in the
protein intake between the 2 groups was not enough to make the
difference in length perceptible in such a short time span. In a
similar study by Miller et al (17), length gains did not differ between
the higher and standard protein groups. In contrast, Olsen et al (18)
in a study on protein fortification showed better length z scores in
neonates who received higher protein (4.6–5.5 g  kg1  day1).
Similar beneficial effects of higher protein fortification on
short-term growth were reported in a recent meta-analysis, which
compared the growth of preterm infants fed standard protein-
fortified human milk (0.7–1.1 g extra protein per 100 mL EBM)
with that containing HMF with higher-than-standard protein con-
tent. The meta-analysis included 5 studies with 352 infants with
birth weight 1750 g and a gestational age 34 weeks. Infants in
the higher-than-standard protein fortifier achieved significantly
greater weight gain, length gain, and head circumference gain at
the study end compared with standard HMF (7).
Although the infants in higher protein group had an increased
weight GV and head growth, but this was far less as compared to the
recommended norms. From estimates of fetal growth and observed
growth of premature infants, targets for growth which have been
suggested are weight gain of 18 to 20 g  kg1  day1, length gain of
1.1 to 1.4 cm per week, and head circumference growth from 0.9 to
1.1 cm per week (19,20). Our growth parameters are far below
the recommendations (11.94 g  kg1  day1 weight gain, 0.77 cm/
TABLE 4. Clinical and biochemical parameters
Group Group
Outcomes A (n ¼ 57) B (n ¼ 55) P

Sepsis (n %) 1 (1.8) 2 (3.6) 0.68

BPD (n %) 9 (15.8) 10 (18.1) 0.80
IVH (grade 2) (n %) 3 (5.3) 3 (5.3) 1.00
1.0 Mortality (n %) 1 (1.8) 1 (1.8) 1.00
1
2 NEC (n %) 1 (1.8) 1 (1.8) 1.00
Proportion of neonates discharged

1-censored 
0.8
2-censored NPO days 0.17 (0.55) 0.13 (0.70) 0.77
Time to regain birth weight, daysy 11 (5–13) 10 (6–16) 0.36
0.6
Time to reach full feeds, daysy 9 (5–11) 8 (5–11) 0.37
Hospital stay, daysy 29.5 (22–45) 29.5 (20–43) 0.89

Prealbumin, mg/dL 8.93 (1.9) 8.71 (2.4) 0.60
0.4 
Calcium, mg/dL 9.4 (0.77) 9.3 (0.90) 0.38

Phosphorus, mg/dL 6.3 (1.6) 6.2 (1.2) 0.73
0.2 
Alkaline phosphatase, IU/L 310.6 (98) 322 (109.9) 0.60

BUN, mg/dL 9.5 (1.6) 7.4 (1.5) 0.03

0.0 Creatinine, mg/dL 0.37 (0.14) 0.38 (0.15) 0.76
0.00 20.00 40.00 80.00 100.00

Days of hospital stay BPD ¼ bronchopulmonary dysplasia; BUN ¼ blood urea nitrogen; IVH ¼
intraventricular hemorrhage; NEC ¼ necrotizing enterocolitis; NPO ¼ nil
FIGURE 2. Kaplan-Meier survival plot showing duration of hospital per oral.

stay in the 2 groups. 1 ¼ group A—high enteral protein; 2 ¼ group B— Mean (standard deviation).
y
standard enteral protein. Median (IQR).

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JPGN  Volume 64, Number 5, May 2017 Effect of Differential Enteral Protein in Infants

TABLE 5. Growth parameters at various time points and neurodevelopmental outcomes in >50% expressed breast milk subgroup

Variables Group A (n ¼ 38) Group B (n ¼ 23) Mean difference, 95% CI P

(a) At discharge
Weight growth velocity, g  kg1  day1 14.06 (3.7) 12.10 (3.4) 1.96 (0.05–3.86) 0.04
OFC gain, cm/wk 0.72 (0.18) 0.61 (0.16) 0.10 (0.01–0.19) 0.02
Length gain, cm/wk 1.05 (0.99) 0.78 (0.30) 0.20 (0.15–0.70) 0.20
(b) At 40 wk
Weight growth velocity, g  kg1  day1 11.96 (2.5) 10.49 (1.68) 1.47 (0.25–2.68) 0.02
OFC gain, cm/wk 0.67 (0.16) 0.59 (0.17) 0.079 (0.009–0.168) 0.07
Length gain, cm/wk 0.78 (0.23) 0.69 (0.27) 0.084 (0.046–0.216) 0.20
(c) At 12–18 mo
Variables Group A (n ¼ 29) Group B (n ¼ 18)
Weight, g 10152 (125) 9733 (112) 418 (311–1148) 0.25
OFC, cm 46.46 (1.45) 46.50 (1.05) 0.04 (0.84–0.75) 0.91
Length, cm 75.87 (1.73) 76.07 (1.35) 0.20 (1.17–0.76) 0.67
MoDQ 91.5 (12.77) 87.2 (14.65) 4.23 (3.93–12.40) 0.30
MeDQ 91.2 (13.76) 90.3 (10.33) 0.97 (6.62–8.57) 0.78

CI ¼ confidence interval; MeDQ ¼ Mental Development Quotient; MoDQ ¼ Motor Development Quotient; OFC ¼ occipitofrontal circumference.
Variables expressed as mean (standard deviation).

week length gain, and 0.66 cm/week OFC increment) even in the
high-protein group. The possible reasons for suboptimal growth in
our population could be lower protein content in breast milk than
assumed or difference in genetic factors determining growth of
these infants.
In our study, the complications of prematurity such as sepsis,
NEC, and mortality appear low (Table 4). We have reported these
complications after enrolment, in infants who could be assessed for
study outcomes at 40 weeks PMA. By study design, infants were
randomized and enrolled once they reached a feed volume of 100
mL  kg1  day1. This further excluded sick and extremely preterm
infants who died during the first few days of life before enrolment as
shown in the study flow chart (Fig. 1).
We also evaluated biochemical parameters in the 2 groups at
discharge. Serum BUN levels are known to be one of the surrogate
markers of protein accretion. We observed that BUN levels,
despite being significantly higher in high-protein group, were
below the normal range usually targeted for adjustable fortification
in VLBW infants (13). Olsen et al have reported higher BUN than
found in our study with no safety concerns (18). There are other
safety issues raised with fortification with high protein like
increase in feed intolerance and NEC. We, however, did not
find any significant increase in incidence of feed intolerance or
NEC in the groups.
Although in our study providing higher protein led to short-
term growth advantage, however, it did not translate into better
long-term growth and neurodevelopmental outcomes. The possible
explanation for the lack of benefit on long-term growth and
neurodevelopment outcome could be that the quantum of difference
in higher protein supplementation and its duration was not signifi-
cant enough. Similarly, Lucas et al (21) also found that breast milk
fortifiers can improve short-term growth (when breast milk intakes
are high) but without beneficial effects on long-term development.
In another study by Biasini et al (22), although the extraprotein
group expressed a higher Griffith Development Mental Scales score
at 3 months corrected age, but no significant difference was
observed at 9 months corrected age.
To compare the effect of high protein fortifier as compared to
standard protein fortifier in infants who received >50% EBM, we
performed a subgroup analysis and found that infants in high-
protein group showed marginally higher motor and mental devel-
opment quotient, although it was not statistically significant.
There are certain strengths and limitations of our study. To
the best of our knowledge, this is the first randomized controlled
trial conducted in developing world comparing the effect of differ-
ential protein fortification on short-term and long-term growth and
neurodevelopmental outcomes in VLBW infants. We, however,
could not measure the amount of protein in the breast milk which is
expected to vary (23) and assumed a protein content of 1.5 g/100
mL EBM (9). The mean duration of fortification was short,
approximately 4 weeks and the usage of human milk was also
low. It would be prudent to evaluate long-term growth and neuro-
developmental outcomes in such infants with higher protein supple-
mentation for a longer period of time and higher rate of human
milk usage.
CONCLUSIONS
Mimicking the intrauterine growth in preterm babies still
remains a challenging task for clinicians. The present study empha-
sizes the need for improving protein intake in preterm VLBW
infants. We conclude that fortification with HMF containing higher
protein content in preterm VLBW infants lead to better gain in the
OFC and better weight GV at 40 weeks PMA without any benefit
of improvement in long-term growth or neurodevelopmental out-
comes at 12 to 18 months corrected age.
Acknowledgments: The authors would like to thank Richard J.
Schanler, MD, Cohen Children’s Medical Center of New York and
North Shore Long Island Jewish Health System for reviewing the
initial draft.
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