Early Human Development 90 (2014) 271–274
Contents lists available at ScienceDirect
Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Leptin, fetal growth and insulin resistance in non-diabetic pregnancies Jennifer M. Walsh, Jacinta Byrne, Rhona M. Mahony, Michael E. Foley, Fionnuala M. McAuliffe ⁎ UCD Obstetrics and Gynaecology, School of Medicine and Medical Science, University College Dublin, National Maternity Hospital, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 28 July 2013 Received in revised form 17 February 2014 Accepted 8 March 2014
a b s t r a c t Background: Interrogation of the association between leptin, insulin resistance and fetal growth may provide a biological link for the fetal programming of later metabolic health. Aims: Our aim was to clarify the relationship between maternal and fetal leptin, insulin resistance and fetal growth. Study design: Maternal leptin, glucose and insulin were measured in early pregnancy and at 28 weeks and the HOMA index calculated. At 34 weeks, ultrasound scan assessed fetal weight and adiposity (abdominal wall width). At delivery birthweight was recorded and cord blood analyzed for fetal c-peptide and leptin. Analysis was performed using a multivariate linear regression model. Subjects: 574 non-diabetic pregnant women. Outcome measures: Fetal growth and maternal and fetal insulin resistance. Results: On multivariate analysis a relationship was identified between maternal and fetal leptin concentrations at each time point and maternal body mass index. Maternal leptin was related to insulin resistance in early pregnancy (β = 0.15, p = 0.02) and at 28 week gestation (β = 0.27, p b 0.001). Fetal insulin resistance correlated with maternal leptin in early pregnancy (β = 0.17, p = 0.004); at 28 weeks (β = 0.12, p = 0.05), and with leptin in cord blood (r = 0.28, p b 0.001). Fetal weight at 34 weeks was related to maternal leptin in early pregnancy (β = 0.16, p = 0.02). Both maternal and fetal leptin correlated with infant size at birth (β = 0.12, p = 0.07 in early pregnancy, β = 0.21, p = 0.004 in cord blood), independent of all other outcome measures. Conclusion: Our findings have confirmed that in a non-diabetic cohort there is a link between maternal and fetal leptin and insulin resistance. We also established a link between maternal leptin in early pregnancy and both fetal and neonatal size. These results add to the growing body of evidence suggesting a role for leptin in the fetal programming of childhood obesity and metabolic dysfunction. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The worldwide prevalence of childhood overweight and obesity increased dramatically in recent decades in both developed and developing countries. If this trend continues, rates are expected to reach 9.1%, or 60 million children worldwide by 2020 [1]. Childhood obesity predisposes to a variety of adverse health complications, linked primarily to metabolic dysfunction, including diabetes, hypertension and premature death [2,3]. There is increasing evidence supporting the developmental origins of health and disease hypothesis, which suggests that the intrauterine environment may program fetuses toward metabolic deregulation and later childhood obesity [4,5]. Infants that are born large for gestational age are more likely to be obese in childhood, adolescence, and early adulthood than other infants, and are at risk of cardiovascular and metabolic complications later in life [6,7]. Maternal
⁎ Corresponding author. Tel.: +353 1 6373216. E-mail address: fi[email protected] (F.M. McAuliffe).
http://dx.doi.org/10.1016/j.earlhumdev.2014.03.007 0378-3782/© 2014 Elsevier Ireland Ltd. All rights reserved.
weight and glucose homeostasis are perhaps the two most important determinants of fetal growth, and certainly are the two most amenable to intervention. Infants of overweight and obese women have increased fat mass in comparison with those of lean or average weight women [8], and there is evidence that fetuses of obese mothers develop insulin resistance in utero [9]. Longitudinal studies have demonstrated higher rates of obesity, impaired glucose tolerance, hypertension, and dyslipidemia in offspring of diabetic women [10]. Since the publication of the HAPO study however [11], it is now apparent that there are continuous associations between maternal glucose levels and a variety of adverse outcomes, including large for gestational age, and that these outcomes are not just confined to diabetic pregnancies. It is, therefore, plausible that fetal programming of metabolic dysfunction and insulin resistance is a continuous relationship not solely occurring in diabetic pregnancies and that a far greater proportion of pregnancies are in fact, at risk. Leptin is an adipokine involved in the regulation of body weight through appetite suppression and the stimulation of energy expenditure [12]. Leptin appears to play a role in a number of processes associated with pathological fetal growth, most notably maternal diabetes
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J.M. Walsh et al. / Early Human Development 90 (2014) 271–274
[13,14]. Most recently, leptin has been proposed as a biomarker of fetal adiposity [15], and, as such, may provide a biological link for the fetal programming of later adult metabolic and cardiovascular health. We hypothesized that even in non-diabetic pregnancies, a relationship between maternal and fetal adiposity, leptin and insulin resistance exists, and that this may allow for identification of otherwise healthy pregnancies that are at risk of fetal programming of metabolic dysfunction. Our objective, therefore, was to clarify the relationship between maternal and fetal leptin, insulin resistance and both maternal and fetal adiposity in a large cohort of healthy, euglycemic women.
Table 1 Baseline maternal demographics.
2. Materials and methods
3. Results
This is a prospective cohort study of 574 mother and infant pairs with institutional ethical approval and written informed maternal consent. Women were excluded if they had any diagnosed underlying medical conditions, if they were less than 18 years of age, if they had previous gestational or pre-existing Type 1 or Type 2 diabetes or if they were unable to give full informed consent. Patients were recruited at first antenatal consultation at 13.8 ± 2.4 weeks. At the initial visit and at 28 weeks all women had measurement of weight, height, and upper arm circumference; fasting serum glucose, insulin and leptin concentrations were measured. At delivery, infant birth weight, infant length and head circumference were recorded and a cord blood sample for fetal glucose, leptin, and C-peptide concentrations taken. Maternal weight was recorded at each antenatal consultation. Gestational weight gain was compared using the Institute of Medicine guidelines for gestational weight gain in pregnancy [16]. Fetal biometry was assessed ultrasonographically at 34 weeks' gestation using a Voluson 730 Expert (GE Medical Systems, Germany). Biparietal diameter, head circumference, abdominal circumference, femur length and anterior abdominal wall width (AAW), a marker of fetal adiposity were recorded. Fetal AAW was measured at the traditional abdominal circumference view, 2–3 cm lateral to the cord insertion, and included fetal skin and subcutaneous tissue [17,18]. Three measurements were obtained and the mean recorded. Multianalyte profiling was performed on the Luminex Magpix system (Luminex Corporation, Austin, USA.). Plasma concentrations of leptin, insulin, and C-peptide were determined by the Human Endocrine Panel. Maternal insulin resistance was calculated using the HOMA index [19]: HOMA score = (fasting insulin μU/mL × fasting glucose mmol/l) / 22.5. Fetal insulin resistance was assessed with cord blood C-peptide estimation. Cord serum C-peptide (secreted in equimolar concentrations with insulin) was used as the index of fetal β-cell function rather than insulin because insulin degradation is increased in the presence of even small amounts of hemolysis, which occurs in 15% of cord samples, and because C-peptide concentration is not altered by hemolysis [20]. Data were assessed for normality using Shapiro Wilk and P-P plot. Positive and negative correlations were assessed using Pearson's correlation coefficient for normally distributed data and Spearman's rho for nonparametric data. Further analysis to adjust for potential confounding was performed using multiple linear regression analysis to produce a multivariate model with leptin at each time point as the dependent continuous variable and co-efficients adjusted for the following outcome measures; maternal BMI, fetal adiposity (AAW), estimated fetal weight at 34 weeks (EFW), birthweight, early pregnancy insulin resistance (HOMA), HOMA at 28 weeks gestation, and cord blood C-peptide. Statistical significance was set at p b 0.05. Statistical analysis was performed using SPSS Windows version 18.0 (SPSS, Chicago, IL).
The baseline maternal demographics are presented in Table 1. The mean maternal age was 31.9 ± 4.2 years and the mean maternal BMI was 26.7 ± 4.9 kg/m2. All women were in their second pregnancies. In total, 37 women had an early pregnancy fasting glucose of greater than or equal to 5.1 mmol/L; these women were excluded from further analysis, leaving 537 in the study group. The mean fasting serum glucose concentration in early pregnancy of the study cohort was 4.4 ± 0.4 mmol/L. A sample size calculation suggested that to detect a correlation coefficient of greater than 0.12 at significance level of 0.05 and a power of 80%, 428 subjects would be required. Maternal leptin at each time-point correlated with fetal leptin (r = 0.42, p b 0.001 in early pregnancy and r = 0.36, p b 0.001 at 28 weeks). The correlations between leptin at each time-point and maternal and fetal size and insulin resistance are presented in Table 2. There was a significant relationship identified between both maternal and fetal leptin concentrations at each time point and maternal body mass index (BMI) (r = 0.52 in early pregnancy, r = 0.42 at 28 weeks, r = 0.16 in cord blood b 0.001 for all). Fetal weight at 34 weeks was related to maternal leptin at each time-point. We also identified a significant relationship between fetal anterior abdominal wall adiposity at 34 weeks gestation and fetal leptin (r = 0.14, p = 0.013) (Table 2). Maternal leptin in early pregnancy and fetal leptin correlated with infant size at birth. Maternal leptin was related to insulin resistance (HOMA) in early pregnancy (r = 0.34, p b 0.001), at 28 weeks gestation (r = 0.39, p b 0.001) and cord C-peptide correlated with maternal leptin in early pregnancy (r = 0.22, p b 0.001) and at 28 weeks (r = 0.12, p = 0.02) and with fetal leptin (r = 0.32, p b 0.001) (Table 2). A multivariate regression model was built to account for potential confounders with leptin at each time point as the dependent continuous variable and adjusted for all other outcome measures, i.e. maternal, fetal and neonatal size, and maternal and fetal insulin resistance. These data are contained in Table 3. On multivariate analysis an independent relationship existed between both maternal and fetal leptin concentrations at each time point and maternal body mass index (BMI) (β = 0.31 in early pregnancy p b 0.001, β = 0.35 at 28 weeks, β = 0.14, p = 0.05 in cord blood). Fetal weight at 34 weeks was related to maternal leptin in early pregnancy (β = 0.16, p = 0.02). Maternal leptin in early pregnancy and fetal leptin correlated with infant size at birth (β = 0.12, p = 0.07 in early pregnancy, β = 0.21, p = 0.004 in cord blood), independent of all other outcome measures. Maternal leptin was related to insulin resistance (HOMA) in early pregnancy (β = 0.15, p = 0.02) and at 28 weeks gestation (β = 0.27, p b 0.001). Fetal insulin resistance as assessed by cord C-peptide correlated with maternal leptin in early pregnancy (β = 0.17, p = 0.004); at 28 weeks (β = 0.12, p = 0.05), and with fetal leptin in cord blood (r = 0.28, p b 0.001) following adjustment (Table 3). Additionally, those with leptin concentrations above the median for the cohort at each time point were significantly more likely to exceed gestational weight gain guidelines as outlined by the Institute of
2.1. Ethics statement Ethical approval was granted by the National Maternity Hospital Ethics Committee (June 2007).
Maternal age (years) Weight (kg) Height (cm) BMI (kg/m2) Arm circumference (cm) Early pregnancy fasting glucose (mmol/l)
Mean
SD
31.9 73.6 166 26.7 29.5 4.4
4.2 14.2 6.4 4.9 3.4 0.4
Baseline maternal characteristics at recruitment.
J.M. Walsh et al. / Early Human Development 90 (2014) 271–274
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Table 2 Correlation of maternal and fetal leptin with maternal and fetal size and insulin resistance.
Maternal BMI (kg/m2) Fetal AAW (cm) EFW (g) Birthweight (g) Early pregnancy HOMA 28 week HOMA Cord blood C peptide
Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value
Early pregnancy leptin
28 week leptin
Cord blood leptin
0.52 b0.001a 0.03 0.5 0.16 b0.001a 0.14 0.001a 0.35 b0.001a 0.26 b0.001a 0.22 b0.001a
0.42 b0.001a 0.07 0.12 0.12 0.008a 0.07 0.1 0.15 0.001a 0.39 b0.001a 0.12 0.02a
0.16 b0.001a 0.14 0.013a 0.11 0.03a 0.28 b0.001a 0.01 0.7 0.07 0.8 0.32 b0.001a
BMI is maternal body mass index at first antenatal consultation. AAW is fetal anterior abdominal wall width; EFW estimated fetal weight and AC abdominal circumference at 34 weeks' gestation. Pearson's correlation coefficient used for parametric and Spearman's rho used for non-parametric data. HOMA = (Fasting insulin μU/mL × fasting glucose mmol/l) / 22.5. a Denotes two-tailed p-value b0.05, considered statistically significant.
Medicine [16] (51% vs. 35%, p b 0.001 in early pregnancy, 54% vs. 34% at 28 weeks, p b 0.001 and 51% vs. 39% at delivery, p = 0.02). 4. Discussion Our findings in a healthy, euglycemic population confirm a relationship between maternal and fetal leptin and insulin resistance. We also established a link between maternal leptin in early pregnancy and both fetal and neonatal size. These results add to the growing body of evidence suggesting a role for leptin in the fetal programming of childhood obesity and metabolic dysfunction. Maternal and fetal leptin concentrations correlated. Maternal leptin from the first trimester of pregnancy was associated with maternal BMI. Mothers with higher leptin concentrations at each time point had significantly higher HOMA indices than those who did not, an effect that was independent of maternal BMI. Similarly, fetal insulin resistance, as assessed by cord C-peptide, correlated with maternal leptin in early pregnancy, at 28 weeks and with fetal leptin in cord blood. With regard to fetal and neonatal size, maternal leptin concentrations in early pregnancy were related to both fetal weight at 34 weeks and to infant birth weight, independent of all other outcome measures, suggesting that any fetal programming of later metabolic dysfunction may begin at an early gestation. Our multiple regression analysis allowed us to determine that the relationships identified between maternal and fetal leptin, insulin resistance and fetal growth were independent of maternal BMI, however not
surprisingly it remained a strong predictor in our model. Leptin is synthesized primarily in white adipose tissue. Its concentrations in the peripheral circulation are directly related to the amount of adipose tissue present. Maternal concentrations of leptin tend to increase in the third trimester of pregnancy, and there is increasing evidence that the placenta, rather than simply maternal adipocytes, is the major source of the increased leptin concentrations during pregnancy [21]. It has been suggested that this increase in maternal leptin may be to enhance the mobilization of maternal fat stores to increase the transplacental transfer of lipids to the fetus during the latter stages of pregnancy [22]. Similar to adult metabolic disorders therefore, it is possible that alterations in maternal and fetal leptin during pregnancy may result in disordered intrauterine growth and programming. It is already established that fetuses of obese mothers develop insulin resistance in utero [9], and that infants of diabetic mothers are more likely to be obese in childhood [23]. Our findings in a non-diabetic population suggest that fetal programming of later metabolic dysfunction represents a continuum, rather than an absolute cut-off, and is not solely confined to diabetic or obese pregnancies. Our findings have significant implications. They have confirmed the detrimental effects of maternal obesity on pregnancy outcome. Those at the higher end of weight distribution in early pregnancy had significantly higher leptin concentrations and higher HOMA indices and ultimately gave birth to infants with higher birth weights. Though leptin concentrations were related to maternal BMI however, our findings relating maternal and fetal insulin resistance and leptin remained significant
Table 3 Multivariate linear regression analysis.
Maternal BMI (kg/m2) Fetal AAW (cm) EFW (g) Birthweight (g) Early pregnancy HOMA 28 week HOMA Cord blood C peptide
Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value
Early pregnancy leptin
28 week leptin
Cord blood leptin
0.309 0.000 0.081 0.2 0.164 0.02 0.121 0.07 0.147 0.02 0.092 0.1 0.173 0.004
0.345 0.000 0.102 0.1 0.102 0.1 0.061 0.3 0.062 0.3 0.269 0.000 0.115 0.05
0.135 0.05 0.075 0.3 0.030 0.7 0.211 0.004 0.04 0.5 0.031 0.6 0.283 0.000
Multivariate regression model with leptin at each time point as the dependent continuous variable and coefficients adjusted for all other outcome measures. BMI is maternal body mass index at first antenatal consultation. AAW is fetal anterior abdominal wall width; EFW estimated fetal weight at 34 weeks' gestation. HOMA = (fasting insulin μU/mL × fasting glucose mmol/l) / 22.5.
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following adjustment for maternal BMI with logistic regression. This suggests that leptin may be considered a biomarker for in-utero insulin resistance, independent of maternal size. Our findings are strengthened by our large cohort, and the fact that these women were healthy and non-diabetic at recruitment. There are conflicting reports in the literature regarding the relationship between maternal and fetal leptin concentrations [24–26]; this large cohort size allows clarification that a significant relationship does indeed exist. A further strength of this study is the longitudinal assessment of leptin and insulin resistance at several time points during pregnancy. This allows us to demonstrate that from as early as the first trimester aberrations in maternal metabolism have potential consequences for fetal growth and insulin resistance. Our findings do have some limitations worthy of consideration. The first is in relation to our marker of fetal adiposity. There are a number of proposed methods to assess fetal fat deposition in utero, and all have limitations. We chose fetal anterior abdominal wall width as we found it to be simple, quick and reproducible marker of abdominal wall fat [17,27]. Undoubtedly assessment of neonatal body composition with a method such as dual-energy X-ray absorptiometry may have provided a more accurate estimation of fetal adiposity, though with a significant resource implication [28]. This may have limited our findings in relation to fetal fat deposition. Additionally we do not, to date, have follow up data on childhood health and obesity rates for this cohort. These findings have confirmed that the intrauterine environment is a vital component of later metabolic risk. Further work is now necessary to ascertain if pregnancy interventions, such as dietary and lifestyle modifications, can alter maternal and fetal leptin concentrations and insulin resistance. It is now clearly apparent that later adult health is, at least in part, mediated by the intrauterine environment. Any attempts to target escalating obesity rates must aim to interrupt any aberrations in fetal insulin and leptin homeostasis, and so future work needs to focus on the critical peri-conceptual and antenatal period. Conflict of interest statement None of the authors has any conflict of interest to declare. Acknowledgments The authors would like to thank the staff at National Maternity Hospital and the mothers who participated in the study. The Health Research Board of Ireland funded this study, with additional financial support from the National Maternity Hospital Medical Fund. None of the funding sources had a role in the study design or manuscript preparation. References [1] de Onis M, Blössner M, Borghi E. Global prevalence and trends of overweight and obesity among preschool children. Am J Clin Nutr 2010;92:1257–64. [2] Franks PW, Hanson RL, Knowler WC, Sievers ML, Bennett PH, Looker HC. Childhood obesity, other cardiovascular risk factors, and premature death. N Engl J Med 2010;362:485–93.
[3] Dietz WH. Health consequences of obesity in youth: childhood predictors of adult disease. Pediatrics 1998;101:518–25. [4] Barker DJP, Osmond C, Simmonds SJ, Wield GA. The relation of small head circumference and thinness at birth to death from cardiovascular disease in adult life. Br Med J 1993;306:422–6. [5] Nolan CJ, Damm P, Prentki M. Type 2 diabetes across generations: from pathophysiology to prevention and management. Lancet 2011;378:169–81 [9]. [6] Boney CM, Verma A, Tucker R, Vohr BR. Metabolic syndrome in childhood: association with birth weight, maternal obesity, and gestational diabetes mellitus. Pediatrics 2005;115:e290–6. [7] Hermann GM, Dallas LM, Haskell SE, Roghair RD. Neonatal macrosomia is an independent risk factor for adult metabolic syndrome. Neonatology 2010;98:238–44. [8] Sewell MF, Huston-Presley L, Super DM, Catalano PM. Increased neonatal fat mass, and not lean body mass is associated with maternal obesity. Am J Obstet Gynecol 2006;195:1100–3. [9] Catalano PM, Presley L, Minium J, Hauguel-de Mouzon S. Fetuses of obese mothers develop insulin resistance in utero. Diabetes Care 2009;32:1076–80. [10] Durnwald C, Landon M. Fetal links to chronic disease: the role of gestational diabetes mellitus. Am J Perinatol 2013 May;30(5):343–6. [11] HAPO Study Cooperative Research Group. Hyperglycemia and adverse pregnancy outcomes. N Engl J Med 2008;358:1991–2002. [12] Stofkova A. Leptin and adiponectin: from energy and metabolic dysbalance to inflammation and autoimmunity. Endocr Regul 2009;43:157–68. [13] Hauguel-de Mouzon S, Lepercq J, Catalano P. The known and unknown of leptin in pregnancy. Am J Obstet Gynecol 2006;194:1537–45. [14] Higgins M, Mc Auliffe F. A review of maternal and fetal growth factors in diabetic pregnancy. Curr Diabetes Rev 2010;6:116–25 [15]. [15] Mantzoros CS, Rifas-Shiman SL, Williams CJ, Fargnoli JL, Kelesidis T, Gillman MW. Cord blood leptin and adiponectin as predictors of adiposity in children at 3 years of age: a prospective cohort study. Pediatrics 2009;123:682–9. [16] Institute of Medicine National Academy of Sciences. Weight gain during pregnancy: re-examining the guidelines; May 28, 2009 [Washington DC]. [17] Higgins MF, Russell NM, Mulcahy CH, Coffey M, Foley ME, McAuliffe FM. Fetal anterior abdominal wall thickness in diabetic pregnancy. Eur J Obstet Gynecol Reprod Biol 2008;140:43–7. [18] Petrikovsky BM, Oleschuk C, Lesser M, Gelertner N, Gross B. Prediction of fetal macrosomia using sonographically measured abdominal subcutaneous tissue thickness. J Clin Ultrasound 1997;25:378–82. [19] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–8. [20] O'Rahilly S, Burnett MA, Smith RF, Darley JH, Turner RC. Haemolysis affects insulin but not C-peptide immunoassay. Diabetologia 1987;30:394–6. [21] Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, et al. Non-adipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 1997;3(9):1029–33. [22] Sattar N, Greer IA, Pirwani I, Gibson J, Wallace AM. Leptin levels in pregnancy: marker for fat accumulation and mobilization? Acta Obstet Gynecol Scand 1998;77(3):278–83. [23] Lindsay RS, Nelson SM, Walker JD, Greene SA, Milne G, Sattar N, et al. Programming of adiposity in offspring of mothers with type 1 diabetes at age 7 years. Diabetes Care 2010;33:1080–5. [24] Laml T, Hartmann BW, Ruecklinger E, Preyer O, Soeregi G, Wagenbichler P. Maternal serum leptin concentrations do not correlate with cord blood leptin concentrations in normal pregnancy. J Soc Gynecol Investig 2001;8:43–7. [25] Oktem O, Dedeoğlu N, Oymak Y, Sezen D, Köksal L, Pekin T, et al. Maternal serum, amniotic fluid and cord leptin levels at term: their correlations with fetal weight. J Perinat Med 2004;32:266–71. [26] Manderson JG, Patterson CC, Hadden DR, Traub AI, Leslie H, McCance DR. Leptin concentrations in maternal serum and cord blood in diabetic and nondiabetic pregnancy. Am J Obstet Gynecol 2003;188:1326–32. [27] Walsh JM, Mahony R, Byrne J, Foley M, McAuliffe FM. The association of maternal and fetal glucose homeostasis with fetal adiposity and birthweight. Eur J Obstet Gynecol Reprod Biol 2011;159:338–41. [28] Catalano PM, Thomas A, Huston-Presley L, Amini SB. Increased fetal adiposity: a very sensitive marker of abnormal in utero development. Am J Obstet Gynecol 2003;189:1698–704.
Contents lists available at ScienceDirect
Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Leptin, fetal growth and insulin resistance in non-diabetic pregnancies Jennifer M. Walsh, Jacinta Byrne, Rhona M. Mahony, Michael E. Foley, Fionnuala M. McAuliffe ⁎ UCD Obstetrics and Gynaecology, School of Medicine and Medical Science, University College Dublin, National Maternity Hospital, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 28 July 2013 Received in revised form 17 February 2014 Accepted 8 March 2014
a b s t r a c t Background: Interrogation of the association between leptin, insulin resistance and fetal growth may provide a biological link for the fetal programming of later metabolic health. Aims: Our aim was to clarify the relationship between maternal and fetal leptin, insulin resistance and fetal growth. Study design: Maternal leptin, glucose and insulin were measured in early pregnancy and at 28 weeks and the HOMA index calculated. At 34 weeks, ultrasound scan assessed fetal weight and adiposity (abdominal wall width). At delivery birthweight was recorded and cord blood analyzed for fetal c-peptide and leptin. Analysis was performed using a multivariate linear regression model. Subjects: 574 non-diabetic pregnant women. Outcome measures: Fetal growth and maternal and fetal insulin resistance. Results: On multivariate analysis a relationship was identified between maternal and fetal leptin concentrations at each time point and maternal body mass index. Maternal leptin was related to insulin resistance in early pregnancy (β = 0.15, p = 0.02) and at 28 week gestation (β = 0.27, p b 0.001). Fetal insulin resistance correlated with maternal leptin in early pregnancy (β = 0.17, p = 0.004); at 28 weeks (β = 0.12, p = 0.05), and with leptin in cord blood (r = 0.28, p b 0.001). Fetal weight at 34 weeks was related to maternal leptin in early pregnancy (β = 0.16, p = 0.02). Both maternal and fetal leptin correlated with infant size at birth (β = 0.12, p = 0.07 in early pregnancy, β = 0.21, p = 0.004 in cord blood), independent of all other outcome measures. Conclusion: Our findings have confirmed that in a non-diabetic cohort there is a link between maternal and fetal leptin and insulin resistance. We also established a link between maternal leptin in early pregnancy and both fetal and neonatal size. These results add to the growing body of evidence suggesting a role for leptin in the fetal programming of childhood obesity and metabolic dysfunction. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The worldwide prevalence of childhood overweight and obesity increased dramatically in recent decades in both developed and developing countries. If this trend continues, rates are expected to reach 9.1%, or 60 million children worldwide by 2020 [1]. Childhood obesity predisposes to a variety of adverse health complications, linked primarily to metabolic dysfunction, including diabetes, hypertension and premature death [2,3]. There is increasing evidence supporting the developmental origins of health and disease hypothesis, which suggests that the intrauterine environment may program fetuses toward metabolic deregulation and later childhood obesity [4,5]. Infants that are born large for gestational age are more likely to be obese in childhood, adolescence, and early adulthood than other infants, and are at risk of cardiovascular and metabolic complications later in life [6,7]. Maternal
⁎ Corresponding author. Tel.: +353 1 6373216. E-mail address: fi[email protected] (F.M. McAuliffe).
http://dx.doi.org/10.1016/j.earlhumdev.2014.03.007 0378-3782/© 2014 Elsevier Ireland Ltd. All rights reserved.
weight and glucose homeostasis are perhaps the two most important determinants of fetal growth, and certainly are the two most amenable to intervention. Infants of overweight and obese women have increased fat mass in comparison with those of lean or average weight women [8], and there is evidence that fetuses of obese mothers develop insulin resistance in utero [9]. Longitudinal studies have demonstrated higher rates of obesity, impaired glucose tolerance, hypertension, and dyslipidemia in offspring of diabetic women [10]. Since the publication of the HAPO study however [11], it is now apparent that there are continuous associations between maternal glucose levels and a variety of adverse outcomes, including large for gestational age, and that these outcomes are not just confined to diabetic pregnancies. It is, therefore, plausible that fetal programming of metabolic dysfunction and insulin resistance is a continuous relationship not solely occurring in diabetic pregnancies and that a far greater proportion of pregnancies are in fact, at risk. Leptin is an adipokine involved in the regulation of body weight through appetite suppression and the stimulation of energy expenditure [12]. Leptin appears to play a role in a number of processes associated with pathological fetal growth, most notably maternal diabetes
272
J.M. Walsh et al. / Early Human Development 90 (2014) 271–274
[13,14]. Most recently, leptin has been proposed as a biomarker of fetal adiposity [15], and, as such, may provide a biological link for the fetal programming of later adult metabolic and cardiovascular health. We hypothesized that even in non-diabetic pregnancies, a relationship between maternal and fetal adiposity, leptin and insulin resistance exists, and that this may allow for identification of otherwise healthy pregnancies that are at risk of fetal programming of metabolic dysfunction. Our objective, therefore, was to clarify the relationship between maternal and fetal leptin, insulin resistance and both maternal and fetal adiposity in a large cohort of healthy, euglycemic women.
Table 1 Baseline maternal demographics.
2. Materials and methods
3. Results
This is a prospective cohort study of 574 mother and infant pairs with institutional ethical approval and written informed maternal consent. Women were excluded if they had any diagnosed underlying medical conditions, if they were less than 18 years of age, if they had previous gestational or pre-existing Type 1 or Type 2 diabetes or if they were unable to give full informed consent. Patients were recruited at first antenatal consultation at 13.8 ± 2.4 weeks. At the initial visit and at 28 weeks all women had measurement of weight, height, and upper arm circumference; fasting serum glucose, insulin and leptin concentrations were measured. At delivery, infant birth weight, infant length and head circumference were recorded and a cord blood sample for fetal glucose, leptin, and C-peptide concentrations taken. Maternal weight was recorded at each antenatal consultation. Gestational weight gain was compared using the Institute of Medicine guidelines for gestational weight gain in pregnancy [16]. Fetal biometry was assessed ultrasonographically at 34 weeks' gestation using a Voluson 730 Expert (GE Medical Systems, Germany). Biparietal diameter, head circumference, abdominal circumference, femur length and anterior abdominal wall width (AAW), a marker of fetal adiposity were recorded. Fetal AAW was measured at the traditional abdominal circumference view, 2–3 cm lateral to the cord insertion, and included fetal skin and subcutaneous tissue [17,18]. Three measurements were obtained and the mean recorded. Multianalyte profiling was performed on the Luminex Magpix system (Luminex Corporation, Austin, USA.). Plasma concentrations of leptin, insulin, and C-peptide were determined by the Human Endocrine Panel. Maternal insulin resistance was calculated using the HOMA index [19]: HOMA score = (fasting insulin μU/mL × fasting glucose mmol/l) / 22.5. Fetal insulin resistance was assessed with cord blood C-peptide estimation. Cord serum C-peptide (secreted in equimolar concentrations with insulin) was used as the index of fetal β-cell function rather than insulin because insulin degradation is increased in the presence of even small amounts of hemolysis, which occurs in 15% of cord samples, and because C-peptide concentration is not altered by hemolysis [20]. Data were assessed for normality using Shapiro Wilk and P-P plot. Positive and negative correlations were assessed using Pearson's correlation coefficient for normally distributed data and Spearman's rho for nonparametric data. Further analysis to adjust for potential confounding was performed using multiple linear regression analysis to produce a multivariate model with leptin at each time point as the dependent continuous variable and co-efficients adjusted for the following outcome measures; maternal BMI, fetal adiposity (AAW), estimated fetal weight at 34 weeks (EFW), birthweight, early pregnancy insulin resistance (HOMA), HOMA at 28 weeks gestation, and cord blood C-peptide. Statistical significance was set at p b 0.05. Statistical analysis was performed using SPSS Windows version 18.0 (SPSS, Chicago, IL).
The baseline maternal demographics are presented in Table 1. The mean maternal age was 31.9 ± 4.2 years and the mean maternal BMI was 26.7 ± 4.9 kg/m2. All women were in their second pregnancies. In total, 37 women had an early pregnancy fasting glucose of greater than or equal to 5.1 mmol/L; these women were excluded from further analysis, leaving 537 in the study group. The mean fasting serum glucose concentration in early pregnancy of the study cohort was 4.4 ± 0.4 mmol/L. A sample size calculation suggested that to detect a correlation coefficient of greater than 0.12 at significance level of 0.05 and a power of 80%, 428 subjects would be required. Maternal leptin at each time-point correlated with fetal leptin (r = 0.42, p b 0.001 in early pregnancy and r = 0.36, p b 0.001 at 28 weeks). The correlations between leptin at each time-point and maternal and fetal size and insulin resistance are presented in Table 2. There was a significant relationship identified between both maternal and fetal leptin concentrations at each time point and maternal body mass index (BMI) (r = 0.52 in early pregnancy, r = 0.42 at 28 weeks, r = 0.16 in cord blood b 0.001 for all). Fetal weight at 34 weeks was related to maternal leptin at each time-point. We also identified a significant relationship between fetal anterior abdominal wall adiposity at 34 weeks gestation and fetal leptin (r = 0.14, p = 0.013) (Table 2). Maternal leptin in early pregnancy and fetal leptin correlated with infant size at birth. Maternal leptin was related to insulin resistance (HOMA) in early pregnancy (r = 0.34, p b 0.001), at 28 weeks gestation (r = 0.39, p b 0.001) and cord C-peptide correlated with maternal leptin in early pregnancy (r = 0.22, p b 0.001) and at 28 weeks (r = 0.12, p = 0.02) and with fetal leptin (r = 0.32, p b 0.001) (Table 2). A multivariate regression model was built to account for potential confounders with leptin at each time point as the dependent continuous variable and adjusted for all other outcome measures, i.e. maternal, fetal and neonatal size, and maternal and fetal insulin resistance. These data are contained in Table 3. On multivariate analysis an independent relationship existed between both maternal and fetal leptin concentrations at each time point and maternal body mass index (BMI) (β = 0.31 in early pregnancy p b 0.001, β = 0.35 at 28 weeks, β = 0.14, p = 0.05 in cord blood). Fetal weight at 34 weeks was related to maternal leptin in early pregnancy (β = 0.16, p = 0.02). Maternal leptin in early pregnancy and fetal leptin correlated with infant size at birth (β = 0.12, p = 0.07 in early pregnancy, β = 0.21, p = 0.004 in cord blood), independent of all other outcome measures. Maternal leptin was related to insulin resistance (HOMA) in early pregnancy (β = 0.15, p = 0.02) and at 28 weeks gestation (β = 0.27, p b 0.001). Fetal insulin resistance as assessed by cord C-peptide correlated with maternal leptin in early pregnancy (β = 0.17, p = 0.004); at 28 weeks (β = 0.12, p = 0.05), and with fetal leptin in cord blood (r = 0.28, p b 0.001) following adjustment (Table 3). Additionally, those with leptin concentrations above the median for the cohort at each time point were significantly more likely to exceed gestational weight gain guidelines as outlined by the Institute of
2.1. Ethics statement Ethical approval was granted by the National Maternity Hospital Ethics Committee (June 2007).
Maternal age (years) Weight (kg) Height (cm) BMI (kg/m2) Arm circumference (cm) Early pregnancy fasting glucose (mmol/l)
Mean
SD
31.9 73.6 166 26.7 29.5 4.4
4.2 14.2 6.4 4.9 3.4 0.4
Baseline maternal characteristics at recruitment.
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Table 2 Correlation of maternal and fetal leptin with maternal and fetal size and insulin resistance.
Maternal BMI (kg/m2) Fetal AAW (cm) EFW (g) Birthweight (g) Early pregnancy HOMA 28 week HOMA Cord blood C peptide
Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value Correlation coefficient p-Value
Early pregnancy leptin
28 week leptin
Cord blood leptin
0.52 b0.001a 0.03 0.5 0.16 b0.001a 0.14 0.001a 0.35 b0.001a 0.26 b0.001a 0.22 b0.001a
0.42 b0.001a 0.07 0.12 0.12 0.008a 0.07 0.1 0.15 0.001a 0.39 b0.001a 0.12 0.02a
0.16 b0.001a 0.14 0.013a 0.11 0.03a 0.28 b0.001a 0.01 0.7 0.07 0.8 0.32 b0.001a
BMI is maternal body mass index at first antenatal consultation. AAW is fetal anterior abdominal wall width; EFW estimated fetal weight and AC abdominal circumference at 34 weeks' gestation. Pearson's correlation coefficient used for parametric and Spearman's rho used for non-parametric data. HOMA = (Fasting insulin μU/mL × fasting glucose mmol/l) / 22.5. a Denotes two-tailed p-value b0.05, considered statistically significant.
Medicine [16] (51% vs. 35%, p b 0.001 in early pregnancy, 54% vs. 34% at 28 weeks, p b 0.001 and 51% vs. 39% at delivery, p = 0.02). 4. Discussion Our findings in a healthy, euglycemic population confirm a relationship between maternal and fetal leptin and insulin resistance. We also established a link between maternal leptin in early pregnancy and both fetal and neonatal size. These results add to the growing body of evidence suggesting a role for leptin in the fetal programming of childhood obesity and metabolic dysfunction. Maternal and fetal leptin concentrations correlated. Maternal leptin from the first trimester of pregnancy was associated with maternal BMI. Mothers with higher leptin concentrations at each time point had significantly higher HOMA indices than those who did not, an effect that was independent of maternal BMI. Similarly, fetal insulin resistance, as assessed by cord C-peptide, correlated with maternal leptin in early pregnancy, at 28 weeks and with fetal leptin in cord blood. With regard to fetal and neonatal size, maternal leptin concentrations in early pregnancy were related to both fetal weight at 34 weeks and to infant birth weight, independent of all other outcome measures, suggesting that any fetal programming of later metabolic dysfunction may begin at an early gestation. Our multiple regression analysis allowed us to determine that the relationships identified between maternal and fetal leptin, insulin resistance and fetal growth were independent of maternal BMI, however not
surprisingly it remained a strong predictor in our model. Leptin is synthesized primarily in white adipose tissue. Its concentrations in the peripheral circulation are directly related to the amount of adipose tissue present. Maternal concentrations of leptin tend to increase in the third trimester of pregnancy, and there is increasing evidence that the placenta, rather than simply maternal adipocytes, is the major source of the increased leptin concentrations during pregnancy [21]. It has been suggested that this increase in maternal leptin may be to enhance the mobilization of maternal fat stores to increase the transplacental transfer of lipids to the fetus during the latter stages of pregnancy [22]. Similar to adult metabolic disorders therefore, it is possible that alterations in maternal and fetal leptin during pregnancy may result in disordered intrauterine growth and programming. It is already established that fetuses of obese mothers develop insulin resistance in utero [9], and that infants of diabetic mothers are more likely to be obese in childhood [23]. Our findings in a non-diabetic population suggest that fetal programming of later metabolic dysfunction represents a continuum, rather than an absolute cut-off, and is not solely confined to diabetic or obese pregnancies. Our findings have significant implications. They have confirmed the detrimental effects of maternal obesity on pregnancy outcome. Those at the higher end of weight distribution in early pregnancy had significantly higher leptin concentrations and higher HOMA indices and ultimately gave birth to infants with higher birth weights. Though leptin concentrations were related to maternal BMI however, our findings relating maternal and fetal insulin resistance and leptin remained significant
Table 3 Multivariate linear regression analysis.
Maternal BMI (kg/m2) Fetal AAW (cm) EFW (g) Birthweight (g) Early pregnancy HOMA 28 week HOMA Cord blood C peptide
Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value Beta coefficient p-Value
Early pregnancy leptin
28 week leptin
Cord blood leptin
0.309 0.000 0.081 0.2 0.164 0.02 0.121 0.07 0.147 0.02 0.092 0.1 0.173 0.004
0.345 0.000 0.102 0.1 0.102 0.1 0.061 0.3 0.062 0.3 0.269 0.000 0.115 0.05
0.135 0.05 0.075 0.3 0.030 0.7 0.211 0.004 0.04 0.5 0.031 0.6 0.283 0.000
Multivariate regression model with leptin at each time point as the dependent continuous variable and coefficients adjusted for all other outcome measures. BMI is maternal body mass index at first antenatal consultation. AAW is fetal anterior abdominal wall width; EFW estimated fetal weight at 34 weeks' gestation. HOMA = (fasting insulin μU/mL × fasting glucose mmol/l) / 22.5.
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following adjustment for maternal BMI with logistic regression. This suggests that leptin may be considered a biomarker for in-utero insulin resistance, independent of maternal size. Our findings are strengthened by our large cohort, and the fact that these women were healthy and non-diabetic at recruitment. There are conflicting reports in the literature regarding the relationship between maternal and fetal leptin concentrations [24–26]; this large cohort size allows clarification that a significant relationship does indeed exist. A further strength of this study is the longitudinal assessment of leptin and insulin resistance at several time points during pregnancy. This allows us to demonstrate that from as early as the first trimester aberrations in maternal metabolism have potential consequences for fetal growth and insulin resistance. Our findings do have some limitations worthy of consideration. The first is in relation to our marker of fetal adiposity. There are a number of proposed methods to assess fetal fat deposition in utero, and all have limitations. We chose fetal anterior abdominal wall width as we found it to be simple, quick and reproducible marker of abdominal wall fat [17,27]. Undoubtedly assessment of neonatal body composition with a method such as dual-energy X-ray absorptiometry may have provided a more accurate estimation of fetal adiposity, though with a significant resource implication [28]. This may have limited our findings in relation to fetal fat deposition. Additionally we do not, to date, have follow up data on childhood health and obesity rates for this cohort. These findings have confirmed that the intrauterine environment is a vital component of later metabolic risk. Further work is now necessary to ascertain if pregnancy interventions, such as dietary and lifestyle modifications, can alter maternal and fetal leptin concentrations and insulin resistance. It is now clearly apparent that later adult health is, at least in part, mediated by the intrauterine environment. Any attempts to target escalating obesity rates must aim to interrupt any aberrations in fetal insulin and leptin homeostasis, and so future work needs to focus on the critical peri-conceptual and antenatal period. Conflict of interest statement None of the authors has any conflict of interest to declare. Acknowledgments The authors would like to thank the staff at National Maternity Hospital and the mothers who participated in the study. The Health Research Board of Ireland funded this study, with additional financial support from the National Maternity Hospital Medical Fund. None of the funding sources had a role in the study design or manuscript preparation. References [1] de Onis M, Blössner M, Borghi E. Global prevalence and trends of overweight and obesity among preschool children. Am J Clin Nutr 2010;92:1257–64. [2] Franks PW, Hanson RL, Knowler WC, Sievers ML, Bennett PH, Looker HC. Childhood obesity, other cardiovascular risk factors, and premature death. N Engl J Med 2010;362:485–93.
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