Elevated plasma concentrations of lipoprotein(a) are associated with increased risk for atherothrombotic diseases. Apolipoprotein(a), the unique glycoprotein component of lipoprotein(a), is characterized by the presence of multiple kringle domains, and shares a high degree of sequence homology with the serine protease zymogen plasminogen. It has been shown that angiostatin, a proteolytic fragment of plasminogen containing kringles 1–4, can effectively inhibit angiogenesis. Moreover, proteolytic fragments of plasminogen containing kringle 5 are even more potent inhibitors of angiogenesis than angiostatin. Despite its strong similarity with plasminogen, the role of apolipoprotein(a) in angiogenesis remains controversial, with both pro- and anti-angiogenic effects reported. In the current study, we evaluated the ability of apolipoprotein(a) to inhibit VEGF- and angiopoietin-induced tube formation in human umbilical cord endothelial cells. A 17 kringle-containing form of recombinant apo(a) (17K), corresponding to a well-characterized, physiologically-relevant form of the molecule, effectively inhibited tube formation induced by either VEGF or angiopoietin-1. Using additional recombinant apolipoprotein(a) (r-apo(a)) variants, we demonstrated that this effect was dependent on the presence of an intact lysine-binding site in kringle V domain of apo(a), but not on the presence of the functional lysine-binding site in apo(a) kringle IV type 10; sequences within in the amino-terminal half of the molecule were also not required for the inhibitory effects of apo(a). We also showed that the apo(a)-mediated inhibition tube formation could be reversed, in part by the addition of plasmin or urokinase plasminogen activator, or by removal of plasminogen from the system. Further, we demonstrated that apo(a) treated with glycosidases to remove sialic acid was significantly less effective in inhibiting tube formation. This is the first report of a functional role for the glycosylation of apo(a) although the mechanisms underlying this observation remain to be determined in the context of angiogenesis. MMP-2, MMP-9, urokinase-type plasminogen activator (uPA), and human fibrinogen were from Calbiochem. Plasmin was obtained from Haematologic Technologies Inc.F-12K medium and fetal bovine serum (FBS) were obtained from the American Type Culture Collection (ATCC). Antibiotic solution  was purchased from ICN Pharmaceuticals. EC growth factor (ECGF) was obtained from Roche. Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) were purchased from R&D. Heparin, thrombin, and 1–4The r-apo(a) variants used in the present study are shown schematically in 2. Cells between passages 13 and 18 were used in these experiments as previously described Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (# CRL-1730). HUVECs were cultured in F-12K medium supplemented with 15% FBS, 1% antibiotic solution, heparin (100 µg/mL), and ECGF (20 µg/mL). Cells were maintained at 37°C in 5% COEndotoxin- and plasminogen-free human fibrinogen was prepared as previously described HUVECs were cultured on the fibrin matrix in the presence of the treatments indicated in the text and in the respective figure legends. At the end of 24 h or 48 h incubation with the indicated treatment, images were captured using a Nikon digital camera (CoolPix 995) and these were analyzed by Image J (Wright Cell Imaging Facility). For each of six randomly pre-selected fields, the total length of capillary-like structures >30 µm was determined; the total area of the residual HUVEC monolayer was determined for the same field. The differentiation index (DI) was calculated as the ratio of total tube length to cell area for each field examined 17K r-apo(a) (10 µg) was incubated with CPS (0.05 units) in 100 µL of phosphate buffer (pH 7.3) overnight at 37°C. The reaction was stopped by heating the reaction mixture at 80°C for 30 min. Deglycosylation of r-apo(a) was confirmed by SDS-PAGE followed by silver staining.p<0.01.Comparisons between data sets were performed using the Student's t-test . Statistical significance was presumed at in vitro tube formation process in cultured human umbilical vein endothelial cells. For this study, two well-established pro-angiogenic factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), were employed to stimulate angiogenesis in a 3-dimentional fibrin gel matrix as previously described We initiated our investigation by examining the ability of 17K r-apo(a), a physiologically-relevant form of apo(a), to modulate the Considering the key role of the plasminogen activation system in angiogenesis and the reported ability of apo(a) to interfere with this system in the context of both fibrin and cell surfaces To further examine the role of the plasminogen activation system in mediating the anti-angiogenic effects of apo(a), additional components of this system were tested. First, we showed that uPA was, like plasmin, capable of rescuing the anti-angiogenic effects of 17K r-apo(a) . Next, w10 and 17KΔV variants to inhibit VEGF-induced tube formation using the methodology described above. While 17KΔLBS10 contains an Asp to Ala substitution at amino acid position 57 in the KIV10 domain, which disrupts the strong LBS present in this kringle, the 17KΔV variant completely lacks sequences corresponding to the KV domain. The data in 10, is required for the apo(a)-mediated inhibition of VEGF-induced tube formation in HUVECs. The inhibitory effect of the KV domain in apo(a) in this process was further confirmed in the tube formation assay by directly comparing the 6K apo(a) variant (containing KIV5–10 as well as the KV and protease-like domains) with the KIV5–10 apo(a) variant  are unable to inhibit VEGF-stimulated HUVEC tube formation. Finally, we tested a recombinant apo(a) variant corresponding to the N-terminal half of apo(a) (KIV1–4) and demonstrated that it had no significant effect on VEGF-induced HUVEC tube formation  on HUVEC tube formation was pursued through analysis of tube formation after treatment with a series of r-apo(a) variants . First,  variant . In orde variant . While bormation .N- and O-linked glycosylation sites present in the molecule Human apo(a) is a highly glycosylated protein (28% carbohydrate by weight) in vitro and in vivo studies have led to conflicting results, with both pro- and anti-angiogenic effects reported for Lp(a)/apo(a), as well as several reports of no effect whatsoever Identification of a physiological role for Lp(a) has remained elusive, largely because of the absence of an animal model expressing this lipoprotein that is amenable to genetic manipulation 1–4 on inhibition of angiogenesis in our studies. This is not consistent with the findings of Schulter and colleagues The present study was motivated to advance the previous work by Schulter and colleagues The plasminogen activation system comprises plasminogen (the proenzyme), plasmin (the active enzyme), several activators and inhibitors that regulate the formation of plasmin and its activity, and a surface (either fibrin or cells) that serves to localize and accelerate plasminogen activation. The serine protease plasmin has been long established as an important player in promoting angiogenesis by pericellular degradation of components of the surrounding ECM, including fibronectin, laminin, and proteoglycans as well as through activation of key matrix metalloproteases In addition to the components of the plasminogen activation system, MMPs represent another important family of proteases that is heavily involved in angiogenesis regulation, especially MMP-2, -9, and -14 in vitro and in vivo angiogenesis 10 domain and the amino-terminal half of apo(a) as necessary elements for apo(a) to maximally inhibit in vitro fibrin-dependent plasminogen activation system 10 and 6K, respectively) were both able to inhibit HUVEC tube formation in the current study. Either the domains in apo(a) that influence plasminogen activation in the current milieu containing HUVECs may be distinct, or the effect of apo(a) is entirely attributable to a decrease in uPA secretion It is noteworthy that we have identified the KV domain as being responsible for apo(a) inhibition of tube formation, considering that this domain shares approximately 80% amino acid sequence identity with the KV domain in plasminogen 9 in this process. Since the domains in apo(a) that modulate HUVEC tube formation in the present study are different, it is likely that the effects of apo(a) in this context also reflect inhibition of fibrin-stimulated plasminogen activation, versus cell-surface-stimulated plasminogen activation.Apo(a) also affects vascular smooth muscle cell responses through an effect on the plasminogen activation system. We and others have shown that apo(a) stimulates smooth muscle cell proliferation and migration by interfering with plasmin-mediated conversion of latent TGF-β to its active form 10 had no effect  on proliferation and to greatly diminish the effect of apo(a) on migration 9-KV fragments. Notably, we found that deglycosylation of apo(a) abolished its ability to inhibit tube formation, suggesting that the glycans on apo(a) may play a direct or indirect (such as influencing apo(a) conformation) role on the plasminogen activation system in this context.A recent study has shown that a peptide derived from apo(a) KV can, at high concentrations, inhibit angiogenesis and tumor growth Despite a profusion of potential mechanisms by which Lp(a) can harm the vasculature, the true biological role of Lp(a), if any, has remained elusive. Lp(a) is unusual in that its plasma levels vary over 1000-fold in the human population. It is important to bear in mind that in the current study apo(a) was primarily tested at a concentration of 100 nM  concentration of ∼8 mg/dL), which is well below the clinical risk threshold identified for Lp(a) (25–30 mg/dL


Apolipoprotein E (apo E) plays a major role in lipid metabolism, obesity and accordingly in development of diabetes and coronary heart disease (CHD). Our main objective was to evaluate the association between apo E gene polymorphism with anthropometric measures.Participants were selected from zone 17 Tehran/Iran. We assessed height, weight, body mass index (BMI), waist circumference (WC), blood pressure, serum fasting blood sugar, total cholesterol and triglycerides. Genotyping for apo E gene polymorphism was carried out using PCR-RFLP technique.Among total study population (n=311), 156 subjects were diabetic. The apo E3/E3 was the most common genotype in our population while E2 and E4 alleles had lower frequencies, respectively. After adjustment for diabetes, the apo E2 and E4 alleles were significantly associated with hypercholesterolemia and WC, respectively . This association was also related to sex and age. The probability of having abdominal obesity in E4 allele carriers was increased from 0.22 to 8.12 in women and to 3.08 in age ≥ 50 years.Apo E polymorphism had significant influences on WC and total cholesterol level in patients with type 2 diabetes. This study highlights the importance of lifestyle modifications which may be more beneficial in hypercholesterolemic women carriers of E2 and E4 alleles concomitant central obesity. The human apo E is polymorphic and its genetic variation can affect its anti atherosclerotic effects . The comIt has been suggested that the apolipoprotein E (apo E) gene polymorphism may be associated with predisposition to diabetic complications (both micro- and macrovascular complications). This association was shown between apo E gene polymorphism and diabetic nephropathy, but not for diabetic retinopathy in type 1 diabetes . But in Recent evidence from both animal and human studies showed the important role of apo E gene in the development of obesity and insulin resistance . ObesityStudy subjects were recruited from zone 17 of Tehran/ Iran. The inclusion criteria were all of subjects who interest to enroll in the study. This study comprised of 311 subjects; 155 non-diabetics and 156 diabetics from the similar area. T2DM was diagnosed according to American Diabetes Association criteria [2). Obesity was defined as BMI ≥ 30 kg/m2 according to WHO recommendations [Body Mass Index (BMI) was calculated based on the following formula; body weight /square height . DNA was extracted from leukocytes using salting-out method . GenomicStrength of association between different variables and apo E gene polymorphism was estimated by Chi-square and logistic regression using SPSS software, version 11.5. P ≤ 0.05 was considered as significant in statistical analysis.The age of the participants in the study was 25–65 years and mean age was 46 ± 13 years. In this study 32.2% of subjects were male and 67.8% were female.The frequency of smokers, dyslipidemic, hypertensive, diabetic subjects, and past history of CHD or T2DM in the participants were 10.7, 55.5, 60.2, 50.2, 10 and 33%, respectively. Baseline characteristics of participants (n=311) are shown in Table The frequency of different polymorphism of apo E gene in our population is shown in Table Coefficients of univariate and multivariate regression models between independents variables  and dependent variables  are shown in Table BMI: The influence of sex on BMI ≥ 30 was independent of diabetes or apo E gene polymorphism. Central obesity: Sex, aging, and apo E4 allele had significant influence on central obesity which these effects were dependent to diabetes.Hypertension: Aging had a significant influence on hypertension dependent to diabetes.Hypercholesterolemia: Sex, aging, and apo E2 allele had significant influence on hypercholesterolemia which these effects were independent of diabetes.Hypertriglyceridemia: We didn’t find any significant association between independent variables and hypertriglyceridemia.The details of all above mentioned results are shown in Table The complex polygenic diseases such as diabetes could be complicated by phenomena such as variable age at onset disease, or environmental triggers . AlthougThe role of apo E gene polymorphism in anthropometric measures has been studied in different populations. Although this study is conducted in a small sample size, this is the first study on association between apo E gene polymorphism and anthropometric measures in Iranian population. In this study Apo E3 was the most common allele in our population, and E2 followed by E4 allele have been found less frequent both in diabetics and nondiabetics. These findings are in agreement with previous reports ,24. So, In subgroup of patients with abnormal WC in our study, all components of anthropometric measures were higher than subgroup that had normal WC. Previous evidence also supports our finding -34 whichIn our study, apo E2 allele was significantly associated with hypercholesterolemia both in women and in patients aged > 50 which was independent of diabetes. Also, lipid levels did not significantly differ between subjects with apo E3/E3 and apo E4/E3. This is in agreement with our previous results and also results by Pedro-Botet et al. who found significantly higher level of total cholesterol in patients with E2 isoform ,29. In cAn association between apo E2 allele and hypertriglyceridemia has been consistently reported in healthy populations -43. DallIn our study, the probability of being obese (BMI ≥ 30) was increased significantly in women, which was independent of apo E polymorphism or diabetes. This parameter is known to influence lipid metabolism or insulin resistance and may interfere with genetic factors. The influence of lifestyle on apo E polymorphism was clearly shown in two parts of Belgium . AlthougIn this study, we observed that there were sex-specific effects on anthropometric measures. For example, abnormal WC was more prevalent in females both in diabetics and nondiabetics. Also, our data has shown that the probability of having obesity, central obesity, and hypercholesterolemia are increased significantly in diabetic women independent of apo E polymorphism. The sex-specific distribution of apo E gene was not differing significantly between two groups. This might be as a result of deviation in female/male ratio in our population. However, gender related differences are difficult to determine due to variations of hormonal status during pre- and post menopause in women and their effects on plasma lipid profile .In our patients the probability of having central obesity, hypertension and hypercholesterolemia was increased significantly in diabetics according to age (≥ 50 years). When we assessed the effect of apo E2 allele on hypercholesterolemia, this trend was equal 0.36 which by considering the effect of age, this figure was changed to 2.05. The trend for increasing central obesity in E4 allele carriers was 0.22 without considering the effect of age and changed to 3.08 in presence of age > 50 years. These results showed the influence of apo E polymorphism on abdominal obesity and hypercholesterolemia was significantly changed in presence age > 50 years. One study was evaluated this effect on postprandial triglycerides . They foAs it is expected, we found a significant increase in blood pressure just at the presence of diabetes and aging. A positive correlation between total serum cholesterol level and blood pressure  had beenIn this study, there was no information about diet of our patients therefore we don’t have any precise explanation for these differences. Because our study was carried out in a small population, therefore the effects observed might not be subject to generalization. The possible contribution of such association to the observed relationship between apo E4 and abdominal obesity also remains to be clarified by prospective studies.In summary, the present study shows low frequency of the E2 and E4 alleles in our study population and indicates that apo E polymorphism might play a role in determining plasma lipids levels. The significant inter-relation between the apo E polymorphism, abdominal obesity, and change in total cholesterol levels supports epidemiological findings.Although lifestyle modification is beneficial in all people, it may be especially recommendable in women carriers of E2 allele to modify potentially elevated fasting cholesterol, and also in carriers of E4 allele to control weight. In addition, apo E4 allele might be a predictor for CHD in diabetics by influencing central obesity. Our data suggest that this risk factor probably exert its atherogenic effects through diverse mechanisms.aNational Cholesterol Education Program (Adult Treatment panel III).The authors had no conflict of interest.OT-M conceived of the study, wrote draft the manuscript and performed the statistical analysis. HF conceived of the study, and participated in its design and coordination. MQ performed the statistical analysis. PA carried out the molecular genetic assessment. BL conceived of the study. JT-B conceived of the study. MMA conceived of the study, carried out the molecular genetic assessment and helped to draft the manuscript. All authors read and approved the final manuscript.This study was performed in Endocrinology & Metabolism Research Center of Tehran Universityof Medical Sciences without financial support.


Stunting affects one-third of children in developing countries, but the causes remain unclear. We hypothesized that enteropathy leads to low-grade inflammation, which suppresses the growth hormone-IGF axis and mediates stunting.We conducted a case-control study of 202 HIV-unexposed Zimbabwean infants who were stunted (height-for-age Z-score (HAZ) <−2; cases) or non-stunted  at 18 months. We measured biomarkers of intestinal damage (I-FABP), inflammation  and growth hormone-IGF axis  in infant plasma at 6 weeks and 3, 6, 12 and 18 months, and in paired maternal-infant plasma at birth. Adjusted mean differences between biomarkers were estimated using regression models. Multivariate odds ratios of stunting were estimated by logistic regression.10 levels of CRP ; P = 0.008) and AGP ; P = 0.087) during infancy were associated with stunting. There were no associations between levels of I-FABP, IL-6, sCD14 or EndoCAb and stunting.At birth, cases were shorter (median (IQR) HAZ −1.00  vs 0.03 ; P<0.001) than controls and their mothers had lower levels of IGF-1 (adjusted mean difference (95%CI) −21.4  ng/mL). From 6 weeks to 12 months of age, levels of CRP and AGP were consistently higher and IGF-1 and IGFBP3 lower in cases versus controls; IGF-1 correlated inversely with inflammatory markers at all time-points. I-FABP increased between 3–12 months, indicating extensive intestinal damage during infancy, which was similar in cases and controls. In multivariate analysis, higher login utero and was associated with low maternal IGF-1 levels at birth. Inflammatory markers were higher in cases than controls from 6 weeks of age and were associated with lower levels of IGF-1 throughout infancy. Higher levels of CRP and AGP during infancy were associated with stunting. These findings suggest that an extensive enteropathy occurs during infancy and that low-grade chronic inflammation may impair infant growth.Stunting began  However, few longitudinal studies have evaluated the mechanisms underlying poor growth among infants in developing countries. We hypothesized that an important cause of child stunting is exposure to chronic, low-grade inflammation during fetal and postnatal life, which suppresses production of IGF-1 Stunting may therefore be driven by intestinal damage and chronic inflammation in addition to dietary inadequacy. Furthermore, since stunting begins The original ZVITAMBO trial and this case-control study were approved by the Medical Research Council of Zimbabwe, Johns Hopkins Bloomberg School of Public Health Committee on Human Research, and Montreal General Hospital Ethics Committee.We designed a case-control study using archived plasma samples and data from HIV-negative mother-infant pairs enrolled in the ZVITAMBO vitamin A supplementation trial, conducted between 1997–2001. The trial tested the effects of a single high-dose vitamin A supplement given to mothers and/or infants during the immediate postpartum period on several infant health outcomes, as previously reported HIV-negative mothers were encouraged to exclusively breastfeed for six months. Data on feeding practices obtained at 6 w and 3 mo were used to categorize exclusive, predominant or mixed breastfeeding, as previously described http://www.who.int/childgrowth/en). Among HIV-negative mothers, there was no effect of vitamin A supplementation on infant linear growth .Maternal height and mid-upper arm circumference (MUAC) were measured within 96 h of delivery; maternal weight was measured at 6 w postpartum. Infant weight and height were measured using an electronic scale , and length board , respectively, at each visit. Anthropometry was undertaken using methods described by Gibson Blood was collected from all enrolled mothers and infants at baseline and from a representative subsample  of mother-infant pairs at all follow-up visits. Samples were centrifuged and plasma removed within 2 h of blood collection; aliquots were stored in −80C freezers with automatic generator backup. Mothers underwent HIV testing at baseline using two parallel ELISA assays. Women who tested HIV-negative at baseline were re-tested at every subsequent visit to detect HIV seroconversion.Cases (stunted) and controls (non-stunted) for this study were selected based on HIV-exposure status and height at 18 mo of age. Eligible infants were born to mothers who remained HIV-negative throughout follow-up, and for whom anthropometric data and archived plasma of sufficient volume (≥0.2 mL) at ≥4 time-points between 6 w and 18 mo of age were available. Cases had height-for-age Z-score <−2.0, and controls had height-for-age Z-score >−0.5 at 18 mo.Of 14110 enrolled infants, 9249 remained HIV-unexposed by 18 mo of age. From these, 132 stunted and 101 non-stunted infants fulfilled our selection criteria at 18 mo . To maxiWe used commercially available ELISA kits to assess small intestinal damage ; microbial translocation ; immune activation ; and activity of the growth hormone axis  and insulin-like growth factor binding protein 3 .Baseline characteristics were compared between cases and controls using Chi-squared tests for categorical variables, Wilcoxon rank sum tests for continuous non-normal variables, or two-sample t-tests for continuous normal variables. Because of baseline differences in important covariates for stunting between cases and controls, adjusted differences in plasma biomarker concentrations were estimated at each time-point. For normally distributed biomarkers, unadjusted differences between means were compared using the two-sample t-test; adjusted differences between means were calculated using an ordinary least-squares regression model. For non-normally distributed biomarkers, unadjusted differences between medians were compared using the cendif command in STATA Data from this study are not available in a public repository but may be made available upon request.2); 28% of mothers of cases and 39% of mothers of controls were overweight or obese (BMI>25 kg/m2). Mothers of infants who became stunted had fewer years of education than mothers of non-stunted infants.Baseline characteristics of infants and mothers are shown in At every time-point between birth and 18 mo, infants who were stunted at 18 mo (cases) had significantly lower height-for-age  and weigTo test our hypothesis that stunting arises due to suppression of the growth hormone-IGF axis during infancy, we measured IGF-1 levels at each time-point between birth and 18 mo. IGF-1 levels at birth were similar in cases and controls, but from 6 weeks of age IGF-1 was significantly lower in cases than controls (adjusted mean difference (95%CI) −10.5  ng/mL; Given the clear differences in IGF-1 between cases and controls throughout infancy, we next measured levels of IGF binding protein 3 (IGFBP3), the principal binding protein of circulating IGF-1. Similar to our findings for IGF-1, levels of the binding protein were similar between groups at birth, but by 6 weeks of age IGFBP3 levels were lower in cases compared to controls (adjusted mean difference (95%CI) −211.9  ng/mL) and remained lower at every time-point through 18 mo .10 mean (SD) 3.12 (0.32) pg/mL in cases; 3.06 (0.26) pg/mL in controls), before declining between 12–18 mo , a component of the gram-negative bacterial membrane. Soluble CD14, a marker of monocyte/macrophage activation produced predominantly in response to LPS stimulation, steadily rose throughout the first year of life in both groups, then fell between 12–18 mo of age . SolubleTo assess inflammatory status, we compared levels of IL-6, CRP and AGP over the first 18 mo of life . IL-6 isGiven the significant differences in inflammatory status between stunted and non-stunted infants we hypothesized that, even in these apparently healthy infants, low-grade inflammation inhibits hepatic production of insulin-like growth factor-1 (IGF-1), as has been shown for chronic inflammatory diseases 10 levels of CRP ; P = 0.008) and AGP ; P = 0.087) between 6 w–12 mo were associated with increased odds of stunting . We ran a multivariate model for each biomarker, adjusted for infant gender, birth weight and birth length, and maternal education, height and MUAC to calculate adjusted odds ratios for stunting at 18 mo . Given tstunting . There wstunting .in utero, we next measured biomarkers in maternal plasma collected within 96 h of delivery, to test the hypothesis that maternal inflammation and IGF-1 levels influence fetal growth. Mothers of cases compared to controls had lower levels of IGF-1 (adjusted mean difference (95%CI) −21.4  ng/mL; Because stunting began We next evaluated maternal inflammatory status at birth, reasoning that mothers of cases would have higher levels of inflammation than mothers of controls. However, after adjusting for baseline differences between groups, there were no significant differences in any inflammatory biomarker between mothers of cases and controls . Finallyin utero IGF-1 levels; and, second, impaired postnatal growth which was associated with chronic inflammation, starting very early in infancy. These findings highlight the importance of the first thousand days from conception to 2 years of age, during which childhood stature and ultimate developmental potential are determined, and provide an insight into the biomedical pathways that may need to be targeted to reduce the high prevalence of stunting in developing countries.We show here an association between low-grade inflammation in the first year of life and perturbation of the growth hormone-IGF axis, which is associated with stunting in apparently healthy Zimbabwean infants. We observed two important periods of poor linear growth: first, impaired intrauterine growth, which may be influenced by maternal health and Levels of IGF-1 were generally low compared to reported values from European infant cohorts Although we selected cases and controls at 18 months of age, stunted infants were already growth restricted at birth. Median height-for-age Z-score at birth among these infants was −1.00 and almost one-quarter of infants in the stunted group had low birth weight. Birth weight was related to infant IGF-1 at birth, which in turn was associated with the inflammatory status of the mother-infant dyad. The infant inflammatory milieu was closely related to the level of maternal inflammation at birth. We speculate, based on these associations, that inflammation during pregnancy may ‘set’ the infant inflammatory axis, which in turn influences the level of IGF-1 in early life. Optimizing the health of pregnant women may be essential to impact antenatal and postnatal stunting. Infants who became stunted were born to mothers who themselves were shorter than mothers of non-stunted infants. However, it was striking that over one-quarter of mothers of cases were overweight or obese. The relationship between maternal and fetal nutritional status is therefore complex and requires further investigation, particularly in view of the emerging obesity epidemic in countries that are experiencing the nutrition transition Intestinal damage during infancy was common in both cases and controls. There is no single, well-validated enteropathy biomarker available; most prior studies have used non-invasive markers of intestinal permeability because intestinal biopsies are difficult to obtain in childhood There are several limitations to this study. First, women were recruited shortly after delivery, so we were unable to characterize maternal inflammation or IGF-1 levels during pregnancy, and biomarker levels may have been influenced by parturition; however, we assume that levels of biomarkers measured shortly after delivery at least partly reflect the antenatal milieu. Second, we did not measure LPS levels because plasma was not collected into endotoxin-free tubes. Rising levels of soluble CD14 and EndoCAb during infancy indirectly indicate chronic exposure to LPS In summary, our data suggest that stunting is influenced by both maternal and infant factors. Antenatally, maternal nutritional and inflammatory status may impact fetal growth, leading to intrauterine stunting and low birth weight; postnatally, low-grade inflammation early in life is associated with stunting. The reasons for higher levels of CRP and AGP in stunted compared to non-stunted infants are unclear and require further investigation. We show here that infants have extensive small intestinal injury and evidence of exposure to LPS during infancy, but we found no evidence that gut damage was associated with stunting. However, future studies of prospective cohorts should use a more extensive panel of markers to characterize enteric dysfunction, as recently proposed Table S1Biomarker levels in stunted and non-stunted infants. Biomarker levels with unadjusted and adjusted differences in values between stunted and non-stunted infants, between birth and 18 months of age. Number of samples available for each measurement are shown in square brackets.(DOCX)Click here for additional data file.


Intake of colostrum after birth is essential to stimulate intestinal growth and function, and to provide systemic immunological protection via absorption of Immunoglobulin G (IgG). The birth order and weight of 745 piglets (from 75 litters) were recorded during a one-week period of farrowing. Only pigs weighing greater than 0.68 kg birth weight were chosen for the trial. Sow colostrum was collected during parturition, and piglets were bled between 48 and 72 hours post-birth. Piglet serum IgG and colostral IgG concentrations were determined by radial immunodiffusion.Sow parity had a significant (P < 0.001) effect on sow colostral IgG concentration, being 5% higher in multiparous females. Sow colostral IgG concentration explained 6% and piglet birth order accounted for another 4% of the variation observed in piglet serum IgG concentration (P < 0.05); however, birth weight had no detectable effect. Piglet serum IgG concentration had both a linear (P < 0.05) and quadratic effect (P < 0.05) on % survival. Piglets with 1,000 mg/dl serum IgG or less (n=24) had a 67% survival; whereas, piglets with IgG concentrations between 2250 to 2500 mg/dl (n=247) had a 91% survival. Birth order had no detectable effect on survival, but birth weight had a positive linear effect (P < 0.05). Piglets weighing 0.9 kg (n = 107) at birth had a 68% survival rate, and those weighing 1.6 kg (n = 158) had an 89% survival.We found that the combination of sow colostrum IgG concentration and birth order can account for 10% of the variation of piglet serum IgG concentration and that piglets with less than 1,000 mg/dl IgG serum concentration and weight of 0.9 kg at birth had low survival rate when compared to their larger siblings. The effective management of colostrum uptake in neonatal piglets in the first 24 hrs post-birth may potentially improve survival from birth to weaning. Modern swine genotypes have been selected for increased litter size over the last 10–15 years which has resulted in greater heterogeneity of piglet birth weight. ColostrOur objective was to examine relationships among birth weight, birth order, serum IgG concentration and their correlation with piglet growth and mortality in a commercial swine facility.All protocols were carefully supervised and approved by the corporate licensed veterinarians. All standard operating procedures for animal treatment and care were in agreement with published guidelines for animal care. The expSow colostrum was manually collected immediately after sows began farrowing. All teats were sampled into a single composite, and the approximate amount collected per sow was 100 ml. Colostrum was harvested in plastic cups and immediately refrigerated. Piglets were bled between 48 to 72 hours post-birth using a 22 × 1.5” gauge needle and a 6 ml (13 × 100 mm) serum vacutainer tube . Blood samples were refrigerated and allowed to clot overnight. Serum was collected after centrifugation  and stored at −20 C until further analysis. Sow colostrum was centrifuged similarly and the defatted fraction containing the IgG was stored at −20 C until further analysis.A radial immunodiffusion test was used to determine IgG content in the piglet serum and sow colostrum,15. MeasTotal protein concentration in the serum samples was determined using the bicinchoninic acid (BCA) protein assay . ProteinThe data were analyzed using the GLM and REG procedures of SAS in order to establish relationships among sow parity, sow IgG colostral concentrations, birth order, and piglet birth weight to piglet plasma IgG and protein concentration. We used a weighed logistic regression analysis in order to determine the effects of pig IgG concentration, piglet birth order and birth weight on % of survival at weaning, with number of piglets per point being the weighting factor. Individual pig was used as the experimental unit.Sow colostral IgG concentration Figure  explainePiglet serum IgG concentration was strongly correlated (P < 0.0001) with total serum protein concentration Figure . Piglet Piglet serum IgG concentration had both a positive linear and a negative quadratic effect Figure  on % surBirth order had no detectable effect on % survival at weaning (data not shown). Piglet birth weight had a linear effect (P < 0.05) on % survival at weaning Figure . PigletsThe most significant finding of this study was that the combined effects of sow parity, sow colostral IgG concentration and birth order only explained 13% of the total variability found in piglet serum IgG concentration. We expected that these factors would have accounted for a greater percentage of the total variability found in piglet serum IgG concentration. Another significant finding was that, contrary to what we would have expected, birth weight had no effect on piglet serum IgG concentration. This lack of effect could be explained by the fact that we physically aided each new born piglet to suckle from their dam immediately after birth.Piglet serum IgG concentration and birth weight had the greatest effect of any of the variables measured on survival percent at weaning. Stone and Leymaster used a sWe selected piglets with birth weight greater than 0.68 kg because very low birth weight piglets die to a much greater extent than their heavier littermates and this would have limited our blood sampling at 2 d of age. A low-birth weight piglet is disadvantaged when competing with its littermates a) because of its size, b) it hBlasco et al., reportedWe specifically chose IgG as the measuring index of circulating maternal antibodies because it is the most abundant immunoglobulin transferred from the blood to the mammary gland in swine. In sow colostrum, all of the IgG, most of the IgM and 40% of the IgA originate from the maternal blood. PigletsWe attribute the significant effect of birth order on piglet serum IgG concentration to the rapid changes in colostrum quality and quantity between onset of parturition and last piglet being born in each litter. Not only does the colostral IgG concentration change in the first 24 h of life, but so does other bio-active compounds  as well. Our resKlobasa and co-workers also repMachado-Neto et al. showed tWe hypothesized that piglet serum IgG concentration is highly correlated with pre-weaning mortality. We found both a linear and quadratic response when correlating piglet serum IgG concentration and piglet survival at weaning. Logistic regression was shown to be an appropriate and useful technique for analysis of factors affecting piglet survival. The advantages of logistic regression are that it can be applied to continuous as well as discrete predictor variables and the fitted regression coefficients are readily translated into the odds ratio, giving a mathematical quantification to observed differences. We founWe argue that while systemic serum IgG concentration might indicate an animals’ general immune status, circulating immunoglobulin content gives no indication as to the specificity of the immunity that may be present or that may have developed in the tissue immune system , which is important in resisting certain diseases common in young piglets. We recognized that colostrum contains others substances (i.e. energy) besides immunoglobulins that also are important for piglet survival. Finally, a reduced consumption of immunoglobulins may predispose piglets to selected kinds of infectious diseases.The reason we placed 11 pigs per sow was due to the interaction between the number of piglets in a litter and the stimulation they provide to sow’s milk output. When there are fewer piglets suckling, the longer the pre-ejection massaging needed to cause a milk ejection. The weiBirth weight has been widely known to be a very important economic trait in swine production. Birth weight drives pre- and post-weaning piglets’ growth. Piglets’ ability to have high colostrum intake is determined by their body weight at birth, which is also the main factor of their vitality and their ability to stimulate the udder to extract colostrum. Our resWe found that sow colostrum, pig birth order and sow parity collectively had a small impact on piglet serum IgG concentration and piglet birth weight did not. Piglet serum IgG concentration was highly correlated with piglet serum total protein concentration. Piglet serum IgG concentration and birth weight had the greatest effect of any of the variables measured on % survival at weaning. Collectively, these data indicate that pigs with ≤ 1,000 mg/dl IgG at 2–3 days of age have reduced survivability and may benefit from IgG supplementation early in life. The number of piglets in this category was low in this study (24/637= 3.8%) but this number is biased downward because we excluded pigs weighing less than 0.68 kg from the experiment. If an IgG-rich supplement were directed to low birth weight piglets, piglets of high birth order and/or piglets from low-parity sows, survivability may be improved. In addition to IgG, the role of colostrum’s bioactive compounds such as cells and growth factors in the development of the immune system merits further investigation.IgG: Immunoglobulin G; IgA: Immunoglobulin A; SAS: Statistical Analysis System; GLM: General Linear Model; REG: Regression; Mg: Milligram; Dl: Deciliter; Kg: Kilograms; Ml: Milliliter; Hr: Hour; FASS: Federation of Animal Science Societies; ID: Identification; CHG: Chlorhexidine gluconate; C: Celsius; M: Molar; BCA: Bicinchoninic Acid; BSA: Bovine Serum Albumin; VIP: Vasoactive Intestinal Polypeptide; BW: Body Weight; ADG: Average Daily Gain.Full financial support for this trial was provided by American Protein Corporation (APC), Ankeny, IA 50021. Dr. Joy Campbell (one of the author in this manuscript) serves as Director of Research and Development for American Protein Corporation. APC is the world’s largest producer of functional proteins. Functional proteins are a complex mixture of biologically active proteins that help support and maintain normal immune function. APC’s functional proteins are derived from unique components found in bovine and porcine blood, and are consumed by millions of animals around the globe. No products from APC were used in this trial.RAC as the lead author was in charge of designing the research protocol, obtaining all materials needed for it, executing it, carrying out all the lab work, data collection and analysis and finally writing the manuscript. XL was instrumental on guiding the lead author on the statistical analysis and lab methods. JC gave significant contribution on the importance of colostrum and immunoglobulins and their effects on neonatal survival. She assisted in designing the study and provided excellent suggestions to the final version of the manuscript. JO as a co-advisor to the lead author, he was involved in the design, execution and interpretation of the results. He also assisted with blood collection and statistical analysis. AJM as a co-advisor to the lead author, he was involved in the design and interpretation of the results. All authors read and approved the final version of the manuscript.RC holds a PhD in Animal Nutrition from North Carolina State University (NCSU). His area of research is neonatal survival, nutrient digestibility and gastrointestinal health of swine. In 2001, he was awarded the “Innovative Award Applied Research” by National Pork Producer Council (NPPC) at the Midwest Animal Science Meeting in Des Moines, Iowa. He is a member of the North Carolina Pork Council and the American Society of Animal Science. He currently serves as Director of Swine Technical Services for Huvepharma USA, Inc. XL holds a PhD in Animal Nutrition from China Agricultural University. His main areas of research are neonatal survival and lipid metabolism primarily focused on the regulation of fatty acid oxidation during neonatal development and epigenetic regulation of fetal development and placenta growth. He is also interested in the role of polyunsaturated fatty acid in the development of neonates. He is a Research Assistant professor at the Department of Animal Science at NCSU and a member of American Society of Animal Science. JC has a PhD in Nutritional Sciences from the University of Illinois Urbana-Champaign. She works with functional proteins (such as plasma) and their impact on gastrointestinal health. Her main research interests are Nutrition and Animal Health. She serves as Director of Research and Development North America for American Proteins Corporation (APC) in Ankeny, IA and is a member of American Society of Animal Science. She received the ASAS/ADSA Outstanding Young Agribusiness Award from American Society of Animal Science. AJM holds a MS in Swine Nutrition, a PhD in Gastrointestinal Physiology and a Doctor of Veterinary Medicine (DVM) all from NCSU. His main area of research is to study basic mechanisms of stress-induced intestinal dysfunction. Stress is an important contributing factor to enteric disorders of veterinary species and humans however, the mechanisms are poorly understood. His work has focused on the role of mucosal mast cells in psychological stress-induced disturbances in intestinal mucosal barrier function. He believes that this work will have important implications in the understanding of stress-related gut disorders such as infectious diarrhea, Inflammatory Bowel Disease, and Irritable Bowel Syndrome, and will facilitate the design of novel preventative and treatment strategies for veterinary and human patients suffering from these disorders. He is an assistant professor of GI physiology and swine medicine at NC State College of Veterinary Medicine. He is member of several professional societies including the American Physiological Society, American Association of Swine Veterinarians, and American Gastroenterological Association (AGA). He has over 34 peer-reviewed publication focused mainly in the area of gastrointestinal health in swine. JO has a PhD in Nutritional Biochemistry from the University of Wisconsin. As a Williams Neal Reynolds Professor in the Department of Animal Science at NCSU, his research interests are molecular and metabolic regulation of lipid digestion and metabolism; neonatal nutrition; intestinal growth and metabolism in normal and pathophysiological states. His program is focused on using the young piglet as a model for the human infant in nutrition and digestive physiology. He also has teaching responsibilities in the areas of nutrition and biochemistry. His most recent awards include “Williams Neal Reynolds Distinguished Professor” and “The Outstanding Graduate Instructor” both given by the College of Agriculture and Life Science at North Carolina State University, the “Animal Growth and Development Research” given by the American Society of Animal Science. He was a member of the National Research Council (NRC) committee which recently published the new 2012 Nutrient Requirement of Swine. He is an Associate Editor in Advances in Nutrition (American Society for Nutrition) and the Journal of Animal Science and Biotechnology.


There is a lack of information on the optimal timing of food supplementation to malnourished pregnant women and possible combined effects of food and multiple micronutrient supplementations (MMS) on their offspring's growth. We evaluated the effects of prenatal food and micronutrient interventions on postnatal child growth. The hypothesis was that prenatal MMS and early invitation to food supplementation would increase physical growth in the offspring during 0-54 months and a combination of these interventions would further improve these outcomes.In the large, randomized MINIMat trial , Bangladesh, 4436 pregnant women were enrolled between November 2001 and October 2003 and their children were followed until March 2009. Participants were randomized into six groups comprising 30 mg Fe and 400 μg folic acid (Fe30F), 60 mg Fe and 400 μg folic acid (Fe60F) or MMS combined with either an early (immediately after identification of pregnancy) or a later usual  program invitation to food supplementation. The anthropometry of 3267 children was followed from birth to 54 months, and 2735 children were available for analysis at 54 months.There were no differences in characteristics of mothers and households among the different intervention groups. The average birth weight was 2694 g and birth length was 47.7 cm, with no difference among intervention groups. Early invitation to food supplementation  reduced the proportion of stunting from early infancy up to 54 months for boys (p = 0.01), but not for girls (p = 0.31). MMS resulted in more stunting than standard Fe60F (p = 0.02). There was no interaction between the food and micronutrient supplementation on the growth outcome.Early food supplementation in pregnancy reduced the occurrence of stunting during 0-54 months in boys, but not in girls, and prenatal MMS increased the proportion of stunting in boys. These effects on postnatal growth suggest programming effects in early fetal life.ISRCTN16581394ISRCTN:  Epidemiological and experimental studies indicate fetal under-nutrition affects large numbers of infants in developing countries, with adverse consequences for their immediate survival and lifelong health ,2. StudiA Cochrane review  concludeThere is a lack of information, however, about the effect of the timing of food supplementation during pregnancy on birth size and subsequent child growth. Animal experiments indicate the timing of under-nutrition in pregnancy produces different responses to size at birth, placental size, and development of endocrine responses . AlthougThe Maternal and Infant Nutrition Interventions in Matlab  is a randomized trial conducted in a rural community in Bangladesh. This trial evaluates the effect of early or later invitation to prenatal food supplementation, combined with one of three different micronutrient supplements, on anthropometry and gestational age at birth and under-five mortality (primary outcomes). The effect on child growth from birth to 54 months is reported in this paper.The hypothesis was that prenatal multiple micronutrient supplementations and early invitation to food supplementation would increase physical growth in the offspring during 0-54 months, and a combination of these interventions would further improve these outcomes. A factorial design was used to determine the effect of a combination of prenatal micronutrient supplementation  and early (around week 9) or later usual standard invitation  to food supplementation on physical growth (weight and length/height) in the offspring during 0-54 months in a poor rural population in Bangladesh.The study, Maternal and Infant Nutrition Interventions in Matlab (MINIMat), was conducted in Matlab, a rural sub-district 57 km southeast of the capital, Dhaka, Bangladesh. In this area, most people depend on agriculture, fishing, or day labor. Breast-feeding is almost universal during infancy. According to the national policy, children from 6-59 mo should be supplemented with vitamin A every 6 months and mothers should receive a vitamin A dose within 2 months after delivary. In Matlab, ICDDR,B maintains a Health and Demographic Surveillance System that has recorded health and demographic information on a monthly basis since 1966. In half of the area ICDDR,B is service provider for maternal and child health. All pregnant women in the ICDDR,B service area were eligible for enrolment. If a woman reported to the Community Health Research Worker (CHRW) that she was pregnant, she was offered a pregnancy test. She was enrolled in the MINIMat study if the following eligibility criteria had been met: fetus was viable, gestational age was < 14 weeks confirmed by ultrasound examination, no severe illness, and consent to participate. In total, 4436 pregnant women were enrolled in the MINIMat study between November 2001 and October 2003, and their children were followed-up until March 2009.Written informed consent was obtained from all participating mothers, and the study was approved by the Research and Ethical Review Committees of ICDDR,B.2 were supposed to receive the food supplements. The food supplement contained 80 g roasted rice powder, 40 g roasted pulse powder, 20 g molasses and 12 mL (6 g) soybean oil, and was provided in plastic packets that were to be mixed with water. The supplement provided 608 kcal (2.85 mJ) and 18 g vegetable protein and was continued up to end of pregnancy. The three types of micronutrient supplements were distributed in special pill bottles that looked identical and contained 35 capsules and were distributed in the homes during monthly home visits by the interviewers. The micronutrient supplements were offered to the enrolled women during a clinic visit at 14 weeks of gestation.Details of the food and micronutrients supplementation are described elsewhere . In brieAt every monthly home visits, the interviewers asked a series of questions to assess compliance with food supplementation in the previous 30 days. The micronutrient bottles in the study were equipped with a microprocessor inside the cap (eDEM) that recorded the date and time of every opening of the cap. This information was downloaded into a computer when the bottles were collected from women. The capsule-counting eDEM is regarded as the best available method for measuring adherence .The enrolled pregnant women were individually randomized into one of six groups of food and micronutrient intervention by a computer-generated register of study identity numbers and random assignment of food and micronutrient groups. The micronutrient supplementation was double-blinded but the food supplementation was allocated randomly and not masked.Sample size calculations were primarily done in relation to a birth weight outcome that had a standard deviation of 400 g, based on previous data from Bangladesh, and the minimum important difference was determined to be 70 g. The estimated sample size with a power of 0.90 (Type II error of 0.10) and 95% confidence level (type I error of 0.05) was 686 in each group, i.e., 4116 in the six food and micronutrient supplementation groups. Adjusting for 5% refusal, 11% loss during pregnancy, and 9% loss during infancy due to death and out-migration, the total sample size required was 5300. This required enrolment over two years at the known rates of pregnancy occurrence in this study population.Because the MINIMat study was designed to evaluate the impact of nutritional interventions on birth weight, the sample size calculations were made on the basis of finding a difference in birth weight. This paper reports a secondary outcome (child growth). We calculated the differences in child growth that we could detect with the adequate sample size (n = 2735) in any supplementation group in our analyses. For this sample size, 80% power and 95% probability we were able to detect a difference of 0.2 SD score (or difference of 0.12 SD score for 2 food groups) between 6 food and micronutrient supplementation groups.http://www.who.int/childgrowth/software/en/). Underweight was defined as <-2 Z-score for weight-for-age, stunting was defined as <-2 Z-score for length/height-for-age, and wasting was defined as <-2 Z-score for weight-for-length/height.Anthropometry at birth was part of the primary outcome of this trial, and in this paper subsequent child growth is reported. Weight and length/height were measured every month up to 1 year, then every 3 months up to 24 months, and again at 54 months of age. Birth anthropometry was performed usually within 72 hours after birth. All birth weights were measured by SECA electronic or beam scales  to a precision of 10 grams. Maternal weight and height were measured on enrollment at around the eighth week of gestation. Maternal weight was measured by electronic scales , with a precision of 100 grams, and height was measured to the nearest 0.1 cm with a stadiometer. The recumbent length of the newborn was measured by locally manufactured, collapsible length boards, with a precision of 1 mm. Weighing equipment was calibrated daily with standard weights, and refresher training was conducted periodically on data collection methods and anthropometric measurements. At the 54 month follow-up, body weight was recorded to the nearest 0.1 kg with a digital scale  and with the child in light clothing and bare feet. The scale was checked on each study day with a standard 20 kg weight. After removing shoes, height was measured to the nearest 0.1 cm with a daily-calibrated freestanding stadiometer Leicester Height Measure Seca 214, UK). Weight and length/height measurements were converted to weight-for-age, length/height-for-age, and weight-for-length/height Z-scores (SD scores), according to the WHO Multicentre Growth Reference Study child growth standards [4, UK. WeAll socioeconomic information was collected during a home visit at the time of enrollment. This information included household structure and family characteristics such as age of mother, parity, parental education, and employment. Data collectors had at least 10 years of education. Data were collected with structured questionnaires, which included both pre-coded and open ended questions and reviewed once every 2 week by the responsible investigators. Refresher training of the interviewers on methods to collect data and anthropometric measurements were repeated every 3 month.An initial dataset was selected with those having the birth and 54 month measurements. Measurements at 1 and 2 months were excluded from analysis because data were missing for one-third of the cases. The data was interpolated linearly if one, or maximum two, consecutive measurements were missing. For example, if the 4-month weight was missing between the 3- and 5-months measurements, it was imputed from the average of the 3- and 5-month weights. For two consecutive missing values, the values were imputed from the weighted average of the previous and subsequent measurement values. Data imputation was done for missing values for continuous covariates such as weight and length/height and the SD scores were calculated. Finally, the data set included data for 16 anthropometric assessments: birth, and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, 24 and 54 months.The baseline and follow-up characteristics, including socioeconomic status indicators, maternal literacy and education, occupation, maternal nutritional status, age, and parity were compared across intervention groups. All singleton newborns were included in the intention-to-treat analysis. Means and standard deviations (SD) were calculated for continuous variables, and proportions were calculated for categorical variables. Histograms were used to confirm that continuous dependent variables were Gaussian, and the standard deviations of intervention groups were compared to ensure that they were similar. Differences between categorical variables were compared by chi-square tests. T-tests and analysis of variance with post hoc Bonferroni corrections were used to compare group differences. General linear modeling of repeated-measurements ANOVA was used to compare the different supplementation groups, mean height-for-age Z-scores and occurrence of stunting throughout the 54 months follow-up. Stunting prevalence ranged from about 17% (at birth) to 52% (month 21), and the linear response function could be used instead of the logistic response function. Occasion was the within-subject factor and the food and/or micronutrient supplementation group was the between-subject factor. Most statistical analyses were performed with SPSS , while GraphPad  software was used for the graphs. We considered results where p < 0.05 statistically significant and p = 0.05 as marginally significant.et al. manuscript submitted). Losses to follow-up after birth anthropometry, other than mortality, did not differ across the six intervention groups .There were 3267 singleton infants with birth anthropometry born by the 4436 women enrolled into the MINIMat trial. Losses before measurement of birth anthropometry included early fetal loss (n = 347), stillbirth (n = 89), out-migration n = 188), and refusal to participate (n = 129). Birth anthropometry was missing for 358 children and 72 were excluded from analysis of birth anthropometry, as they were twins . After imputation, an average of 15.4 (± 3.78) anthropometric measurements were available for analysis, and the number of measurements per child did not differ across the intervention groups (p = 0.60). For the longitudinal analysis, anthropometric information was available for 1634 children. The analyzed and non-analyzed groups were compared, and there was no evidence of selective dropout (p = 0.19) among the six intervention groups (data not shown). By design, early invitation to food supplementation consumed more packages of supplement than usual invitation to food supplementation (mean difference 30 packages from enrolment to wk 30 examination). On average the participants had taken 77 micronutrient capsules from wk 14 to 30. MMS groups had taken a few less capsules than the other groups (p = 0.025) and the early invitation food group also had a few less micronutrient capsules on the average (p = 0.015) (data not shown).There were no significant differences in the characteristics of mothers and households across the different intervention groups Table . The aveAnthropometric indices at birth were low, gradually worsened up to 18 months, and then improved slightly at 54 month of age. For example, at birth, height-for-age Z-score (HAZ) was mean -0.91 and proportion of stunting was 17%; at 18 months, HAZ was mean -1.94 and stunting 49%; and, at 54 months mean was -1.49 and stunting 31%. There was no significant difference in mean weight-for-age, weight-for-height, or height-for-age (n = 1634) in the cross-sectional analyses across intervention groups Table . FurtherA longitudinal analysis of linear growth was performed using repeated-measures analysis Table . There wWeight-for-age and weight-for height Z-scores as continuous and dichotomous variables were also compared in repeated-measures analysis, and we found neither any interactions between food and micronutrient groups nor any significant main effects on growth (data not shown).The effect of early vs. usual invitation to food supplementation on frequency of stunting was significantly shown for boys , but not for girls , Figure The analysis was also stratified by maternal BMI using the median BMI 19.7 as cut-off level. Among mothers with higher BMI (BMI > = 19.7) stunting was less frequent  in early invitation food group , while this was not significant among mothers in the lowest half of the BMI distribution . The increased occurrence of stunting in the MMS group (in comparison with Fe60F) was found only among mothers in the lowest half of the BMI distribution , Figure In this large randomized MINIMat trial in Bangladesh, children born to mothers receiving an early invitation to food supplementation were less likely to be stunted during the first five years of life. Children born to mothers who received multiple micronutrients rather than the standard iron-folate program, however, had a higher occurrence of stunting during the first five years of life: these effects were primarily in boys.In MINIMat trial, the interviewers were unaware of the intervention groups and the mothers were unaware of their micronutrient supplement. By design, there was a difference in the number of food supplements of 18,000 kcal (30 packets) between the early and usual invitation groups. Allocation to food and micronutrient supplementation was according to MINIMat protocol, and masking was maintained for the micronutrient supplementation groups until the completion of intention-to-treat analysis. There was no difference among the groups in loss to follow-up, although there were minor differences in the number of capsules consumed across the groups. The pregnant mothers received almost 4 months of daily micronutrient supplements.Sex differences in fetal growth are reported , but theEarly food supplementation had an effect on early child growth. In Indonesia, maternal nutrition during pregnancy influenced the growth of offspring in early childhood, and high-energy supplementation produced less stunting throughout the first five years . In thisThe causes of stunting are complex and involve poor quality diet, breastfeeding practices, long-term burden of infectious disease morbidity, and chronic adverse environmental exposure; these factors are difficult to measure. The biological basis for the effects of multiple micronutrient supplements and the role of specific micronutrients in human biology is a complex mechanism ,32. MultThe World Health Organization (WHO) recommends iron and folic acid supplementation to women during pregnancy as part of routine antenatal care. The effect of multiple-micronutrient supplementation with iron and folic acid supplementation was assessed, and we observed no significant added benefit of multiple-micronutrient supplements over iron and folic acid supplementation for post-natal growth. There is a lack published data presenting similar findings of more stunted children in a maternal MMS group than in iron+folic acid supplementation groups. This study was conducted in a low-income country, and further studies in different populations are needed to assess the consistency of the findings. The study was not designed to examine the potential risks of excess micronutrient supplementation or to examine potential adverse interactions between the micronutrients, which need addressing in future studies. As an early start to supplementation during pregnancy is beneficial for early child growth, future food supplementation during pregnancy should be implemented at the beginning of pregnancy. The randomized, double-blind design was a major strength of this study. As a placebo group could not be added due to ethical reasons, it is not appropriate to conclude micronutrient supplementation does not have any beneficial effect on birth outcome or early child growth.It may be pertinent to ask the effect size of different nutrition interventions that improve child growth, particularly stunting. Our data suggest that early food supplementation during pregnancy had a positive impact on preventing stunting. Effects of nutrition-related interventions on stunting in 36 country analysis showed that nutrition education and counseling of complementary feeding and other supportive strategies could reduce stunting by 19.8% at 12 months, 17.2% at 24 months, and 15.0% at 36 months . In our Early food supplementation in pregnancy reduced the occurrence of stunting during 0-54 months in boys, but not in girls, and prenatal MMS increased the proportion of stunting in boys. The results of this study indicate food supplementation to women during early pregnancy reduces early childhood malnutrition, especially stunting. These effects on postnatal growth suggest programming effects in early fetal life.The authors declare that they have no competing interests.LÅP, IK and AIK: study design; LÅP, SEA, and EAF: designed the MINIMat study; AIK, KÅM, DSA, MU, SEA, and LÅP: supervised data collection; AIK, LÅP, and EAF: statistical analysis; LÅP: supervised data analysis and interpretation; AIK: writing of first draft of the manuscript. All authors contributed to subsequent discussions and revisions. All authors read and approved the final manuscript.


Background. Increased childhood weight gain has been associated with later adiposity. Whether excess early postnatal weight gain plays a role in childhood abdominal fat is unknown. Design. In the ongoing Wheezing Illnesses Study Leidsche Rijn (WHISTLER), birth cohort weight and length from birth to age 3 months were obtained. In the first 316 five-year-olds, intra-abdominal and subcutaneous fat were measured ultrasonographically. Individual weight and length gain rates were assessed in each child. Internal Z-scores of weight for length gain (WLG) were calculated. Multiple imputation was used to deal with missing covariates. Results. Per-1-unit increase in Z-score WLG from birth to 3 months, BMI, waist circumference, and subcutaneous fat were significantly higher; 0.51 kg/m2, 0.84 cm, and 0.50 mm, respectively. After multiple imputation, a trend towards significance was observed for intra-abdominal fat as well (0.51 mm/SD). In the associations with 5-year adiposity, no interaction between postnatal Z-score WLG and birth size was found. Conclusion. Excess early postnatal weight gain is associated with increased general and central adiposity, characterized by more subcutaneous and likely more intra-abdominal fat at 5 years of age. Obesity is one of the major cardiovascular disease (CVD) risk factors and is a common health problem both in adults and in children . Not onl Growth patterns in early life have been suggested to be important for later fat distribution. Several studies determined an association between weight gain in the first 2-3 years of life and central adiposity, mostly assessed by larger waist circumference, in both adulthood  and earl We set out to measure intra-abdominal and subcutaneous fat ultrasonographically in healthy 5-year-old children, to study whether differences in growth in the first 3 postnatal months, in particular increased weight gain accounting for length gain, are associated with increased central adiposity in early childhood. n = 1124 on April 26th, 2011) were invited according to the last-known telephone number and address, for follow-up measurements. 215/1124 (19%) subjects were lost to follow-up, due to incorrect telephone numbers and addresses, and 54/1124 (5%) were not yet contacted. Of the remaining 855 subjects, 285 (33%) declined to take part and 570 (67%) were willing to participate of whom 517 were measured before April 26th 2011. Abdominal ultrasonography was performed and intra-abdominal fat and subcutaneous fat were measured successfully in 434/517 (84%) and 463/517 (90%) subjects, respectively. In the remaining participants the focus was solely on respiratory measures. Complete data on postnatal weight gain relative to length gain and abdominal fat distribution were available for 360/517 children (70%), and in addition on birth size for 316/517 (61%). An overview of the study population is presented in The present study is part of the Wheezing Illnesses Study LEidsche Rijn (WHISTLER) study, an ongoing population-based birth cohort on determinants of wheezing illnesses, initiated in 2001 . HealthyWHISTLER-Cardio was approved by the paediatric Medical Ethical Committee of the University Medical Center Utrecht. Written informed parental consent was obtained.Parents visited the clinic when their offspring was approximately four weeks of age for lung function measurements, not further described here . Birth w2) was calculated. In addition, intra-abdominal and subcutaneous fat were measured using ultrasound according to a previously described procedure [Methods of the follow-up measurements have been described previously . Childrerocedure , 27 withIn addition, information on child and parental characteristics, like smoking habits among others, with respect to the previous years was collected by questionnaires.We used weight gain rate, length gain rate, and particularly weight gain rate adjusted for length gain rate as measures of growth in the first 3 postnatal months. These measures were studied separately, as length gain might reflect lean mass accumulation, while weight gain, and particularly weight gain accounting for length gain, might reflect fat mass accumulation.Z-scores internal to our study population for weight gain, conditioned on length gain, by using the standardized residuals from the linear regression model with weight gain as the dependent variable and length gain as the independent variable. A Z-score of +1 SD WLG indicates that the weight gain of a certain child is one standard deviation larger than the mean weight gain in the population based on the length gain of that child. Furthermore, size at birth was assessed by calculating internal Z-scores for birth weight, adjusted for birth length, gestational, age and gender.For each child with at least two anthropometric measurements available in the first 3 postnatal months, weight gain and length gain rates were estimated using the monthly anthropometrics, to reduce an effect of measurement error and individual variation over time (regression to the mean). Since the number of weight and length measurements (median count: 3) and the age at which these were measured differed per child, the association between age, and both weight and length was assessed using linear mixed modelling, allowing for individual variation in birth weight or height and growth rate. Subsequently, to obtain weight gain rates for each child individually, linear regression modelling on the predicted weight and length values of the linear mixed model was performed stratified by child. The same steps were taken to assess individual length gain rates. Because of the small time window, weight and length gain rate were assumed constant in the first 3 postnatal months. WLG was assessed by deriving Z-score WLG. Differences by tertiles of Z-score WLG were tested using analysis of variance, or Kruskal Wallis test in case of skewed data, for continuous variables and Chi-squared tests or Fisher's exact tests for frequencies  and corresponding 2 (interquartile range (IQR): 14.3–16.1), 52.5 mm (IQR: 50.5–54.8), 6.3 mm (IQR: 5.0–7.9), and 36.4 ± 6.5 mm, respectively. Children with higher postnatal WLG were relatively thin at birth. No differences in infant feeding type and parental characteristics were determined across growth tertiles, except for parental smoking habits and maternal gestational diabetes: parents of children with lower WLG smoked more often in the 5 years after birth and all three infants of the mothers who have had gestational diabetes belonged to the highest Z-score WLG tertile. Postnatal WLG was positively associated with weight and height of the children at the follow-up visit at 5 years of age . Again, no interaction between size at birth and postnatal growth on body fat distribution was present. After multiple imputation of birth length, to have complete data on size at birth for all 360 children in which an ultrasound was successfully performed and growth data was available, the associations between postnatal WLG and BMI, waist circumference, and subcutaneous fat strengthened. Regarding intra-abdominal fat, a trend towards significance was observed: per 1 unit increase in The present study contributes to current knowledge in showing that excess weight gain relative to length gain in the first 3 months after birth is associated with central adiposity. Strong associations with BMI, waist circumference, and abdominal subcutaneous fat were observed. In addition, these results suggest an increase in intra-abdominal fat as well.Z-scores for weight gain rate conditioned on length gain rate, as references values for comparable Z-scores are not available. Since data on fetal growth have not been collected as part of the present study, birth size was used as proxy. Birth weight is determined by gestational age, growth potential, and intrauterine environment. Therefore, birth weight adjusted for birth length and gestational age might be a better indicator of intrauterine exposures. Besides age, gender, current height, and sonographer, we did not consider other factors, like parental BMI, infant feeding, tobacco smoke exposure among others as confounders, as these factors might precede the studied association and might therefore be in the causal pathway. Whether or not to adjust for achieved height is topic of discussion [ Some remarks have to be made. WHISTLER is a population-based cohort and families with a healthier lifestyle might be more willing to participate. Moreover, children with follow-up measurements tended to have a higher birth weight and were larger at birth (data not shown) compared to those lost to follow-up. Since data on birth size was not available for all children, birth length was imputed using multiple imputation. Missing birth length was associated with lower gestational age and the imputed birth lengths were smaller than those nonimputed. Multiple imputation strengthened the observed associations between postnatal growth and adiposity measures in young childhood, indicating an effect of selection on the results from the complete case analyses. We consider our study population to be representative of a healthy pediatric population, as the mean weights and lengths in infancy are in line with the WHO growth charts . We deriscussion , 30. Howscussion , 20. Sinscussion , ultrasoscussion . When thscussion , 27. Morscussion , 33 and scussion , 35 and scussion . Althougscussion , it shouPreviously, associations between weight gain in the first 2 years of life and childhood BMI , 9, waisThe observed differences in adiposity associated with excess weight gain in early infancy might be explained by increased accumulation of fat mass in the early postnatal period. Weight gain for length gain in the first months after birth primarily reflects accumulation of fat mass, since fat mass increases from approximately 13% of total body weight to 31% between 0.5 and 3–6 months of age . AccordiWe found some evidence for an association between excess early postnatal weight gain and intra-abdominal fat. Besides these differences in quantity of intra-abdominal fat, the previously observed adverse metabolic consequences later in life associated with increased postnatal growth , might bWhile the observed differences in fat distribution are relatively small from an individual perspective, they are relevant from a population perspective. However, we should be careful in extrapolating these finding to growth interventions, as rapid postnatal growth might have benefits as well –41. MoreIn conclusion, variations in postnatal growth are associated with abdominal fat in early childhood. Over the whole range of birth size, excess early postnatal weight gain relative to length gain is associated with increased general and central adiposity, characterized by higher BMI, larger waist circumference, more abdominal subcutaneous fat, and likely more intra-abdominal fat at 5 years of age.