Pediatrics and Neonatology (2014) 55, 449e454

Available online at www.sciencedirect.com

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journal homepage: http://www.pediatr-neonatol.com

ORIGINAL ARTICLE

Changes in Preterm Breast Milk Nutrient

Content in the First Month
Ya-Chi Hsu a, Chao-Huei Chen b,c,d,*, Ming-Chih Lin b,c,
Chi-Ren Tsai b, Jiin-Tsae Liang e, Teh-Ming Wang b
a

Department of Pediatrics, Chiayi Branch, Taichung Veterans General Hospital, Chiayi, Taiwan
Division of Neonatology, Department of Pediatrics, Taichung Veterans General Hospital, Taichung,
Taiwan
c
Department of Pediatrics and Institute of Clinical Medicine, National Yang-Ming University, Taipei,
Taiwan
d
School of Medicine, China Medical University, Taichung, Taiwan
e
Pathology and Laboratory Medicine Department, Taichung Veterans General Hospital, Taichung,
Taiwan
b

Received Aug 29, 2013; received in revised form Feb 28, 2014; accepted Mar 24, 2014

Available online 24 May 2014

Key Words
calcium;
human milk;
immune component;
macronutrient;
phosphate;
preterm infant

Background: The primary aim of the study was to investigate the changes in composition
of breast milk from mothers with preterm infants (gestation age < 35 weeks) during the first
4e6 weeks of lactation.
Methods: Breast milk from 17 mothers who had delivered preterm infants was collected longitudinally for 4e6 weeks. Breast milk from 15 mothers of full-term infants was also collected at the
1st week and 4th week. Fat, protein, lactose, energy, minerals (calcium and phosphate), and immune components [secretory immunoglobulin A (IgA), leptin, lysozyme, and lactoferrin] content
were measured weekly in each participant. A mid-infrared human milk analyzer was used to measure the protein, fat, and lactose contents. Calcium and phosphate components were checked via
spectrophotometry. The concentrations of major immune components (secretory IgA, lactoferrin, lysozyme, and leptin) were quantified using enzyme-linked immunosorbent assay kits.
Results: Eighty samples from 17 preterm mothers were collected. The mean gestational age was
29.88
2.39 weeks. There were significant changes in nutrient components during these periods,
with increases in lactose (p < 0.001), lipid (p Z 0.001), calorie (p Z 0.012), and phosphate
(p Z 0.022) concentration and decreases in protein (p < 0.001) and secretory IgA (p < 0.001) concentration. There were no differences in calcium (p Z 0.919), lactoferrin (p Z 0.841), leptin
(p Z 0.092), and lysozyme (p Z 0.561) levels. Furthermore, there were no significant differences
in most components of breast milk between full-term and preterm mothers.
Conclusion: The longitudinal study revealed significant changes in macronutrient contents and
secretory IgA concentration in preterm milk over the 4e6 week period, which is compatible with

* Corresponding author. Division of Neonatology, Department of Pediatrics, Taichung Veterans General Hospital, Number 1650, Taiwan
Boulevard, Section 4, Taichung 40705, Taiwan.
E-mail address: joy1477@gmail.com (C.-H. Chen).
http://dx.doi.org/10.1016/j.pedneo.2014.03.002
1875-9572/Copyright 2014, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved.

450

Y.-C. Hsu et al
the results of previous studies. The quantification of phosphate in preterm breast milk was lower
than the normal range, suggesting that close monitoring of body bone mass may be indicated.
More studies are warranted to evaluate the clinical significance of alterations of major milk components during the postnatal stage.
Copyright 2014, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights
reserved.

1. Introduction
Human milk is the standard for infant feeding and nutrition
according to the statement of the American Academy of Pediatrics.1 It contains various nutrients, enzymes, hormones,
host defense agents, and so on. These components are influenced by many factors, including gestational age,2 lactation
stage,2,3 mothers body mass index (BMI),4 parity number,5
diurnal variations,6 and even the material of containers.7
It is important to provide sufficient nutrients to support
the extrauterine rate of growth and adequate neurodevelopment of preterm infants.8 Recent studies on the effect of early postnatal nutrition reveal that the rate of body
weight gain of preterm infants is affected by the quantity of
calories in the diet; the gains in head circumferences and
body length are influenced by the amount of protein given.9
Other studies have proved that human milk provided to
preterm infants has numerous beneficial effects, such as
improvements in host defense, gastrointestinal function,
and neurodevelopment.8 In clinical practice, most clinicians
suggest fortification of human milk with artificial supplement, such as the human milk fortifier, to fulfill the increased
nutritional needs of preterm infants.10 However, because of
the gastrointestinal immaturity of premature infants, it is
important to consider whether the composition of these
supplementary nutrients, as well as the osmolality, are
appropriate.11 Therefore, it is important to precisely quantify the nutrient components given to preterm infants.
Longitudinal analyses of these nutrient components are
scarce and diverse. Thus, the primary aim of the present
study was to investigate the changes in the composition of
breast milk from mothers with preterm infants whose
gestational age was <35 weeks during the first 4e6 weeks
of lactation. We analyzed the levels of macronutrients,
calcium and phosphate, and the bioactive proteins,
including leptin, secretory IgA, lactoferrin, and lysozyme.

2. Materials and methods

The study was conducted at Taichung Veterans General
Hospital, Taichung, Taiwan, between September 2012 and
January 2013. It was approved by the Institutional Review
Board of Taichung Veterans General Hospital. Informed
consent was obtained from all participants before the
study.

infants, gestational age < 35 weeks and birth

weight < 2000 g. Each participant gave 20 mL breast milk
each time on Days 5e7, Days 12e14, Days 19e21, Days
26e28, Days 33e35, and Days 39e42 after delivery, as long
as she was lactating. Milk was obtained by hand or pump
expression. After each expression, the samples were immediately poured into the container provided and stored in the
refrigerator in order to prevent the difference between
foremilk and hindmilk. Each participant was taught the
collecting techniques by our nurses who were all qualified as
lactation consultants. Thirty fresh human milk samples were
also collected from 15 healthy lactating full-term mothers on
Days 3e7 and Days 28e35 after delivery and served as the
control group. All samples were processed and analyzed
within 24 hours. In order to keep a good relationship with the
volunteer mothers, we kept contacting them by telephone at
the collect period and the first author would visit the mother
after they were discharged.

2.2. Procedures
Each human milk sample was divided into three parts. Calcium
and phosphate components were analyzed by spectrophotometry of 5 mL milk. Another 5 mL milk sample was used to
measure the components of fat, protein, lactose, and energy
by the mid-infrared human milk analyzer, as described in our
previous study.7 Before using the analyzer, the breast milk was
heated to 40 C in a water bath and homogenized in the
container. Each sample was analyzed twice, and the average
value was used for further analysis. A sample of the breast milk
(10 mL) was used to check the concentrations of secretory
immunoglobulin A (IgA), lactoferrin, lysozyme, and leptin
using the same methods described previously.12

2.3. Statistical analysis

The Kruskal-Wallis test was used to test the differences
between groups. Infants gestational age and birth weight
were included in the analysis and changes in nutrient
components were determined by a multiple regression test.
All statistical analyses were performed using SPSS version
13 (SPSS Inc., Chicago, IL, USA). Significant differences
were defined as p < 0.05.

3. Results

2.1. Milk samples

3.1. Demographic characteristics of mothers

A total of 80 fresh human milk samples were collected from

17 healthy lactating mothers who gave birth to preterm

A total of 110 milk specimens were collected in the study,

including 80 samples from preterm mothers and 30 samples

Preterm breast milk nutrient content

Table 1
infants.

451

Demographic characteristics of mothers and

p*

Variable

Prematurity
(n Z 17)

Full-term
(n Z 15)

Maternal age, y
BMI
Race: Taiwan (n)
Parity
Gestational age, wk
Birth weight, g

30.35
5.01
24.24
2.69
17
1.65
0.99
29
2.39
1282
468.47

32.67
5.24
0.313
24.20
2.38
0.941
15
1.87
1.30
0.602
38
0.88
< 0.001
3281
307.14 < 0.001

BMI Z body mass index.

Data are presented as mean
SD.
* Mann-Whitney U test.

from full-term mothers. The demographic characteristics of

the participants are compared in Table 1. There were no
significant differences between these two groups of
mothers in age, BMI, parity, and race. All of the participants
were healthy, without systemic disease and in good nutritional condition. The main reason for preterm delivery was
preeclampsia.

3.2. Changes in concentrations of breast milk

nutrients in preterm infants with different stages
of lactation
There were significant changes in the nutrient components
during the lactation period, with increases in lactose
(p < 0.001), lipid (p Z 0.001), calorie (p Z 0.012), and
phosphate (p Z 0.022) content, and decreases in protein

(p < 0.001), and secretory IgA (p < 0.001) concentrations.

There were no differences in calcium (p Z 0.919), lactoferrin (p Z 0.841), leptin (p Z 0.092), and lysozyme
(p Z 0.561) levels (Figures 1 and 2).

3.3. Changes in concentrations of breast milk

nutrients in full-term infants with different stages
of lactation
There were significant reductions in the concentrations of
protein and secretory IgA from Days 3e7 to Days 28e35
(both p < 0.001). The concentration of lactose significantly
increased (p Z 0.004). The concentrations of fat, calcium,
phosphate, and bioactive proteins did not change significantly (Figures 1 and 2).

3.4. Comparisons between pre- and full-term

breast milk in the same stage of lactation
Two preterm mothers missed giving the milk sample in the
4th week of lactation. Thus, in the part of the study with
comparisons between pre- and full-term breast milk in the
same stage of lactation, there were only 15 mothers in the
preterm group. There were no significant differences in the
composition of breast milk between the two groups (Tables
2e5).

3.5. Factors influencing the concentration of

nutrients in preterm milk
The concentrations of nutrients were significantly influenced by infants gestational age and birth weight. The

Figure 1 Changes in concentrations of breast milk macronutrients and electrolytes in preterm and full-term infants at different
weeks of lactation. The Kruskal-Wallis test was used to examine the relationships between the changes in nutrients and the number
of postpartum weeks. *p < 0.05 for the preterm group. yp < 0.05 for the full-term group.

452

Y.-C. Hsu et al

Figure 2 Changes in concentrations of bioactive proteins in the breast milk of preterm and full-term infants at different weeks of
lactation. The Kruskal-Wallis test was used to examine the relationship between the changes in nutrients and the number of
postpartum weeks. *p < 0.05 for the preterm group. yp < 0.05 for the full-term group.

milk of mothers with a lower birth weight preterm infant

contained more protein. As birth bodyweight or the gestational age increased, the level of milk calcium was higher.
With regard to bioactive proteins, the lysozyme level was
found to be inversely proportional to the birth weight
(Table 6).

4. Discussion
This is the first study to monitor changes in all macronutrients, calcium, phosphate, and bioactive proteins of
preterm milk for at least 4 weeks in an Asian population.
We confirmed that lactose and fat concentration of human
milk increased gradually during the postpartum weeks of
lactation, whereas the protein level of human milk
decreased slowly, in accordance with results of previous
studies.5,6 The lipid content reached a plateau at the 2nd
postpartum week in our study, but the lactose concentration increased weekly, which was not shown in previous
research.13 Mean calories in the milk samples scored highest in the 2nde3rd postpartum week up to 70.0 kcal/dL,
which may be related to the lipid content in the same
stage.

The multivariate analysis revealed an inverse relationship between protein content and birth weight, indicating
that the components of breast milk were likely adjusted
according to the needs of the infants.
In this study, there was no correlation between calcium
levels and number of postpartum weeks, which was
consistent with previous findings,6 but the phosphate level
increased with the number of postpartum weeks. The
phosphate level was lower in our investigation, with a
calcium to phosphate ratio of 4.5e5.5:1. The ratio was
higher than that reported in previous studies.14 However,
none of our cases had fractures or osteopenia. A previous
study showed that a higher whole body bone mass was
associated with human milk intake, despite its low mineral
content. This may be related to non-nutritive factors in
breast milk.15 We postulate that preterm infants fed with
human milk may need long-term follow-up of their bone
mineral content.
Bioactive proteins in breast milk, such as secretory IgA,
lactoferrin, lysozyme, and leptin, serve as immunomodulatory factors and are involved in the regulation of the
neuroendocrine axis.2,16e19 In our study, secretory IgA

Table 2 Differences in the concentrations of breast milk

nutrients between groups in the first week of lactation.

Table 3 Differences in the concentrations of breast milk

nutrients between groups in the fourth week of lactation.

Variable

Variable

Lactose (g/dl)
Protein (g/dl)
Lipid (g/dl)
Calorie (kcal/dl)
Calcium (mg/dl)
Phosphate (mg/dl)

Concentrations of
human milk nutrients

Concentrations of human
milk nutrients

Preterm group Full term group p*

(n Z 15)
(n Z 15)

Preterm group Full term group p*

(n Z 15)
(n Z 15)

M
SD

M
SD

6.365
1.685
2.650
59.50
31.25
4.306

M
SD
0.47
0.46
0.72
6.00
8.33
3.06

Data are presented as mean
SD.

* Kruskal-Wallis test.

6.643
1.567
2.757
60.47
31.54
5.400

0.37
0.32
0.60
4.63
8.82
2.65

0.253
0.261
0.740
0.639
0.901
0.097

Lactose (g/dl)
Protein (g/dl)
Lipid (g/dl)
Calorie (kcal/dl)
Calcium (mg/dl)
Phosphate (mg/dl)

6.793
0.925
3.996
68.61
28.43
5.779

M
SD
0.39
0.27
0.95
8.34
6.70
2.29

Data are presented as mean
SD.

* Kruskal-Wallis test.

6.970
0.917
3.347
63.57
28.76
5.880

0.45
0.12
1.09
8.03
5.88
1.20

0.146
0.452
0.125
0.146
0.787
0.836

216.71 1058.21
300.05 0.576

0.24
0.907
0.58
0.395

M
SD

281.34
75.15

239.09
45.12

0.290

18.38
10.96

14.16
6.96

0.468

672.01
319.06
0.739
0.51

651.72
264.50
0.732
0.49

0.724
0.648

sIgA Z secretory immunoglobulin A.

Data are presented as mean
SD.
* Kruskal-Wallis test.

Phosphate
Calcium
Calorie
Lipid

0.014/0.88
0.122/0.015**

M
SD

0.102/0.35
0.088/0.113

Full term group

(n Z 15)

Protein

p*

Preterm group
(n Z 15)

B/p-value*

Lactoferrin
(mcg/ml)
Lysozyme
(mcg/ml)
sIgA (mcg/ml)
Leptin (ng/ml)

Concentrations of
human milk nutrients

Variable

Variable

Factors that influence the concentrations of the nutrients in preterm milk.

Table 5 Differences in the concentrations of breast milk

immune components between groups in the fourth week of
lactation.

Table 6

levels decreased as the number of postpartum weeks

increased, which was in accordance with the findings of
other studies.2 Due to the increase in the daily amount of
milk, the total IgA intake may remain the same and protect
the suckling infants from microbial infection.20 The other
bioactive proteins remained at relatively constant levels in
preterm human milk.21
In most previous studies, preterm breast milk had
considerable quantitative differences in levels of various
nutrients, especially protein concentration, compared to
corresponding values in term milk.6,22 However, we did not
find significant differences in protein content between
preterm human milk and term milk in the 1st lactation week
and 4th lactation week. The protein level in our preterm
group was lower than that in other studies.23 In a study
conducted in India, no difference in protein level of breast
milk between term or preterm groups was reported.24 The
discrepancy between previous findings and those of the
present study might be due to ethnic differences, maternal
nutritional status, small sample size, or different milk

Lactoferrin

Leptin

sIgA Z secretory immunoglobulin A.

Data are presented as mean
SD.
* Kruskal-Wallis test.

BW Z birth weight; GA Z gestational age; sIgA Z secretory immunoglobulin A.

Data are presented as mean
SD.
* Multiple linear regression.
** p < 0.05.

0.468

38.225/0.68
18.602/0.69

15.71
7.78

0.122/0.191
0.505/0.29

7.88

9.421/0.67
3.129/0.78

0.633

0.319/0.660
0.352/0.34

263.99
52.62

4.661/0.040
3.115/0.008**

M
SD
40.72

3.366/0.195
2.571/0.05

M
SD
Lactoferrin
274.09

(mcg/ml)
Lysozyme
18.03

(mcg/ml)
sIgA (mcg/ml) 1039.46

Leptin (ng/ml)
0.676

p*

0.444/0.140
0.259/0.091

Full term group

(n Z 15)

sIgA

Preterm group
(n Z 15)

GA
BW

Concentrations of
human milk nutrients

Lactose

Variable

coefficients/p-value

Table 4 Differences in the concentrations of breast milk

immune components between groups in the first week of
lactation.

1.891/0.57
3.956/0.023

453

Lysozyme

Preterm breast milk nutrient content

454
sample collection methods. Further studies should be done
to analyze the reasons for differences among studies on
human milk using the current normal standards. In addition,
the levels of bioactive proteins were also similar in preterm
and term human milk, which was compatible with the
findings of previous review studies.25,26 We tried to analyze
if there was any circadian changes of those nutrients. The
distributions of circadian changes between pre- and fullterm groups were not significantly different either in the
1st week or the 4th week. Further study using regression
model analysis with a bigger sample size may be needed.
There were some limitations in our study. We did not
analyze the daily milk volume in this study, which may
potentially influence some of our tested components.6,13
The other limitation was the small sample size. However,
the sample size of this study was estimated to have 80%
power to detect a difference of mean between groups
greater than one standard deviation. Minor differences
between groups might need a larger scale study to detect.
The design of the present study did not provide an ideal
model for evaluating whether the changes of nutrient
contents in the postpartum period were associated with
real baby growth and development in clinical practice.
Further studies are needed to investigate this phenomenon.
In conclusion, significant changes of preterm breast milk
nutrient content, especially macronutrients, were found in
the present study. Fortification of breast milk should be individualized and time-dependent. In our population, a lower
level of phosphate content in breast milk was noted, and thus,
monitoring of an infants body bone mass may be indicated.

Y.-C. Hsu et al

6.

7.

8.

9.
10.
11.

12.

13.

14.

15.

16.

Conflicts of interest
17.

All authors declare no conflicts of interest.

18.

Acknowledgments
We are grateful to Bond Biotech Inc. Taichung, Taiwan for
conducting the enzyme-linked immunosorbent assay and
enzyme immunoassay in this study. We also express our
most sincere appreciation to the volunteer mothers for
their participation and commitment to the research
project.

19.
20.

21.

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