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LIFE-COURSE: Original Article

Adipokines in Human Milk and Risk of Overweight in Early Childhood

A Prospective Cohort Study

Weyermann, Maria; Brenner, Hermann; Rothenbacher, Dietrich

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doi: 10.1097/EDE.0b013e3181567ed4
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As the obesity epidemic is still on the rise, there is an urgent need to understand the underlying mechanistic pathways. There is growing evidence that fetal and neonatal life are critical periods for the development of the metabolic syndrome and obesity, and that nutrition during these periods may be especially important in determining later risk of obesity.1–3 Although multiple studies have suggested that breast-feeding may reduce the risk for obesity,4,5 results have not been consistent.6 Specific constituents of human milk may play a role, but have rarely been considered.

Leptin and adiponectin are members of a growing group of recognized adipose-secreted proteins known as adipokines. Leptin levels in serum directly correlate with body fat mass, and the primary role of leptin is to regulate food intake and energy utilization.7 Adiponectin plays a role in the modulation of glucose and lipid metabolism in insulin-sensitive tissues,8 and has strong antiatherogenic and antiinflammatory effects.9,10

In addition to regulation of whole-body metabolism, leptin and adiponectin are known to be produced within the intrauterine environment.11–16 The high levels of expression of both leptin and adiponectin in the placenta and the fetus suggest that they may play a role in fetal development.17,18

Leptin and adiponectin are also present in maternal milk,19–21 and some studies have described a possible association between leptin in human milk and obesity in infants.19,22 Furthermore, recent studies on the effects of orally administrated leptin on neonatal rats showed that several metabolic adaptations occur in leptin-supplemented animals during lactation.23

To our knowledge, no previous study has assessed the relation between adiponectin in human milk and childhood weight. At present, the role of adiponectin in intrauterine or neonatal growth is unclear. There is a well-established negative association between adiponectin serum levels and obesity in adults. In contrast, studies of the association between cord blood adiponectin and neonatal birthweight or body mass index (BMI) have found inconsistent results.14,24–26 This difference between adults and neonates may indicate age-specific differences in production or regulation of this adipokine. Given that nutrition during neonatal life may be important in determining later risk of obesity, and that adiponectin is considered a key adipokine in metabolic syndrome,8 it may be of special interest to analyze the impact of adiponectin in human milk and risk of overweight in early childhood.

The purpose of this study was to evaluate the role of adiponectin and leptin levels in human breast milk on childhood overweight at the age of 2 years.


Study Design and Study Population

We screened all women who presented to the Department of Gynecology and Obstetrics at the University of Ulm between November 2000 and November 2001 for the delivery of their baby. The primary purpose of the study was to investigate intrafamilial transmission of Helicobacter pylori infection, and details of study methodology have been described elsewhere.27 We excluded women who delivered before 32 gestational weeks, or whose baby was less than 2500 g birthweight or was transferred to inpatient pediatric care immediately after delivery. Overall, 1066 mother-infant pairs were included in this study (67% of all 1593 eligible families who fulfilled the inclusion criteria). During this time, the Department of Gynecology and Obstetrics at the University of Ulm was the only major department of obstetrics in the study area, and served most of childbearing women in the city of Ulm and the nearby communities.

Active follow-up of all children included in the baseline examination was performed at ages 6 weeks, 12 months, and 24 months. Participation was voluntary, and informed consent was obtained from the parents in each case. The study was approved by the Ethics Boards of the Universities of Ulm and Heidelberg and of the Physicians’ Boards of the states of Baden-Wuerttemberg and Bavaria.

Data Collection

All mothers underwent standardized interviews conducted by trained interviewers in the hospital after delivery. Interviews included detailed questions about family demographics, socioeconomic status, housing and living conditions, medical history, and health status during pregnancy including self-reported height and weight at the beginning of pregnancy. A standardized form was used to collect laboratory and anthropometric data during pregnancy from the mother's pregnancy health chart. Mothers were contacted by phone 6 weeks after delivery and were asked whether they were breast-feeding. As shown in Figure 1, 1024 mothers (96%) were successfully recontacted. At this time 786 (77%) were breast-feeding their infants, and of these, breast milk samples were collected from 767 (98%). A trained study nurse visited all women who were breast-feeding and collected 10 mL of human milk, which was immediately cooled and frozen at −80°C within 24 hours. Collection date ranged between 33 and 71 days postpartum (mean = 44 days; median = 43 days; 10th-90th percentile = 39–48 days). In almost all cases, milk samples were collected by the study nurse from both breasts by manual expression before feeding. In rare cases, milk samples were collected manually or by breast pump by the mothers themselves.

Flow diagram showing participation and collection of milk sampling among breast-feeding mothers.

At the follow-up examinations at age 12 and 24 months, the parents again filled out a detailed questionnaire including infant feeding practices. Also, the child's pediatrician provided a detailed medical history for the first and second year of life, including the children's height and weight as measured in regular health screening examinations. (In Germany, health examinations are offered free of charge around the first and second birthday.) BMI was calculated as weight (kg)/height2 (m2). Overweight was defined as BMI above the 90th age- and sex-specific percentile of the German reference population, and severe overweight was defined as BMI above the 97th age- and sex-specific percentile.28

The present analysis is restricted to ever-breast-fed children with available milk samples collected 6 weeks postpartum and with known BMI at the age of 2 years, leading to a final sample size of 674 mother-infant pairs (Fig. 1).

Measurement of Adiponectin and Leptin

Adiponectin and leptin concentrations in breast milk were determined by a commercially available ELISA (R&D Systems, Wiesbaden, Germany). Because milk fat can interfere with the measurement of leptin in whole milk,29,30 we used the fat-free phase for determining adiponectin and leptin. Measurements were conducted according to the manufacturer's instructions with slight modification of the protocols: to remove fat, we centrifuged breast milk for 5 minutes at 12,350 g in a Heraeus Biofuge (Kendro Laboratory Products, Langensebold, Germany), and we used the fat-free phase for determining adiponectin and leptin. The intra- and interassay coefficients of variation were both less than 7.0%.

Statistical Analyses

We first carried out descriptive analyses concerning sociodemographic factors as well as maternal and children's BMI and the duration of breast-feeding. Then we described distributions of adiponectin and leptin in human milk according to potential determinants. Because of the skewness of distributions, we also calculated geometric means. To investigate the role of potential covariates, we describe the prevalence of children's overweight at the age of 2 years, according to sociodemographic and lifestyle characteristics of mothers, anthropometric measurements of the neonates [birthweight and ponderal index at birth (kg/m3)] as well as duration of breast-feeding.

To describe the independent association between adiponectin and leptin in human milk (independent variables) and children's overweight at the age of 2 (dependent variable), we estimated odds ratios (ORs) and their 95% confidence intervals (CIs) by unconditional logistic regression. The following potential confounders were considered as covariates: age of mother (years), school education of mother (≤9 years; ≥10 years), nationality of mother (German; Turkish; other), BMI of mother at the beginning of pregnancy (kg/m2), smoking status of mother during follow-up (no; yes), and birthweight (grams). In addition to analyses in the overall sample, specific analyses were carried out according to duration of breast-feeding (<6 months; ≥6 months).

We included 65 children from whom weight and height at the age of 2 years was available by the parents report only. Results were very similar if considering only children from whom height and weight were taken by the pediatricians during the routine health examinations, and therefore results of the overall sample are shown.

All analyses were carried out with the SAS statistical software package (SAS Institute, Cary, NC).


Almost half of the infants were breast-fed for at least 9 months and 45% were exclusively breast-fed for at least 6 months (Table 1). Exclusive breast-feeding was defined as no other nutrition than breast milk and eventually tea. According to the definition of Kromeyer-Hausschild et al,28 56 children (8.3%) were overweight and 19 (2.8%) were obese at age 2 years.

Baseline Characteristics, Breast-Feeding Duration, and Body Mass Index of Children at Age 2 Years (n = 674)

Figure 2 shows the distribution of adiponectin and leptin in milk collected 6 weeks postpartum. Adiponectin concentration ranged from 0.8 to 110 ng/mL (median 10.9 ng/mL), and leptin concentration ranged from 0 to 4119 pg/mL (median 175 pg/mL). Adiponectin and leptin levels in milk were positively associated (Spearman’ ρ: 0.20; P < 0.0001).

Distribution of (A) adiponectin and (B) leptin levels in human milk.

Adiponectin level was higher in milk of mothers who did not smoke 6 weeks after birth compared with smoking mothers, and decreased with the number of cigarettes smoked per day (Table 2). Other potential covariates showed no clear relationship with adiponectin levels in milk.

Breast Milk Adiponectin and Leptin, According to Maternal/Child Characteristics

Leptin levels in human milk were lower in women who gave birth to a boy compared with women who gave birth to a girl (geometric mean 147 pg/mL vs. 178 pg/mL) (Table 2). Also, leptin levels in milk were lower in women with German nationality compared with mothers with Turkish or other nationality, and higher in women with fewer years of education. In addition, leptin levels increased strongly with increasing prepregnancy BMI.

Table 3 shows the associations of overweight in 2-year-olds with known or suspected determinants of childhood overweight. Prevalence of overweight increased with increasing birthweight as well as with increasing ponderal index at birth, and decreased with increasing duration of breast-feeding. Prevalence of overweight was higher in children whose mothers smoked during follow-up compared with children whose mother did not smoke (14% vs. 7%). Other potential covariates showed no clear relationship with overweight.

Prevalence of Overweight Among Children at the Age of 2 Years According to Maternal/Child Characteristics and Breast-Feeding

Table 4 shows the crude and adjusted ORs for overweight in children at the age of 2 years according to quartiles of adiponectin and leptin levels in human milk. Compared with children breast-fed with milk containing the lowest quartile of adiponectin, the risk for overweight was somewhat higher for children in the 3 higher quartiles. Compared with children breast-fed with milk containing the lowest quartile of leptin, the risk for overweight was increased only for children in the upper quartile of distribution, and the increase was modest.

Risk of Overweight of Children at Age 2 Years, According to Human Milk Adiponectin and Leptin Levels

Table 5 shows the risk for overweight in 2-year-olds per unit increase log as transformed milk adiponectin and leptin levels. The adjusted OR for overweight among children ever breast-fed was 1.6 (95% CI = 1.0–2.6) per unit increase of log-transformed adiponectin in human milk (corresponding to a 2.7-fold increase of adiponectin). Risk for overweight with increasing log adiponectin milk levels was elevated among children who were breast-fed for at least 6 months [adjusted OR = 2.1 (95% CI = 1.1–4.2) per unit increase of log adiponectin]. Among ever-breast-fed children, there was no increased risk of overweight with increasing log leptin levels in milk. Considering time of breast-feeding did not change this relationship.

Risk of Overweight of Children at Age of 2 Years per Unit Increase of Log Adiponectin and Log Leptin in Human Milk, by Duration of Any Breast-Feeding and of Exclusive Breast-Feeding

Considering duration of exclusive breast-feeding, the risk for overweight with increasing log adiponectin and log leptin levels was highest among children who were exclusively breast-fed for at least 6 months [1.7 (0.9–3.4) per unit increase of log adiponectin; 1.4 (0.8–2.6) per unit increase of log leptin].

Excluding children whose mothers were of Turkish nationality did not change the results (data not shown). Furthermore, simultaneous consideration of both adipokines in 1 model did not change the pattern meaningfully; the adjusted OR for overweight among children who were breast-fed for at least 6 months was 2.5 (1.1–5.5) per unit increase of log adiponectin and 1.1 (0.7–1.6) per unit increase of log leptin.


In this prospective study of breast-feeding children in Germany, we found among children who were breast-fed for at least 6 months an increased risk for overweight at the age of 2 with increasing breast milk adiponectin levels; this risk persisted after adjustment for covariates. This finding might have implications for interpreting the inconsistent results regarding the association between breast-feeding and overweight in childhood.4–6 We found no clear relationship between risk of overweight and breast milk leptin levels.

Leptin in Human Milk

Experimental studies have suggested the ability of the mammary epithelial cells to transfer leptin from the blood and to synthesize it before its secretion.29 Our results show an enormous variation in milk leptin levels, ranging from 0 to 4119 pg/mL; the mean level of leptin in milk collected 6 weeks postpartum in our study population is comparable to that of former reports of leptin in skim milk among relatively small populations.30–33

Uysal et al22 found no association between breast milk leptin concentrations and BMI of infants (n = 50), and Bielicki et al19 found only a moderate correlation of milk leptin levels with infant weight or BMI. However, the small sample sizes of these studies limited their power.

The strong positive association seen between milk leptin level and prepregnancy BMI is in agreement with earlier studies.22,32,34 Breast-fed infants nursed by overweight mothers may be exposed to higher amounts of leptin than infants nursed by lean mothers, and much higher amounts than those fed with infant formulas, which do not contain leptin.20 High levels of serum leptin in adults cause a suppression of appetite, but as studies in mice and rats suggest, leptin may have a different role in neonates. Exogenous acute administration of leptin to rat neonates altered the expression of neuropeptides known to affect appetite in adults, but did not alter appetite in neonatal rats.35

In contrast to our results, which indicate no clear relationship between milk leptin levels and risk of overweight, Miralles et al36 recently found a negative correlation between milk leptin concentration and infant BMI during the first 2 years of age among a group of 28 nonobese women and their infants from Spain. In our much larger study, there was no correlation between milk leptin concentration and BMI at age 2 (Spearman's ρ: 0.06; P = 0.11).

The effects of leptin in milk on the human neonate are not known. In 1997, Casabiell et al31 demonstrated that leptin in nursing rats can be transferred via milk to the stomach and can be found afterward in the neonatal circulation. Recent studies on the effects of orally administrated leptin on neonatal rats showed that several metabolic adaptations occur in leptin-supplemented animals during lactation: there is lower food intake, lower leptin production in the stomach and the subcutaneous adipose tissue, and lower thermogenic capacity,23 suggesting that oral leptin is absorbed by the immature gastric epithelium of the neonate. However, these metabolic adaptations in leptin-supplemented animals occurred during lactation without affecting body weight, and experiments were stopped at the end of the suckling period. Longer-term effects of high amounts of orally administrated leptin during lactation period on body weight have not yet been investigated.

Adiponectin in Human Milk

Very recently, Martin et al21 demonstrated the presence of adiponectin in human milk among 2 small populations from Cincinnati and Mexico. They reported adiponectin levels in skim milk samples determined by a radioimmunoassay to be between 4.2 and 87.9 ng/mL, comparable to our study. Further studies are needed to investigate the origination of adiponectin in human milk.

In contrast to leptin, adiponectin levels showed no significant associations with maternal prepregnancy BMI or with other sociodemographic characteristic of the mothers, but there was an inverse association with maternal smoking habits 6 weeks postpartum (when milk samples were collected). Pardo et al37 reported a decreased adiponectin level in cord blood among neonates born to mothers who smoked during pregnancy compared with neonates born to nonsmoking mothers. They speculated that this association is possibly linked with smoking-induced pathogenesis, and can potentially contribute to development of metabolic dysfunctions seen in infants born to smoking mothers.


The following limitations should be kept in mind. We did not measure leptin and adiponectin in fore- and hindmilk, but Ucar et al34 reported that leptin levels did not differ in these components. Weaning influences the concentrations of certain constituents of milk as well as total milk production. However, in our study population most of mothers were still breast-feeding their children exclusively 6 weeks postpartum and had not started weaning. Therefore, it is unlikely that weaning influenced the concentrations of leptin and adiponectin. Also, we were not able to take the time of sample collecting into account and therefore we are not able to provide information about possible circadian cycles of both adipokines in human milk.

Furthermore, we here analyzed adiponectin and leptin levels in milk samples collected only at 6 weeks postpartum. We have also assayed adiponectin and leptin levels in 42 randomly selected milk samples collected 6 months postpartum and found they were in comparable ranges (adiponectin: 1.6 to 50.2 ng/mL, leptin 5.8 to 723.0 pg/mL). The agreement was quite high compared with milk samples collected 6 weeks postpartum for (adiponectin, Spearman's ρ = 0.47 [P = 0.002]; for leptin, Spearman's ρ = 0.64 [P < 0.0001]).38

Despite these limitations, our data provide evidence that the possible protective effect of breast-feeding against childhood obesity might depend, at least in part, on low levels of breast milk adiponectin. To the extent that early nutrition is important in determining later risk of obesity, breast-feeding may hold a key position with regard to prevention of obesity-related diseases, especially in countries with high prevalence of breast-feeding. Breast-feeding mothers, however, did not constitute a uniform group. The composition of breast milk showed marked individual variations, which may contribute to the inconsistent success of breast-feeding in reducing the risk of obesity and obesity-related diseases. In conclusion, breast-fed children may benefit most from prolonged breast-feeding with respect to prevention of obesity when milk contains low levels of adiponectin.


We thank Gisela Breitinger (Department of Clinical Epidemiology and Aging Research, The German Cancer Research Center) for the collection of milk samples, and Wolfgang Koenig and Gerlinde Trischler (Department of Internal Medicine II-Cardiology, Medical Clinic Ulm) for the measurement of adiponectin and leptin in breast milk.


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