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ADC-FNN Online First, published on January 20, 2018 as 10.1136/archdischild-2016-312572
Original article

Preterm human milk macronutrient concentration is

independent of gestational age at birth
Jan Maly,1 Iva Burianova,2,3 Veronika Vitkova,2 Eva Ticha,1 Martina Navratilova,1
Eva Cermakova,4 On behalf of PREMATURE MILK study group

1
Department of Paediatrics,
Division of Neonatology, Faculty
of Medicine, Charles University,
University Hospital Hradec
Kralove, Hradec Kralove, Czech
Republic
2
Department of Neonatology,
Thomayer Hospital, Prague,
Czech Republic
3
Department of Paediatrics,
Second Faculty of Medicine,
Charles University, Prague,
Czech Republic
4 loss of lactation. 24-Hour milk collection was not done.
Faculty of Medicine, Computer
Technology Centre, Charles
University, Hradec Kralove,
Czech Republic
Correspondence to
Dr Jan Maly, Department
of Paediatrics, Division of
Neonatology, Faculty of
Medicine Charles University,
University Hospital in Hradec
Kralove, Hradec Kralove 500 05,
Czech Republic; ​malyj@​lfhk.​
cuni.c​ z
Received 28 December 2016
Revised 13 December 2017
Accepted 19 December 2017
To cite: Maly J, Burianova I,
Vitkova V, et al. Arch Dis Child
Fetal Neonatal Ed Epub
ahead of print: [please
include Day Month Year].
doi:10.1136/
archdischild-2016-312572
Copyright Article author (or their employer) 2018. Produced by BMJ Publishing Group Ltd (& RCPCH) under licence.
Abstract
Objective  To evaluate the amount of macronutrients in
aggregate of human milk samples after preterm delivery
during the first 2 months of lactation.
Methods  Analysis of the donated single milk samples,
gained by complete emptying of the whole breast at the
same daytime between 24+0 and 35+6 gestational age
(GA), was designed as prospective observational cohort
trial. Two milk samples were analysed every postnatal
week up to the discharge from the hospital, week 9 or
Analysis was performed using the MIRIS Human Milk
Analyser (MIRIS AB, Uppsala, Sweden).
Results  A set of 1917 human milk samples donated by
225 mothers after preterm labour was analysed. Group A
(24–30 GA) contains 969 milk samples; group B (31–35
GA) contains 948 milk samples. No difference in milk
composition between the groups was identified. Median
of true protein content decreased from 1.6 g/dL in group
A and 1.5 g/dL in group B in the first week of life, to
1.1 g/dL in both groups at the end of week 3, and then
remained stable up to week 9. Content of carbohydrates
and fat was stable during the whole observation, with
interindividual differences.
Conclusion  Human milk does not differ as a function
of degree of prematurity. Protein content of preterm
human milk is low and decreases during the first 3 weeks
of lactation. Recommended daily protein intake cannot
be achieved with routine fortification in majority of milk
samples.
Introduction
Breast feeding (or human milk feeding) is generally
accepted as the most suitable form of nutrition for
all newborns and infants during the first months
of life.1 2 Its unique combination of essential nutri-
ents and non-nutritive bioactive components leads
to short-term and long-term benefits. Feeding of a
preterm newborn represents a distinct challenge in
the clinical settings especially in neonates with birth
weight below 1500 g, where breast milk feeding
has an even deeper impact on survival and preven-
tion of serious morbidity.3–5 Nutritional deficits and
postnatal growth restriction represent a common
problem in neonatal intensive care units6 despite
decreased prevalence of postnatal growth failure
seen in the last years.7–9 It is generally accepted
that malnutrition leads to short-term health risks in
this critical period of development, and it increases
the possibility of death and late sequelae including
brain functions.6 10–12
Maly J, et al. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F7. doi:10.1136/archdischild-2016-312572
What is already known on this topic?
►► Macronutrients content of breast milk changes
during lactation with significant interindividual
variability.
►► Slow postnatal growth of preterm newborns
may be associated with low protein content in
expressed breast milk.
►► Protein content of preterm human milk may
differ depending on the term of delivery.
What this study adds?
►► Human milk composition is not influenced by
the degree of prematurity.
►► Protein content of preterm human milk
decreases during early lactation.
►► Most preterm breast milk contains less than
1.5 g/dL of protein.
Breast milk is recommended worldwide for
feeding of preterm neonates despite the fact that
breast milk feeding does guarantee neither suffi-
cient nor standard nutrients intake. Several authors
described high interindividual and intraindividual
differences of human milk nutrients content,
particularly regarding protein and fat, and also
decreasing amount of protein content within weeks
and months after the birth.13–18 This fact, coupled
with high nutritional needs and many patholog-
ical conditions that may impair postnatal growth,
predisposes premature infants to important nutri-
tional risks. Despite routine use of breast milk
fortifiers, it may be quite complicated to meet the
recommended macronutrients (especially protein)
content. Moreover, the quality and quantity of
growth in preterm infants fed with fortified human
milk or preterm formulas differ significantly from
normal fetal growth.19–22
Target breast milk fortification brings a new
concept of an ‘individually-tailored’ strategy that has
potential to optimise feeding of preterm newborns,
improve their growth and perhaps positively affect
neurodevelopmental outcomes.23 24 Unfortunately,
because of considerable variability of nutrients in
human milk, a direct, repeated and frequent milk
analysis is required, which may be costly, laborious
and not available at all.25 The literature contains
information regarding the mean content of energy,
lipids and carbohydrates in human milk after
   F1
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Original article
preterm birth. These facts serve as the base for the development
Table 1  Characteristics of the study population
of commercially available breast milk fortifiers, notwithstanding
different results of breast milk composition.18 Similarly, we have 24–30 GA 31–35 GA
n=85 n=140 P value
limited and conflicting data about the changes of macronutri-
ents content during lactation after preterm birth,13 16 17 which Samples (n)  969  948
may be the decisive phenomena explaining why growth of many Age of mother (median 0.93
preterm infants is not ideal.8 9 The first goal of our study was to years, range) 31 (20–43) 31 (21–44)
evaluate the amount of protein, fat and carbohydrates in aggre- Pregnancy (median, range) 2 (1–8) 2 (1–9) 0.75
gate of human milk samples using midinfrared human milk anal- Parity (median, range) 1 (1–4) 1 (1–8) 0.875
yser after preterm birth during the first 2 months of lactation. In vitro fertilisation 6 (7%) 21 (15%) 0.09
The second aim of this study was to describe the changes that Caesarean section 63 (74%) 78 (56%) 0.007
happen with regard to the degree of preterm delivery and dura- Birth weight (g) (median, *
tion of lactation. range) 1105 (385–1870) 1955 (1070–2970)
GA (median, range) 28 (24–30) 33 (31–35) *
The non-parametric Mann-Whitney U test was used for continuous variables, which
Methods are presented as median values (range). Categorical variables were compared using
The study was designed as a prospective observational cohort Fisher’s exact test, which are presented as numbers (%). Statistically significant
trial and was realised by two research groups (Hradec Králové results are marked in bold.
and Prague) from May 2014 to May 2015. All milk samples were *Not performed for obvious reasons.
analysed in these two institutions. Other four participating level GA, gestational age.
III centres were responsible for recruitment of milk samples. All
the participating mothers signed their informed written consent.
Study material represented a set of the donated milk samples samples were analysed using the built-up mode of the device for
after consecutive preterm births between 24+0 and 35+6 gesta- analysis of homogenised human milk. When the samples were
tional age (GA). Age of the mother, parity and route of the analysed immediately after breast milk expression (without
birth were recorded. Breast milk expressed at the hospital was freezing and thawing period), routine homogenisation was not
by manual expression or an electric pump; only hospital-ex- performed and analysis was done by using the unhomogenised
pressed milk samples were included. Milk collection was over- built-up mode of the instrument according to the manufactur-
seen by an experienced nurse/lactation consultant following the er’s recommendation.
study design during the day shift between 07:00 and 16:00. The demographic characteristics were compared using the
Aliquots for analysis (2–3 mL) were taken immediately after non-parametric Mann-Whitney U test for continuous variables,
complete emptying of the breast and mild stirring of the whole and data are presented as median values (range). Categorical
volume of the expressed milk to minimise possible preanalytical variables were compared using Fisher’s exact test as appro-
fault.26 24-Hour milk collection was primarily declined because priate and are presented as numbers (%). Wilcoxon signed-
of possible interference with availability of the mother’s milk rank test and Mann Whitney U test were used for comparison
especially in extremely preterm neonates. The first two milk of macronutrients content between the groups during the first
samples were obtained in the first week after preterm birth, weeks of life, and gained data are presented as median values
precisely between the days 4 and 7, with the rule one day-one (range). Differences were considered statistically significant at
sample only. The other samples were taken during the next P<0.05. All P values were obtained from two-sided tests, and
consecutive weeks up to the ninth postnatal week or discharge all statistical analyses were performed using NCSS V.9 (J Hintze
from the hospital or loss of lactation. So, ideally, we could (2013), NCSS, Kaysville, Utah, USA; www.​ncss.​com) or Statis-
analyse 18 milk samples taken in 18 different days from each tica V.13 (Dell (2015), Dell Statistica (data analysis software
one donor after completing 9 weeks of the study. Samples were system);  ​software.​dell.​com).
divided into two groups according to GA. Group A covered milk
samples after preterm labour from 24+0 to 30+6 GA. Group B Results
covered milk samples after preterm labour from 31+0 to 35+6 During the study period, a total of 1917 human milk samples
GA. donated by 225 mothers after preterm labour were analysed.
All milk samples were labelled by a unique code and imme- Group A (24–30 GA) contains 969 milk samples, while group
diately analysed (Hradec Králové), or frozen (−18°C) and B (31–35 GA) contains 948 milk samples (table 1). The median
transported for analysis (Prague+participating centres) in the of donated samples per mother was 12 in group A (range 1–18)
regular order. Analysis was performed using the MIRIS Human and 6 (range 1–18) in group B. We observed change of colostrum
Milk Analyser (MIRIS AB, Uppsala, Sweden). This device uses to mature milk between the first few days of life (samples 1 and
midinfrared transmission spectroscopy to provide values for 2) with increase of energy, carbohydrates and fat content and
fat, protein, carbohydrates and calculated energy content with decrease of protein amount similarly in both groups (table 2).
previously described good linearity and precision.27 28 The No difference in milk composition between the groups was
ability of the analyser to calculate the ‘true’ protein amount identified during the first 3 weeks of lactation (table 3). Change
(free of non-nutritive nitrogen substances) was taken into in macronutrients content in preterm milk during the study is
consideration, and all results have been presented as the ‘true’ described in detail (tables 2–4 and figures 1–3). Contrary to the
nutritionally usable protein. Prior to the analysis, frozen proteins, content of carbohydrates and fat (table 4, figure 3)
aliquots were slowly thawed in the refrigerator, then warmed after the first week of lactation was stable during the whole
to 37°C in a water bath and homogenised using a sonicator observation, with interindividual differences. Almost half of all
(VCX 130 Chemical Instruments AB, Sollentuna, Sweden) the analysed samples (925–48.3%) had energy content less than
according to the manufacturer’s recommendation. Homogeni- 67 kcal/dL, and the protein content was less than 1.5 g/dL in
sation was performed in all originally frozen samples; then the 1512 samples (78.9%).
F2 Maly J, et al. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F7. doi:10.1136/archdischild-2016-312572
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Original article
1512 samples (78.9%), which used to be the values considered
Table 2  Comparison of macronutrients content and change in the
as standard for preterm milk composition.30
first week of lactation
Guidelines on nutrition of preterm infants recommend breast
Characteristics Day 24–30 GA P value* 31–35 GA P value† milk for enteral nutrition.1 2 However, breast milk feeding does
Energy (kcal/dL) 4–6 69 (45–91) 0.01 67 (42–104) 0.002 not guarantee a sufficient nutrient intake, particularly with
5–7 74 (45–103) 71 (47–112) regard to protein and energy. Previous studies revealed higher
Protein (g/dL) 4–6 1.7 (1–5) 0.002 1.6 (1.1–3.6) <0.0001 content of protein and lower content of lactose in preterm milk
5–7 1.6 (0.8–3.8) 1.5 (0.9–5.3) as compared with the milk of mothers delivering at term.31–33
Carbohydrates 4–6 6.25 (3.5–7.2) 0.0007 6.4 (0.6–7.1) 0.0006 Up to now, only a few studies have investigated macronutrient
(g/dL) 5–7 6.5 (4.2–7.6) 6.6 (0.4–7.2) content of extremely premature milk.16 17 29 33
Fat (g/dL) 4–6 3.6 (0.9–6.1) 0.005 3.4 (1.6–7) 0.0004 Bauer and Gerss16 detected high values of protein content
5–7 4.25 (1.1–7) 4 (1.6–7.4) associated with the degree of prematurity in the first month of
lactation. Protein concentration of 2.7–3.0 g/dL was detected in
Variables were compared using Wilcoxon signed-rank test and presented as median
values and range. Statistically significant results are marked in bold.
the milk of mothers delivering at gestational weeks 23–24, but
*P value, a comparison between sample 1 (earlier) and sample 2 (later) from the only 1.8–2.3 g/dL in advanced gestation (32–33 GA). It should be
newborns delivered at GA 24–30 weeks. mentioned that this study evaluated only 22 mothers in extremely
†P value, a comparison between sample 1 (earlier) and sample 2 (later) from the preterm milk group and the total number of analysed samples is
newborns delivered at GA 31–35 weeks. not known. Besides, it is not obvious whether the results were
GA, gestational age.
presented as total or true protein content. Another study by
Faerk and coworkers29 analysed 476 preterm milk samples from
Discussion 101 mothers delivering before 32 GA on a weekly basis until
The key findings of the presented study are as follows: (1) We 36 GA. The true protein concentration decreased significantly
showed the content of macronutrients and energy of preterm in time to a level equivalent to the term milk, and similar to
breast milk is highly inconsistent, although with no difference our findings the authors did not describe any association of the
in milk composition between the groups of mothers delivering macronutrients content with GA. A Danish study17 has analysed
before and after the 30th gestational week. (2) Protein content the content of macronutrients in preterm milk below 32 GA
was generally higher in the early postnatal period. (3) Protein (736 samples). Similar to our study midinfrared transmission
content declined until the end of third week after birth and then spectroscopy was employed. True protein content varied from
remained stable between 4 and 9 weeks. This observation is well 1.06 to 2.96 g/dL with a mean value of 1.76 g/dL, and decreased
comparable with studies of Faerk et al,29 Zachariassen et al17 significantly until 8 weeks after birth (mean 1.33 g/dL), which
and a meta-analysis by Gidrewicz and Fenton.18 (4) Almost half are also data supporting our results.
of all the analysed samples (925–48.3%) had energy content less Systematic review and meta-analysis by Gidrewicz and
than 67 kcal/dL and protein content did not reach 1.5 g/dL in Fenton18 included 41 published analyses of breast milk (26

Table 3  Macronutrients content and change in the first 3 weeks of lactation between the groups
Characteristics Energy (kcal/dL) Protein (g/dL) Carbohydrates (g/dL) Fat (g/dL)
Week 1, sample 1, GA 24–30 69 (45–91) 1.7 (1.0–5.0) 6.3 (3.5–7.2) 3.6 (0.9–6.1)
Week 1, sample 1, GA 31–35 67 (42–104) 1.6 (1.1–3.6) 6.4 (0.6–7.1) 3.4 (1.6–7.0)
P value* 0.59 0.28 0.14 0.76
Week 1, sample 2, GA 24–30 74 (45–103) 1.6 (0.8–3.8) 6.5 (4.2–7.6) 4.3 (1.1–7.0)
Week 1, sample 2, GA 31–35 71 (47–112) 1.5 (0.9–5.3) 6.6 (0.4–7.0) 4.0 (1.6–7.4)
P value† 0.38 0.35 0.20 0.59
Week 2, sample 3, GA 24–30 75 (50–116) 1.3 (0.7–2.4) 6.6 (4.3–7.3) 4.3 (2.0–9.0)
Week 2, sample 3, GA 31–35 72 (45–105) 1.3 (0.9–4.5) 6.7 (3.6–7.4) 4.1 (1.0–7.0)
P value‡ 0.72 0.51 0.37 0.65
Week 2, sample 4, GA 24–30 74 (55–107) 1.3 (0.6–2.3) 6.7 (5.2–7.5) 4.4 (2.2–7.5)
Week 2, sample 4, GA 31–35 71 (47–107) 1.2 (0.7–3.0) 6.8 (5.2–7.4) 4.1 (1.3–7.9)
P value§ 0.20 0.17 0.48 0.24
Week 3, sample 5, GA 24–30 73 (51–100) 1.2 (0.6–2.2) 6.8 (4.8–7.5) 4.3 (1.9–7.1)
Week 3, sample 5, GA 31–35 68 (47–120) 1.1 (0.7–3.6) 6.8 (2.2–7.5) 3.9 (1.7–9.0)
P value¶ 0.09 0.03 0.84 0.08
Week 3, sample 6, GA 24–30 70 (51–99) 1.1 (0.6–1.8) 6.8 (5.2–7.4) 4.1 (1.8–6.9)
Week 3, sample 6, GA 31–35 67 (46–105) 1.1 (0.6–5.0) 6.9 (2.8–7.5) 3.8 (1.4–6.9)
P value** 0.19 0.44 0.35 0.26
Continuous variables were compared using a non-parametric Mann-Whitney U test with Bonferroni adjustment and are presented as median (range).
*P value, a comparison between sample 1 (earlier sampling, week 1) from the newborns delivered at GA 24–30 weeks and at GA 31–35 weeks.
†P value, a comparison between sample 2 (later sampling, week 1) from the newborns delivered at GA 24–30 weeks and at GA 31–35 weeks.
‡P value, a comparison between sample 3 (earlier sampling, week 2) from the newborns delivered at GA 24–30 weeks and at GA 31–35 weeks.
§P value, a comparison between sample 4 (later sampling, week 2) from the newborns delivered at GA 24–30 weeks and at GA 31–35 weeks.
¶P value, a comparison between sample 5 (earlier sampling, week 3) from the newborns delivered at GA 24–30 weeks and at GA 31–35 weeks.
**P value, a comparison between sample 6 (later sampling, week 3) from the newborns delivered at GA 24–30 weeks and at GA 31–35 weeks.
GA, gestational age.

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Table 4  Macronutrient content regarding the duration of lactation after preterm birth irrespective of gestational age
Energy (kcal/dL) Protein (g/dL) Carbohydrates (g/dL) Fat (g/dL)
Week Sample Median (IQR) Median (IQR) Median (IQR) Median (IQR)
1 1 65 (57–74) 1.7 (1.4–2.1) 6.4 (5.9–6.7) 3.2 (2.5–4.3)
2 70 (62–82) 1.5 (1.3–1.7) 6.5 (6.1–6.8) 3.8 (3–4.9)
2 1 71 (65–82) 1.3 (1.1–1.5) 6.6 (6.35–6.9) 4 (3.4–5.1)
2 71 (64–80) 1.2 (1–1.5) 6.7 (6.5–7) 4 (3.3–4.95)
3 1 70 (64–78) 1.1 (0.9–1.3) 6.8 (6.6–7) 4 (3.4–4.8)
2 68 (62–76) 1.1 (0.9–1.2) 6.9 (6.7–7.1) 3.85 (3.1–4.8)
4 1 68 (63.5–76.5) 1 (0.9–1.2) 6.9 (6.7–7) 3.8 (3.3–4.7)
2 70 (63–78) 1 (0.8–1.2) 6.8 (6.6–7) 4 (3.3–4.9)
5 1 70 (61.5–76.5) 1 (0.8–1.1) 6.9 (6.6–7) 4 (3.1–4.7)
2 68 (61–74) 1 (0.9–1.1) 6.9 (6.6–7) 3.7 (3.1–4.7)
6 1 68 (59–75) 0.9 (0.8–1.2) 6.9 (6.6–7.1) 3.65 (2.8–4.5)
2 66 (62–74) 0.9 (0.8–1.1) 6.8 (6.6–7.1) 3.6 (3.1–4.4)
7 1 66 (61–72) 0.9 (0.8–1.1) 6.9 (6.6–7.1) 3.7 (3.1–4.4)
2 64 (58–71) 0.9 (0.8–1.1) 6.8 (6.5–7) 3.4 (2.9–4.4)
8 1 64 (57.5–73) 0.9 (0.75–1) 6.8 (6.7–7.1) 3.5 (2.85–4.3)
2 65 (61–75) 0.9 (0.7–1) 6.8 (6.7–7.1) 3.6 (3.1–4.6)
9 1 66 (58–71.5) 0.9 (0.75–0.9) 7 (6.8–7.1) 3.5 (2.9–4.2)
2 66.5 (65–73) 0.8 (0.7–1) 7 (6.8–7.1) 3.8 (3.5–4.3)
Data are presented as median values. IQR, interquartile range.

studies of preterm milk and 30 of term milk). This review pointed
out some similarities between preterm and term breast milk as
to energy, fat, oligosaccharides, calcium and phosphorus. They
concluded that protein content in preterm milk decreases within
the first postnatal week similarly to the trend seen in term milk.
Protein content of the preterm breast milk (mean 2.7 g/dL) was
higher than the term milk in the very first days of lactation and
then decreased to 1.0 g/dL. Breast milk macronutrients compo-
sition revealed relatively stable values between 2 and 12 weeks.
Some studies reported carbohydrates to increase during the
first month of life,33–35 and our data also supported this trend.
We recorded change of colostrum (with lower carbohydrates
content) to mature preterm milk during the first week after
delivery. Later carbohydrates content was very stable and inter-
individual differences did not differ from previous studies.17 18
Maas and coworkers33 and also another study36 described higher
concentration of carbohydrates with increased milk production
(24-hour milk volume). Unfortunately we cannot support these
findings in our results because precise 24-hour milk volume
production was not known in the presented study. The main
reason was the fact that mothers were not hospitalised together
with their children at the nurseries all the time (up to 9 weeks),
and second only hospital-expressed milk was included in the
study material (eg, sample given by mother during her visit at

Figure 1  Energy content of breast milk after preterm birth during 9 consecutive postpartum weeks irrespective of gestational age. Data are
presented in percentile curves (3rd, 25th, 50th, 75th and 97th). 50th percentile curve is marked in bold.
F4 Maly J, et al. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F7. doi:10.1136/archdischild-2016-312572
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Figure 2  True protein content of breast milk after preterm birth during 9 consecutive postpartum weeks irrespective of gestational age. Data are
presented in percentile curves (3rd, 25th, 50th, 75th and 97th). 50th percentile curve is marked in bold.

the nursery). Fat content in preterm milk has been reported to
increase during the first and second week after delivery.17 18 29 35
We observed increase in fat concentration during the change of
colostrum to mature milk in the first few days after delivery;
however, mature milk was stable and fat did not differ between
the groups at any point in time. Generally, fat content is the most
variable macronutrient of the human milk and very liable for
losses, for example, during feeding or whatever manipulation.
This is why 24-hour milk collection is the preferred method for
fat content analysis. Unfortunately pooled-milk analysis may
interfere with the mother’s own milk availability for extreme
preterm neonates especially during the very first weeks of life.
Despite using bedside analyser, only minimal amount of breast
milk for analysis and refusal of 24-hour milk collection because
of reasons mentioned above, we present results comparable with
previous studies.17 18 33 37 We can assume that interindividual
difference of the fat amount in milk samples constitutes also the
difference of energy, for energy content was calculated automat-
ically by the analyser and not directly measured.
In general, our data agree with the results of recently published
studies17 18 29 (except the study by Bauer and Gerss16) and support
the theory that there is no significant difference in the milk

Figure 3  Fat content of breast milk after preterm birth during 9 consecutive postpartum weeks irrespective of gestational age. Data are presented
in percentile curves (3rd, 25th, 50th, 75th and 97th). 50th percentile curve is marked in bold.
Maly J, et al. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F7. doi:10.1136/archdischild-2016-312572 F5
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Original article
composition between severe and moderate/mild prematurity.
Energy and especially protein contents are low in the majority of
samples and routine fortification of preterm human milk fails to
meet the recommended nutritional intake.37 This fact, together
with the data obtained from previous studies, enhances the need
for target fortification of preterm milk and perhaps the devel-
opment of new breast milk fortifiers with appropriately higher
content of protein and energy.
The major advantage of our study lies in the sample size and
longitudinal analysis of severely preterm (969 samples) and
moderately/mild preterm (948 samples) breast milk, which
seems useful from a clinical point of view. In all likelihood, the
recorded changes of nutrient content in this vulnerable period
have a detrimental effect on the newborn’s growth, and these
data may perhaps serve as a ground for more appropriate nutri-
tional intervention. The mean values and percentile curves may
provide useful estimates of preterm milk macronutrients content
in cases when concentrations cannot be directly measured.
It should be noted that we determined macronutrient
content from samples obtained from two different scenarios: by
analysing samples of native preterm milk and by analysing frozen
samples collected from participating centres. This strategy was
chosen deliberately to achieve a collection of a large number
of milk samples in both groups. We are aware of the fact that
manipulations of breast milk during expression, storage and
transportation are additional factors that influence the vari-
ability of expressed breast milk composition. In order to solve
this problem, mothers and participating nurses were instructed
to follow guidelines as proposed by the study methods. Analysis
of single milk samples was chosen to minimise interference with
lactation, handling with obtained milk and thus at least partially
decrease the possibility of preanalytical fault. 24-Hour pooled
batched analysis was not performed. This approach may be
criticised as an important weakness of the study, for 24-hour
milk collection yields the most precise results of energy and fat
content. On the other hand, the most important fact, regarding
the enteral feeding of preterm newborn from clinical point of
view, is the true digested nutrients content, which is in reality
probably quite different from the results of pooled batched anal-
ysis or other sophisticated laboratory methods.
Infrared spectroscopy has been proven to be a useful analytical
method for determining the macronutrient content of human
milk.27 28 In this study we employed the MIRIS Human Milk
Analyser, which requires only 2 mL of breast milk for investi-
gation, making it feasible for bedside use. Silvestre et al27 and
Fusch and coworkers28 tested the reliability of the infrared milk
analysers (including MIRIS Human Milk Analyser) using refer-
ence milk analysis, and despite some limitations they stated
that midinfrared measurement of human milk macronutrients
revealed good linearity and precision and reached acceptable
results for clinical use.
Conclusions
There was no difference in the breast milk composition as a
function of degree of premature delivery. Protein content of
preterm human milk decreases during the first 3 weeks of lacta-
tion. Interindividual difference of breast milk macronutrients
makes feeding of premature neonates problematic in terms of
adequate nutrients intake.
Acknowledgements  The authors appreciate the collaboration with PREMATURE
MILK study group. The authors would like to thank Eva Jirouskova, Helena Krizova,
Jiri Bronsky and Marian Kacerovsky for their enthusiastic support, and Jan and Emily
Hammer for language corrections.
F6 Maly J, et al. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F7. doi:10.1136/archdischild-2016-312572
Collaborators  K Borakova (Institute for Care of the Mother and Child, Prague,
Czech Republic), T Brozova (Institute for Care of the Mother and Child, Prague, Czech
Republic), L Kantor (Division of Neonatology, Palacky University, Faculty of Medicine
and University Hospital, Olomouc, Czech Republic), J Kudlackova (Department of
Neonatology, Thomayer Hospital, Prague, Czech Republic), T Matejek (Department
of Paediatrics, Division of Neonatology, Charles University, Faculty of Medicine
and University Hospital in Hradec Kralove, Czech Republic), M Panek (Division of
Neonatology, Krajska zdravotni a.s., Usti nad Labem, Czech Republic), J Pankova
(Division of Neonatology, Krajska zdravotni a.s., Usti nad Labem, Czech Republic),
E Vokurkova (Department of Obstetrics and Gynaecology, Division of Neonatology,
Charles University, First Faculty of Medicine, General University Hospital, Prague,
Czech Republic), I Vranova (Division of Neonatology, Palacky University, Faculty of
Medicine and University Hospital, Olomouc, Czech Republic) and B Zlatohlavkova
(Department of Obstetrics and Gynaecology, Division of Neonatology, Charles
University, First Faculty of Medicine, General University Hospital, Prague, Czech
Republic).
Contributors  JM was primarily responsible for the study design, samples
analysis, data analysis and interpretation, and drafted the final manuscript. IB
was the coauthor of the study design; she was responsible for sample analysis,
data analysis and interpretation, and manuscript preparation. VV, ET and MN
were responsible for collecting the samples and analysis. EČ was responsible for
statistical analysis.
Funding  This work was supported by Nutricia a.s., Czech Republic, and PRVOUK
programme P 37/12.
Competing interests  None declared.
Ethics approval  The study was approved by the Ethics Committee/Institutional
Review Board of Charles University, Faculty of Medicine, Hradec Kralove, Czech
Republic (reference number 2 01 312 S21 P).
Provenance and peer review  Not commissioned; externally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the
article) 2018. All rights reserved. No commercial use is permitted unless otherwise
expressly granted.
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Preterm human milk macronutrient

concentration is independent of gestational
age at birth
Jan Maly, Iva Burianova, Veronika Vitkova, Eva Ticha, Martina
Navratilova and Eva Cermakova

Arch Dis Child Fetal Neonatal Ed published online January 20, 2018

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