Multiple Correlations Between Cord Blood Leptin Concentration and Indices of Neonatal Growth

Archives of Medical Research 41 (2010) 26e32

ORIGINAL ARTICLE

Multiple Correlations Between Cord Blood Leptin Concentration and Indices of Neonatal Growth Ali Awsat Mellati,a Seideh Mazloomzadeh,a Afagh Anjomshoaa,a Mohsen Alipour,b Fatemeh Karimi,c Sahar Mazloomi,a and Seyed Ali Naghi Kazemia a

Zanjan Metabolic Disease Research Center, Zanjan University of Medical Sciences, Zanjan, Iran b Physiology Department, Zanjan Medical School, Zanjan, Iran c Pediatric Department, Movsavi Hospital, Zanjan, Iran Received for publication June 12, 2009; accepted October 30, 2009 (ARCMED-D-09-00266).

Background and Aims. The discovery of a role for leptin in controlling fetal and neonatal growth suggests a fetal origin of some adult chronic diseases and has stimulated research into the mechanisms of action of leptin early in life. The aim of this study was to determine umbilical cord blood leptin levels and to evaluate their association with newborn growth indices. Methods. Two hundred healthy newborns (89 males, 110 females, and one of undetermined gender; gestational ages ranging from 34e43 weeks) and their healthy mothers were enrolled in this study conducted at Moovsavi Hospital in Zanjan, Iran. The body size index of each newborn was determined in terms of birth weight, birth length, head circumference, body mass index (BMI) and ponderal index. Umbilical cord blood leptin levels were measured by ELISA. Results. Umbilical cord leptin concentration was found to positively correlate with birth weight (r 5 0.322; p !0.0001), neonatal BMI (r 5 0.247; p !0.0001), ponderal index (r 5 0.206; p 5 0.04), and gestational age (r 5 0.221; p 5 0.002). There was no significant correlation between cord leptin and birth length or umbilical glucose concentration. Umbilical cord leptin concentrations (15.20  12.3 vs. 12.08  11.7; p 5 0.01) were significantly greater in female as compared to male newborns, respectively. Linear regression analysis indicated that umbilical cord leptin levels correlated with birth weight, umbilical triglyceride concentration, neonatal gender, and method of delivery. Conclusions. Our findings confirm the association of leptin concentrations with weight gain in fetal and newborn infants. Ó 2010 IMSS. Published by Elsevier Inc. Key Words: Leptin, Newborns, Growth indices, Gestational age.

Introduction Leptin is a 16 kD polypeptide hormone comprising 167 amino acids and encoded by the obesity (ob) gene (1,2). The primary source of leptin is adipose tissue (white and brown), but it is also a known product of the placenta (3e5) and several fetal tissues (6). It circulates at a concentration that is proportional to fat mass in both humans and rodents (7,8). Therefore, leptin appears to serve as a metabolic signal of adiposity to the Address reprint requests to: Seyed Ali Naghi Kazemi, MD, Associate Professor, Zanjan Medical School, Pediatric Department, Mousavi Hospital, Shahrak Karmandan, Zanjan, Islamic Republic of Iran; Phone: 00982414130000; Fax: 00984249553; E-mail: [email protected]

neuroendocrine and reproductive systems. Leptin plays a role in a multiplicity of events in human physiology but, in particular, regulates body fat mass through a negative feedback loop between adipose tissue and the hypothalamic centers of satiety, causing a decrease in food intake and an increase in both body temperature and energy expenditure (9,10). In pregnant women, placental leptin mRNA concentrations approach those present in adipose tissue. In vitro studies have revealed that |95% of placental leptin is distributed to maternal circulation (11). Cord blood leptin is derived from fetal tissue. It is detectable by gestational week 18 but only increases to significant levels after week 34, which coincides with the development of fetal adipose tissue (12). Whereas

0188-4409/10 $esee front matter. Copyright Ó 2010 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2009.12.001

Cord Blood Leptin in Neonatal Growth

much maternal leptin is produced by the placenta, most cord leptin is produced by fetal adipose tissue (6,12). Maternally circulating leptin does not cross the human placenta in significant amounts; hence, there is no correlation between maternal leptin levels and fetal weight (13,14). In contrast, cord leptin levels exhibit a very strong, positive correlation with the fat mass or ponderal index of neonates (6). The correlation of cord blood leptin concentration with body weight and other anthropometric characteristics of newborn infants remains controversial. Several studies have demonstrated a positive correlation between leptin concentration in cord blood and newborn birth weight (15e18), but some reports have indicated a higher correlation of leptin levels with other anthropometric indices to the exclusion of birth weight (19,20). Furthermore, the suggestion that leptin plays an intrinsic role in fetal growth and development is still a matter of debate because patients with leptin gene mutations have normal birth weights and lengths (21,22). The positive association of leptin levels with birth weight may reflect either a single relationship with adipose tissue mass or a more complex and active role for leptin in fetal growth. Leptin receptors have been discovered in numerous tissues (23), and umbilical cord leptin levels increase sharply with progressing gestational age, particularly from the 34th gestational week to term. This increase in leptin concentration corresponds to the gradual increase of the BMI throughout gestation, which is followed by a rapid decline after birth (24). Together these observations suggest the involvement of leptin in the regulation of fetal adiposity, growth, and development. However, the precise physiological role of leptin in fetal and neonatal development is presently unknown. Neonates who have either high (25,26) or low leptin levels (27) have a higher risk of developing obesity and type 2 diabetes mellitus as compared with children who have normal leptin levels at birth (28). Thus, leptin levels may predispose an individual to the development of chronic degenerative diseases such as obesity, cardiovascular diseases, diabetes mellitus, cancer, and osteoporosis in later life. In this report we describe cord serum leptin concentrations in 200 newborn infants born to healthy mothers and link them to gestational age, gender, term, neonate anthropometry at birth (birth weight, birth length, BMI, ponderal index, head circumference), some maternal characteristics (maternal age, delivery, previous parity), and various biochemical parameters in umbilical cord blood. Also, by stepwise linear regression analysis, we determined the newborn variables that strongly correlate with serum cord leptin level.

Subjects and Methods Subjects This study was carried out on 200 healthy newborns (89 males, 110 females, and one of undetermined gender,

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ranging in gestational age from 34 to 43 weeks) born to healthy mothers at Valieasr University Teaching Hospital from July to August 2008. Approval from the ethical committee of Zanjan Medical School was obtained prior to the start of the study. The purpose of this study was fully explained to every pregnant woman, and written informed consent was obtained before enrollment. Subsequently, maternal data was collected by an investigator (B.S.) using special questionnaire forms; clinical and auxological data were recorded in a standardized form (Table 1). All mothers were healthy and did not require special medical attention after delivery. Mothers with a history of gestational insulin and non-insulin-dependent diabetes mellitus, eclampsia, or preeclampsia were excluded. Infants with dysmorphic features, major congenital malformations, intrauterine infections, organic disorders, and neurological or genetic defects were also excluded. Gestational ages were determined by means of last menstrual period, obstetric ultrasound, or both. Newborns were divided into two groups: ‘‘term,’’ if gestational age was $37 weeks, and preterm if gestational age was !37 weeks. We considered infants to be large for gestational age (LGA) if their birth weight was O4000 g or appropriate for gestational age (AGA) if their birth weight was !4000 and O2500 g or small for gestational age if birth weight was !2500 g, consistent with previous studies (1,17). BMI (kg/m2) and ponderal index were also calculated for each newborn [ponderal index 5 body weight (g)/[body length (cm)3/100]. Blood Sampling Under sterile conditions, 10 cc of blood was drawn from the umbilical vein after double clamping of the umbilical cord at birth by an investigator. Blood samples were centrifuged at 4  C to obtain sera, which were immediately frozen and stored at 20 C for up to 2 months until assayed. Laboratory Methods Serum leptin concentrations were determined by ELISA (Human Leptin ELISA kit, BioVendor, Modrice, Czech Republic) using a Stat Fax 2100 microplate reader (Awareness Technology, Palm City, FL). Intra- and inter-assay coefficients of variation (CVs) were 5.4% and 6.8%, respectively. The minimum detectable concentration of leptin was 0.17 ng/mL. Cord blood glucose was measured colorimetrically on the day of blood collection using glucose oxidase. Total cholesterol (TC) and triglyceride (TG) levels were assayed colorimetrically to a level of detection of 5 mg/dL with enzymatic tests using cholesterol esterase and cholesterol oxidase or glycerol phosphate oxidase, respectively (Pars Azmon Kits, Tehran, Iran). High-density lipoprotein cholesterol (HDL-c) was measured after precipitation of apolipoproteins with phosphotungstic acid. Low-density

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Mellati et al./ Archives of Medical Research 41 (2010) 26e32

Table 1. Clinical and paraclinical characteristics of mothers and newborns at birth, plus leptin concentrations and various biochemical parameters from cord blood Group/Parameter Maternal variables Maternal age, years Delivery Cesarean, n (%) Vaginal, n (%) Previous parity 0, n (%) 1, n (%) O1, n (%) Newborn variables n 5 200 Male/Female, n (%) Birth weight, g Birth length, cm BMI, kg/m2 Ponderal index Head circumference, cm 5-min Apgar scores Gestational age, weeks Full term, n (%) Preterm, n (%) AGA, n (%) SGA, n (%) LGA, n (%) Cord blood biochemical variables Leptin, ng/mL Glucose, mg/dL Triglyceride, mg/dL Cholesterol, mg/dL HDL, mg/dL LDL, mg/dL

Mean  SD

Range

26.2  5.93

14e40

n (%)

42 (21) 158 (79) 106 (53) 55 (27.5) 39 (19.5)

89/110 (44.5/55) 3139  443.74 49.09  4.30 13.4  4.0 2.8  1.4 34.90  4.21 9.0  0.1 38.86  1.45

1700e4300 32.0e56.0 9.4e17.4 1.81e10.57 27.0e54.0 8.0e10.0 34.0e43.0 187 13 192 2 6

13.82  12.16 64.16  22.13 96.59  69.75 73.88  27.25 28.68  11.84 29.25  11.74

(93) (6.5) (95.5) (1) (3)

0.3e75.5 18e147 32e812 36e22.9 11e99 15e100

Values are determined as mean  SD or range according to the variable distribution. n 5 number; M, male; F, female; LGA, large for gestational age; AGA, appropriate for gestational age; SGA, small for gestational age; BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

lipoprotein cholesterol (LDL-c) was calculated from serum TC, TG, and HDL-c using Friedwald’s formula (29). Lipid standards (cat. #75935, c.f.a.s./Boehringer Mannheim, Mannheim, Germany) were used to calibrate the Selectra 2 autoanalyzer (Vital Scientific, Spankeren, Netherlands) on each day experiments were performed. Assay performance was checked for 1/20 test intervals using lipid control sera perineum (normal range) and precipath (pathological range) whenever applicable. Inter- and intra-assay coefficients of variation (CV) for the assay (TC or TG) were 1.2% and 1.8% in the lower limit, 1.0% and 0.6% for the upper limit. Statistical Analysis All data were expressed as mean  SD, median, and range. Spearman’s correlation coefficient was used to determine the relationship between variables. Mann-Whitney U test and Kruskal-Wallis test were used for comparisons between variables. Stepwise linear regression analysis was applied to detect independent variables associated with leptin. A p value !0.05

was considered significant. All analyses were performed using SPSS Statistics v.11.5 for Windows (Chicago, IL).

Results Two hundred healthy newborns were included in this study. Gestational ages ranged from 34 to 43 weeks (mean: 38.86  1.45 weeks). Birth weights ranged from 1700e4300 g with a mean of 3139  443.7 g, mean BMI was 13.4  4.0 kg/m2, and head circumferences ranged from 27.0e54.0 cm (mean: 34.90  4.21 cm). Of the 200 newborns included in the study, 110 (55.5%) were female. Most of the 200 infants were full term (93%) and AGA (95.5%). The mean maternal age was 26.2  5.93 years (range: 14.0e40.0 years), and 21% were delivered by Cesarean section. The mean 5-min Apgar score was 9.0  0.1 in newborns. Table 1 summarizes major characteristics of the study population. Leptin was detectable in the cord blood of all newborns in concentrations ranging from 0.3 to 75.5 ng/mL (mean:

Cord Blood Leptin in Neonatal Growth Table 2. Correlation between leptin concentration in cord blood with maternal age along with newborn and cord blood biochemical variables r Maternal age Newborn variables Birth weight Birth length BMI Ponderal index Head circumference Neonatal gestational age 5-min Apgar score Cord blood biochemical variables Glucose Triglycerides Cholesterol HDL-c LDL-c

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Table 3. Back-transformed mean differences and 95% confidence intervals of cord leptin (ng/mL) by nominal variables

p value

0.130

0.069

0.322 0.103 0.247 0.206 0.215 0.221 0.045

!0.0001 0.152 !0.0001 0.004 0.002 0.002 0.534

0.123 0.155 0.095 0.078 0.003

0.086 0.030 0.186 0.275 0.966

Nominal variables Gender Male Female Term Full term Preterm Delivery Cesarean Normal Previous parity 0 1 O1

Geometric mean

Mean difference (backtransformed)

95% confidence interval

p value

0.91 1.06

1.40

1.09e1.81

0.009

1.00 0.88

0.75

0.44e1.27

0.28

1.10 0.97

0.73

0.53e0.99

0.04

0.83 0.86

0.57e1.21 0.57e1.27

0.33 0.44

0.99 0.99 1.06

r, Spearman correlation; BMI, body mass index.

Discussion 13.82  12.16 ng/mL). With respect to maternal variables (Table 2), there was no correlation between cord blood leptin level and maternal age. Mean cord blood leptin concentration was significantly lower in cases of normal delivery than in Cesarean section (12.03  9.2 vs. 20.62  18.4 ng/mL, respectively; p 5 0.004; Table 3). There were significant correlations between cord blood leptin concentration and most measured newborn indices including birth weight (r 5 0.322; p !0.0001), BMI (r 5 0.247; p !0.0001), ponderal index (r 5 0.206; p 5 0.004), head circumference (r 5 0.215; p 5 0.002), and neonatal gestational age (r 5 0.221; p 5 0.002). (Table 2 and Figures 1 and 2). However, there was no detectable correlation between cord blood leptin levels and birth length. There was no relationship between leptin levels in cord blood and Apgar scores measured 5 min after delivery (Table 2). We found a significant difference in mean leptin concentrations between male and female infants (12.08  11.7 vs. 15.20  12.3 ng/mL, respectively; p 5 0.01), but there was no significant difference in mean cord leptin levels between full-term and pre-term infants (Table 3). Correlations between cord blood leptin concentrations and other biochemical cord blood variables are shown in Table 2. Leptin levels showed a significant positive correlation with TG levels (r 5 0.155; p 5 0.030); however, no other significant biochemical relationships were detected. Significant associations between cord blood leptin levels and both maternal and newborn variables were assessed by stepwise linear regression analysis (Table 4). The results showed that four variables: weight ( p 5 0.0001), TG level ( p !0.0001), gender ( p 5 0.009), and method of delivery ( p 5 0.019) were significantly and independently associated with cord blood leptin concentration.

In the present study we determined that umbilical cord leptin levels were considerably elevated in comparison to those reported for adults (30e32) but lower than reported fetal concentrations (33). Reductions of leptin levels during the first few days of life have been reported by several investigators (34e36). Park et al. (37) suggested that the decrease of leptin levels during the first days of life may reflect the transitory loss of weight during that period. Thus, a sudden postpartum drop in the leptin level might limit neonatal body energy expenditure by conserving nutritional reserves for growth and development (38) while stimulating appetite and the initiation of feeding (24). Our findings indicate that there is no significant association between maternal age and cord blood leptin levels. Although we did not acquire data related to maternal

Figure 1. Correlation between leptin concentrations in cord blood and birth weight in newborns. Color version of this figure available online at www.arcmedres.com.

Mellati et al./ Archives of Medical Research 41 (2010) 26e32

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Figure 2. Correlation between leptin concentrations in cord blood and gestational age in newborns. Color version of this figure available online at www.arcmedres.com.

BMI, we speculate that our results support the view that there is no correlation between maternal BMI and cord leptin level an observation recorded by most previous investigators (6,12). In this study we observed higher leptin levels in newborns delivered by Cesarean section as compared to those delivered normally (Table 3). It has been proposed that leptin is a stress-related hormone. However, the ability of leptin to inhibit responses to stress through enhancement of glucocorticoid receptor expression in the central nervous system and sensitization of the glucocorticoid inhibitory feedback mechanism is controversial (8,39). It remains unclear whether the difference in leptin levels between the two methods of delivery is the result of stress. This question will likely remain unresolved until the physiological relationships between leptin concentration and stress are clarified by additional research. The present study showed that the concentration of umbilical cord serum leptin significantly and positively correlated with birth weight (r 5 0.322, p !0.0001), neonatal BMI (r 5 0.247; p !0.0001), head circumference (r 5 0.215; p 5 0.002), and cord serum TG concentration (r 5 0.155, p !0.030) but was not associated with body length or with certain measured biochemical parameters such as cholesterol or glucose concentration. Table 4. Linear regression analysis for variables associated with leptin levels Variable Intercept Weight Triglyceride Gender Delivery SE, standard error.

Regression coefficient 23.43 0.39 0.38 0.16 0.15

SE

p value

5.99 0.002 0.011 1.47 0.37

!0.0001 !0.0001 0.009 0.019

Our findings support the majority of previous studies (18,40,41) that showed a positive and significant correlation between umbilical cord serum leptin level and birth weight. Therefore, it is tempting to speculate that in the fetus, as in later life (23), leptin is signaling the expression of fat stores to the brain and other target tissues. In addition, total body fat at birth is positively associated with birth weight and reflects intrauterine growth (18). Therefore, intrauterine growth-retarded full-term newborns have significantly lower than appropriate umbilical cord leptin concentrations (8). Furthermore, increased neonatal adiposity is associated with childhood obesity (42,43), which in turn is a risk factor for chronic high blood pressure, hyperlipidemia, and metabolic diseases (44). The mechanisms by which leptin may impact birth weight and later-developing phenomena are not yet fully understood. Numerous studies have demonstrated a positive correlation between umbilical cord leptin levels and BMI (8,15,23, 45). Our data also indicate that this is the case (Table 2). However, it is important to note that BMI (weight to height ratio) is not a marker of fat distribution and cannot distinguish fat from muscle mass (46,47). Consequently, it is only a crude measure of body fat mass. Yajnik et al. (48) found that Indian infants, despite their lower birth weight, have a predisposition to central adiposity. Other comparative studies have shown that Indians have a higher percentage of body fat and lower muscle mass for a given BMI as compared to Caucasians and African-Americans (49,50). A report by Deurenberg et al. (51) indicates that for a given BMI, adults of Caucasian ethnicity have a lower percentage of body fat than adults of Chinese ethnicity. By using regression analysis, Martinez et al. (52) discovered that the relationship between leptin levels and birth weight was not independent of body fat percentage (% BF). Taken together, we propose that correlations between cord blood leptin levels and BMI should be assessed in parallel with measurements of newborn body fat percentage (% BF), as determined by bioelectric impedance analysis. Our present findings, similar to most previous studies (33,43,45), show a positive and significant correlation between cord blood leptin and gestational age. These correlations reflect an increase in adipose tissue during gestational progression from 32 weeks to term. No significant correlation was detected between leptin levels measured in fetal cord blood between the age of 14e32 weeks and gestational growth patterns, thus emphasizing the importance of adipose tissue development in defining leptin levels (44). Inconsistent with most previous studies (45,53e55), we found a significant gender difference in cord blood leptin concentrations, namely, that females had higher leptin concentrations than males (15.20  12.3 vs. 12.08  11.7 mg/dL, respectively; p 5 0.01). However, in contrast to our report, a small number of studies did not find differences in cord leptin levels according to gender (15,34). A review by Roemmich et al. (56) states that there

Cord Blood Leptin in Neonatal Growth

is no gender effect on cord leptin concentrations at birth; however, the studies cited by this review used very small sample sizes. There are several views as to why cord serum leptin concentrations may vary with gender. Some investigators (56,57) suggested that these differences may reflect transient elevations in sex steroid levels because androgens are known to suppress, and estrogens promote, leptin expression in vitro and in vivo. However, Matsuda et al. (15) observed that serum concentrations of estradiol and testosterone did not differ between male and female neonates or did it correlate with leptin concentrations. This suggests that the manifestation of this gender difference depends on genetic factors. Kayemba-Kay’s et al. (55) speculated that body weight at birth does not reflect body fat mass alone, and that higher leptin concentrations in girls correlate with additional subcutaneous fat. Like Petridou et al. (41), we believe that the sexual dimorphism in umbilical cord blood leptin concentrations is attributable to differences in fat mass and lean body mass as well as heavier placental weights in females. Ultimately, further studies are needed to provide a clear explanation for this effect. An advantage of this study is our relatively large sample size, which has allowed statistical documentation of several important associations. A disadvantage of this study is the omission of direct body fat mass (% BF) and placental weight measurements, which may have provided additional valuable information. In conclusion, umbilical cord serum leptin levels correlate positively and significantly with newborn body weight, head circumference, and neonatal anthropometric indices such as BMI and ponderal index. On the other hand, no significant correlation was found between leptin levels and body length. In addition, we observed a gender difference in cord leptin concentrations at birth where higher levels were found in females. In both genders, cord leptin concentrations increased with gestation time. Further research is needed to clarify the mechanistic relationships between leptin levels in cord blood and these neonatal variables.

Acknowledgements This study was financially supported by the Zanjan University of Medical Sciences. The authors express appreciation to the staff of the Zanjan Metabolic Research Center, Associate Prof. Dr. Sharifi, Ebrahimi Zohravi L, Miss Arteashdar, and Mr. Zabihian for their valuable help in conducting this study.

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