The participants were divided into four groups based on breastfeeding periods: 1-6 months (n = 43), 6-12 months (n = 47), 12-18 months (n = 50) and 18-24 months (n = 44). There were no significant differences between the groups with respect to demographics or baseline characteristics (Table 1).
Table 1. Characteristics of the Study Population.
|
Breastfeeding
1-6 months
(n = 43)
|
Breastfeeding 6-12 months
(n = 47)
|
Breastfeeding 12-18 months
(n = 50)
|
Breastfeeding 18-24 months
(n = 44)
|
P-value
|
|
Maternal age, y (mean ± SD, median, P25th-75th) 1
|
32.6 ± 3.6,
32, 29-35
|
30.9 ± 4.6,
31, 28-35
|
32.2 ± 4.8,
32, 33-36
|
32.0 ± 4.5,
32, 29-36.7
|
0.28
|
|
Maternal BMI, kg/m2 (mean ± SD, median; P25th-75th) 2
|
23.2 ± 4.5,
22.5, 19.7-24.8
|
22.6 ± 4.8,
21.3, 19-25.1
|
22.3 ± 4.1,
21.6, 20.2-23.8
|
22.3 ± 3.3,
21.7, 19.8-25.1
|
0.78
|
|
Educational level (n, %) 3
|
|
|
|
|
|
|
Primary school
|
9 (20.9)
|
7 (14.9)
|
16 (32)
|
12 (27.3)
|
0.13
|
|
Secondary school/ Certificate
|
0 (0)
|
3 (6.4)
|
4 (8)
|
4 (9.1)
|
|
|
Graduate degree
|
34 (79.1)
|
37 (78.7)
|
30 (60)
|
28 (63.6)
|
|
|
First ANC, months (mean ± SD, median; P25th-75th) 2
|
2.0 ± 1.3,
2, 1-2
|
2.3 ± 1.6,
2, 1-3
|
2.3 ± 1.3,
2, 1-3
|
2.1 ± 1.2,
2, 1-3
|
0.78
|
|
Gestational age, days (mean ± SD, median; P25th-75th) 2
|
270.6 ± 6.4,
272, 266-273
|
271.3 ± 8.1,
272, 266-278
|
271.5 ± 6.4,
273, 266-275
|
272.3 ± 8.3,
272.5, 266-280
|
0.84
|
|
Birth order 3 (n, %)
|
|
|
|
|
|
|
First born
|
27 (62.8)
|
31 (66)
|
33 (66)
|
25 (56.8)
|
0.71
|
|
Second born
|
15 (34.9)
|
15 (31.9)
|
13 (26)
|
16 (36.4)
|
|
|
Third-fifth born
|
1 (2.3)
|
1 (2.1)
|
4 (8)
|
3 (6.8)
|
|
|
Parental status, couple (n, %) 3
|
42 (97.7)
|
47 (100)
|
48 (96.0)
|
44 (100)
|
0.46
|
|
Breastfeeding frequency, times per day (mean ± SD; median, P25th-75th) 2
|
6.4 ± 5.6,
6, 1-10
|
6.0 ± 4.8,
5, 2-9
|
5.7 ± 3.8,
6, 3-8
|
6.1 ± 3.9,
5, 3.2-8
|
0.98
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 One-Way ANOVA test, 2 Kruskal-Wallis test, 3 Fisher’s exact test were used for statistical calculations, and a p-value lower than 0.05 was regarded as significant.
BMI, body mass index; ANC, antenatal care; SD, standard deviation; P, percentiles
All the participants were Thais.
Correlation Between Human Milk Composition and Duration of Lactation
Macronutrients
The fat and energy contents in human milk expressed by mothers who had been lactating from 1-24 months showed a positive correlation with the duration of lactation (r = 0.229, p = 0.002 and r = 0.229, p = 0.002, respectively) (Figure 1b,1c.). There were no significant correlations between protein and carbohydrate concentrations with the length of lactation (r = 0.106, p = 0.15; r = -0.032, p = 0.67, respectively) (Figure 1a,1d).
In the subsequent lactation period (Table 2), the protein concentration in human milk after 18 months postpartum (2.84 ± 0.90 g/dL) increased significantly compared with that observed in human milk collected from 6-12 and 12-18 months postpartum (2.39 ± 0.52 g/dL, p = 0.001 and 2.40 ± 0.75 g/dL, p < 0.001, respectively). The fat and energy contents were significantly higher in human milk collected after 18 months (4.64 ± 1.61 g/dL and 94.64 ± 16.13 kcal/dL, respectively) than those observed in the other groups (1-6 and 12-18 months of lactation, fat concentration 3.67 ± 1.30 g/dL, p = < 0.001 and 3.90 ± 1.32 g/dL, p = 0.03, respectively; energy content 84.86± 12.93 kcal/dL, p = 0.001 and 87.91 ± 13.23 kcal/dL, p = 0.03, respectively).
Table 2. Comparison of Macronutrients, IgA and Total Antioxidant Capacity of Human Milk by Month of Lactation.
Breastfeeding
Duration
|
1-6 months
(n=43) a
|
6-12 months
(n=47) b
|
12-18 months
(n=50) c
|
18-24 months
(n=44) d
|
Mean rank or
Mean diff. (SE)
|
P- value
|
Mean ± SD
|
Median,
P25th-75th
|
Mean ± SD
|
Median,
P25th-75th
|
Mean ± SD
|
Median,
P25th-75th
|
Mean ± SD
|
Median,
P25th-75th
|
Protein (g/dL)
|
2.56 ± 0.62
|
2.53,
2.21-2.92
|
2.39 ± 0.52
|
2.23,
2.05-2.69
|
2.40 ± 0.75
|
2.36,
1.87-2.61
|
2.84 ± 0.90
|
2.66,
2.29-3.13
|
50.49, 40.94 ab
52.22, 42.51 ac
40.23, 47.68 ad
50.09, 47.98 bc
37.34, 55.25 bd
38.87, 57.31 cd
|
<0.001 1,**
0.08 3
0.08 3
0.17 3
0.71 3
0.001 3,**
<0.0013,**
|
Fat (g/dL) 1
|
3.67 ± 1.30
|
3.79,
2.92-4.33
|
3.96 ± 1.36
|
3.53,
3.17-4.83
|
3.90 ± 1.32
|
3.85,
3.17-4.68
|
4.64 ± 1.61
|
4.61,
3.8-5.13
|
41.26, 49.38 ab
42.78, 50.63 ac
34.69, 53.10 ad
49.20, 48.81 bc
40.88, 51.47 bd
41.67, 54.13 cd
|
<0.0011,**
0.14 3
0.16 3
<0.001 3,**
0.94 3
0.06 3
0.03 3,*
|
Energy (kcal/dL) 1
|
84.86 ± 12.93
|
86.09,
77.42-91.55
|
87.77 ± 13.61
|
83.54,
79.94-96.55
|
87.91 ± 13.23
|
86.70,
79.94-95.01
|
94.64 ± 16.13
|
94.28,
86.24-99.48
|
41.26, 49.38 ab
42.77, 50.64 ac
34.67, 53.11 ad
49.20, 48.81 bc
40.88, 51.47 bd
41.67, 54.13 cd
|
<0.0011,**
0.14 3
0.16 3
0.001 3,**
0.94 3
0.06 3
0.03 3,*
|
Carbohydrates (g/dL) 1
|
9.62 ± 1.04
|
9.43,
8.97-10.12
|
9.34 ± 0.59
|
9.27,
8.97-9.71
|
9.31 ± 0.84
|
9.35,
8.95-9.74
|
9.39 ± 0.81
|
9.41,
8.96-9.73
|
48.90, 42.39 ab
49.73, 44.65 ac
46.53, 41.52 ad
48.12, 49.83 bc
44.83, 47.25 bd
47.48, 47.52 cd
|
0.65 1
0.24 3
0.36 3
0.35 3
0.76 3
0.66 3
0.99 3
|
Breastfeeding
Duration
|
1-6 months
(n=43) a
|
6-12 months
(n=47) b
|
12-18 months
(n=50) c
|
18-24 months
(n=44) d
|
Mean rank or
Mean diff. (SE)
|
P- value
|
Mean ± SD
|
Median,
P25th-75th
|
Mean ± SD
|
Median,
P25th-75th
|
Mean ± SD
|
Median,
P25th-75th
|
Mean ± SD
|
Median,
P25th-75th
|
IgA (mg/dL)
|
110.82 ± 14.06
|
111.48,
103-117.63
|
129.59 ± 16.67
|
130.33,
115.03-140.5
|
124.29 ± 10.80
|
125.07,
116.35-131.37
|
127.16 ± 14.59
|
126.06,
115.73-139.57
|
-18.77 (3.18) ab
-13.47 (3.14) ac
-16.34 (3.23) ad
5.31 (3.06) bc
2.43 (3.16) bd
-2.87 (3.12) cd
|
< 0.0012,**
<0.001**
<0.001**
<0.001** 0.08
0.44 bd
0.36 cd
|
TAC
|
1.61 ± 0.94
|
1.62,
1.2-2.04
|
1.61 ± 0.67
|
1.64,
1.89-2.05
|
1.84 ± 0.85
|
1.84,
1.38-2.38
|
1.60 ± 0.97
|
1.27,
0.69-2.09
|
0.03 (0.18) ab
-0.23 (0.18) ac
0.21 (0.18) ad
-0.23 (0.18) bc
0.18 (0.18) bd
0.44 (0.18) cd
|
0.09 2
0.85
0.19
0.25
0.19
0.33
0.01 *
|
|
|
|
|
|
|
|
|
|
|
|
|
1 Kruskal-Wallis test, 2 One-Way ANOVA post hoc, 3 Mann-Whitney U tests were used for statistical calculations.
*p < 0.05, ** p < 0.001
Total Antioxidant Capacity (TAC) unit is mM, Trolox equivalent; SD Standard deviation; P Percentiles; Mean diff. Mean difference; SE Standard error.
Immunoglobulin A (IgA)
The concentration of IgA in human milk showed a positive correlation with lactation duration (r = 0.304, p < 0.001) (Figure 1e). The mean IgA concentration was the lowest from 1-6 months (110.82 + 14.06 g/dL) compared with that observed in the longer duration groups (6-12, 12-18 and 18-24 months of lactation; 129.59 + 16.67, 124.29 + 10.80 and 127.16 + 14.59 g/dL, respectively, p < 0.001).
Total Antioxidant Capacity (TAC)
Similar to the carbohydrate content, there was no significant correlation between the antioxidant capacity of human milk and the length of lactation (r = -0.056, p = 0.45) (Figure 1f).
Factors Affecting Human Milk Composition
Correlations among maternal age, maternal BMI, breastfeeding frequency, and milk composition were tested using Spearman’s Rank correlation coefficient and Pearson’s correlation coefficient (Table 3). Maternal BMI was positively correlated with the fat and energy contents in human milk (r = 0.233, p = 0.001 and r = 0.233, p = 0.001, respectively) and negatively correlated with carbohydrate content (r = -0.193, p = 0.01). Maternal age was positively associated with changes in the carbohydrate concentration (r = 0.148, p = 0.04), while breastfeeding frequency was negative associated with the carbohydrate content of human milk (r = -0.182, p = 0.01) (Table 3). In addition, the multiple regression analysis results indicated that maternal BMI was positively associated with fat and energy contents in human milk (p < 0.001) and negatively associated with carbohydrate content (p < 0.05) (Table 4).
Table 3. Correlation Coefficients of Maternal Age, Maternal BMI and Breastfeeding Frequency with Human Milk Composition.
|
Protein
(g/dL) a
|
Fat
(g/dL) a
|
Energy
(kcal/dL) a
|
Carbohydrate (g/dL) a
|
IgA
(mg/dL) b
|
TAC
(mM) b
|
r
|
P-value
|
r
|
P-value
|
r
|
P-value
|
r
|
P-value
|
r
|
P-value
|
r
|
P-value
|
Maternal age
|
0.02
|
0.78
|
-0.112
|
0.13
|
-0.112
|
0.13
|
0.148
|
0.04*
|
-0.082
|
0.27
|
0.105
|
0.15
|
Maternal BMI
|
0.081
|
0.27
|
0.233
|
0.001**
|
0.233
|
0.001**
|
-0.193
|
0.01*
|
-0.116
|
0.12
|
-0.069
|
0.35
|
Breastfeeding frequency
|
-0.116
|
0.12
|
0.002
|
0.97
|
0.003
|
0.97
|
-0.182
|
0.01*
|
-0.048
|
0.52
|
0.095
|
0.20
|
a Spearman’s rank correlation coefficient was used for statistical calculations, b Pearson’s correlation coefficient was used for statistical calculations, p-value lower than 0.05 was regarded as significant, *p < 0.05, ** p < 0.01
Table 4. Associations between Maternal Age, Maternal BMI, Breastfeeding Frequency and Human Milk Composition Using Multiple Linear Regression.
|
Fat
|
Energy
|
Carbohydrate
|
B
|
SE
|
95 % CI
|
B
|
SE
|
95 % CI
|
B
|
SE
|
95 % CI
|
Maternal age
|
-0.025
|
0.02
|
-0.07, 0.02
|
-0.25
|
0.23
|
-0.71, 0.22
|
0.02
|
0.01
|
-.005, 0.05
|
Maternal BMI
|
0.09
|
0.02
|
0.04, 0.13**
|
0.86
|
0.25
|
0.37, 1.35**
|
-0.03
|
0.01
|
-0.06, -0.005*
|
Breastfeeding frequency
|
0.001
|
0.02
|
-0.04, 0.05
|
0.01
|
0.23
|
-0.45, 0.47
|
-0.01
|
0.01
|
-0.04, 0.01
|
*p < 0.05, ** p < 0.001