Cord blood Advanced Lipoprotein Testing shows an interaction between gestational diabetes and birth- weight: an observational study

Francisco Algaba-Chueca Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Elsa Maymó-Masip Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Mónica Ballesteros Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Albert Guarque Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Olga Freixes Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Núria Amigó Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Sonia Fernández-Veledo Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Joan Vendrell Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII Ana Megia (  amegia.hj23.ics@gencat.cat ) Joan XXIII University Hospital in Tarragona: Hospital Universitari de Tarragona Joan XXIII https://orcid.org/0000-0002-5101-9452


Abstract Background
Abnormal lipid metabolism is observed in gestational diabetes mellitus (GDM) and in neonates with abnormal fetal growth, however, how these alterations speci cally affect the umbilical cord blood lipoprotein pro le is not well understood.

Objective
To assess the impact of GDM on the cord blood lipoprotein pro le across birth-weight categories by using Advanced Lipoprotein Testing.
Methods observational study involving 74 control and 62 GDM pregnant women and their offspring. Newborns were classi ed according to birth-weight as small (n = 39), adequate (n = 50) or large (n = 49) for gestational age (SGA, AGA and LGA, respectively). Two-dimensional diffusion-ordered 1 H-NMR spectroscopy was used to pro le umbilical cord serum lipoproteins. One hundred and three children were available in a two years follow-up study to evaluate associations between cord blood lipid pro le and obesity.

Results
Baseline characteristics of the two groups were similar except for gestational weight gain. The size, lipid content, number and concentration of particles within their subclasses were similar between offspring born to GDM and control mothers. Using two-way analysis of variance, we observed an interaction between GDM and birth-weight categories for IDL-cholesterol content and IDL-and LDL-triglyceride content, and the number of medium VLDL and LDL particles, speci cally in AGA neonates. Small LDL particles were independently associated with offspring obesity at two years.

Conclusions
In this selected cohort, GDM disturbs triglyceride and cholesterol lipoprotein content across birth-weight categories, and AGA neonates born to GDM mothers display a pro le more similar to adults with dyslipidemia and atherosclerosis than to those born to mothers with normal glucose tolerance.

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Fetal growth and development is a particularly vulnerable period in life greatly affected by maternal environment. Prenatal exposure to nutritional stressors has been associated with fetal programming, which can impact both metabolism and physiology and, consequently, predispose to later development of cardiovascular and metabolic diseases, including obesity. (1) In this context, it has been proposed that cardiovascular disease can begin in early in life (2) and that atherosclerosis may originate during the fetal period. (3) Birth-weight is strongly determined by neonatal fat mass and gestational age, and fetal growth disorders can result from impaired maternal and fetal lipid metabolism. In fact, the levels and composition of cord blood lipids, apolipoproteins and lipoproteins are affected by both maternal and fetal factors. (4)(5)(6) Disturbed lipid pro les at birth have been described in small and large for gestational age (SGA and LGA, respectively) neonates. (6)(7)(8)(9) When compared with adequate for gestational age (AGA) peers, SGA neonates show higher levels of triglycerides, triglyceride-enriched very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL), and lower levels of total cholesterol. (6,9,10) By contrast, LGA neonates display higher LDL, HDL and total cholesterol levels than AGA neonates. (8) Diabetic pregnancies are associated with a higher incidence of fetal growth disorders and there is evidence that disturbances in maternal metabolism strongly contribute to these situations.(11) Changes in cord blood lipoprotein concentrations have been reported in mothers with type 1 diabetes mellitus, including an increased cholesterol content of LDL and a decrease in HDL. (12,13) The situation appears more complex in gestational diabetes mellitus (GDM), with some studies showing no differences with normal glucose tolerant mothers and others showing lower HDL-and higher VLDL-and LDL-cholesterol concentrations. (14) Additionally, qualitative changes in HDL remodeling resulting in an altered functionality have been reported in GDM neonates,(15) but it does not seem to affect newborn cholesterol metabolism in both obese and well-controlled GDM mothers.(16) 1 H-nuclear magnetic resonance ( 1 H-NMR)-based analysis of lipoproteins has established that the number of LDL and HDL particles is a more powerful index of cardiovascular risk than classical cholesterol determinations, given the large variability in the amount of cholesterol per particle and in particle size.(17) 1 H-NMR-based tests have also demonstrated the incomplete conversion of VLDL into LDL in diabetes, which results in a higher prevalence of VLDL and small and dense LDL particles. (17) Given the heterogeneity of growth patterns and the inconclusive ndings in the cord blood lipoprotein pro le of infants from GDM mothers, a comprehensive characterization of the main lipoproteins, including the assessment of the size and number of particles, is necessary to identify possible alterations in fetal lipoprotein metabolism and their potential consequences for fetal health. In the present study, we used the Liposcale test, a novel advanced lipoprotein assessment method based on 2D diffusion-ordered 1 H-NMR, (18) in order to examine for correlations between fetal growth disorders and differences in umbilical cord blood lipoprotein pro le. Additionally, we want to explore the potential association with offspring outcomes, including obesity, at two years of age.
according to the formulas: Pre-pregnancy BMI = Pre-pregnancy Weight (kg)/Height (m) 2 and GWG = Final weight-Pre-pregnancy weight.
Infant data included sex, gestational age, way of delivery and anthropometry. Neonatal length and weight were measured after delivery using a measuring board to the nearest 0.1 cm and a calibrated scale to the nearest 10 g. Ponderal index (PI) was calculated by the formula: PI = Birth-weight (g)/length (cm) 3 . Suprailiac skinfold thickness was measured within the rst 48 hours of life and was used to calculate the fat mass percentage. (21) Neonates were classi ed according to gestational age-and sex-speci c growth charts of the World Health Organization (WHO). (22) Infants were considered LGA if the birth-weight was over the 90th percentile while SGA if the birth-weight was under the 10th percentile. The remainders were considered AGA. The distribution of neonates according to birth-weight category was: 25 AGA, 25 SGA and 24 LGA in the control group and 25 AGA, 14 SGA and 23 LGA in the GDM group.

Infant growth and child BMI
Height and weight information from birth at two years of age was collected for 103 children. We de ned obesity as a BMI ≥ 85th percentile according to age-and sex-speci c BMI tables of the WHO growth standards. (22) Umbilical cord blood collection Umbilical cord blood was obtained immediately after delivery. Serum was immediately separated by centrifugation, divided into aliquots and stored at -80ºC until analysis.

Laboratory analysis
Maternal fasting serum samples were obtained between the 33rd and 36th weeks of pregnancy to determine glucose, triglycerides, total and HDL-cholesterol in an ADVIA 2400 (Siemens AG, Munich, Germany) autoanalyzer by standard enzymatic methods (23). LDL-cholesterol was calculated using the Friedewald formula. Plasma insulin was determined by immunoassay in an ADVIA Centaur System (Siemens AG. Munich, Germany). This assay shows a cross-reactivity of 0.1% to intact human proinsulin and the primary circulating split form des 31,32 proinsulin. Insulin resistance was estimated using homeostatic model assessment of insulin resistance (HOMA)-IR, as described. (24). 1 H-NMR spectroscopy-based cord blood lipoprotein pro ling Cord blood serum samples were analyzed using the 2D diffusion-ordered 1 H-NMR-based Liposcale test (Biosfer Testlab, Reus, Spain) (18). This technique has shown to be reliable with samples stored at -80º C for more than a decade (25). The test provides information about size, lipid concentration (cholesterol and triglycerides) number of particles, and concentration of particles within their subclasses (large, medium and small) for the main classes of lipoproteins: VLDL, LDL, intermediate-density lipoprotein (IDL) and HDL. 1 H-NMR spectra were recorded on a Bruker Avance III 600 spectrometer (Bruker BioSpin, Rheinstetten, Germany).

Statistical analysis
Statistical signi cance was set at p value < 0.05. Data were analyzed with SPSS software v20.0 (IBM, Armonk, NY) and presented as percentages for categorical variables, mean (SD) for normally-distributed continuous variables, and median (interquartile range) for non-normally distributed variables. Normality of the data was tested with the Kolmogorov-Smirnov test. Non-normally distributed quantitative variables were used after log10 transformation, when required. For comparisons of proportions, differences between groups were analyzed using the chi-square test, while for comparisons of normally-and nonnormally-distributed quantitative variables an unpaired t-test or Mann-Whitney U test was applied. Oneway analysis of variance (ANOVA) was used to test differences among three or more groups and the twoway ANOVA was used to examine potential interactions between GDM and birth-weight categories, and the Bonferroni procedure for post hoc analyses was performed for multiple comparisons. Spearman's rank correlation coe cients were used for the analysis of the relationships between 1 H-NMR-assessed lipoprotein pro le and maternal and offspring metabolic and clinical variables. To control the false discovery rate (FDR), the Benjamini-Hochberg (B-H) procedure was used and only those values signi cant with the B-H correction were considered (26). Logistic regression was applied to investigate the independence of the association between 1 H-NMR-assessed large LDL and small LDL particles, offspring obesity (percentile ≥ 85th = 1), and the normal weight (percentile < 85th = 0), adjusted for potential confounders (GDM, gestational age at delivery, birth-weight, sex, pre-gestational BMI and gestational weight gain)

Results
Clinical characteristics and cord blood 1 H-NMR-based lipoprotein pro le of the studied population Clinical and metabolic characteristics of the two groups are shown in Table 1. Clinical and laboratory parameters and the 1 H-NMR-lipoprotein pro le (Table 2) were similar between GDM and control groups with the exception of GWG, which was signi cantly lower in the GDM group. As well, GWG (7.2 ± 4.4 vs 10.1 ± 5.1 kg; p = 0.023) and fasting glucose (78 ± 8 vs 89 ± 12 mg/dL mg/dL; p < 0.001) were lower in the GDM group of women treated only with diet compared to those who needed insulin therapy. In the 1 HNMR assessed lipoprotein pro le, no difference was observed between the two groups, except for a lower concentration of medium HDL-P in GDM treated with insulin compared with those treated with diet (10.3 ± 1.2 vs 9.6 ± 1.4; p = 0.026).  One hundred and thirty-six neonates born to GDM (N = 62) and control (N = 74) mothers were categorized into three groups based on birth-weight categories according to age-and sex-weight speci c charts (Table 3). As expected, there were signi cant differences in birth-weight, percentage of fat mass and PI across groups, increasing from the SGA to the LGA group. There were also differences between birthweight groups for GWG and cord blood insulin. To note, in the GDM group the type of intervention (only diet or diet plus insulin) was distributed similarly in the three birth-weight groups (p = 0.321)  LGA: large for gestational age; a: P < 0.05 between SGA and AGA; b: P < 0.05 between SGA and LGA; c: P < 0.05 between AGA and LGA.
Lipoprotein particle size and number was also different across the three groups. The number of VLDLparticles (VLDL-P) was highest in the SGA group and lowest in the LGA group, which was consistent with the differences observed among particle sizes (large, medium and small VLDL-P). The number of LDL-P was lower in the SGA group than in the AGA and LGA groups, and the same distribution was observed for large and small LDL-P. The number of HDL-P was highest in the AGA group and showed an inverse U distribution when compared with the LGA and SGA groups. This phenomenon was observed speci cally for small size particles (Table 4).

GDM alters the cord blood lipoprotein pro le across birthweight categories
While the cord blood lipoprotein pro le was similar in offspring born to GDM and control mothers, we found some interactions when we assessed the effect of both birth-weight categories and GDM. AGA neonates born to GDM mothers had higher IDL-cholesterol and -triglyceride content, and LDL-triglyceride content than the SGA and LGA groups, whereas those of control mothers had lower concentrations. The same pattern was also observed with medium VLDL-P and LDL-P, which followed an inversed U distribution ( Fig. 1 and Tables 5 and 6).

Relationship of 1 H-NMR-assessed lipoprotein pro le with clinical and laboratory parameters
We next examined the relationship of 1 H-NMR-based lipoprotein pro le and maternal clinical and laboratory variables and neonatal outcomes, separately in both GDM and control groups. These results are shown in Fig. 2. Only correlations that remain signi cant after FDR correction are marked in the heat map with an asterisk. In the GDM group, maternal LDL-cholesterol was negatively associated with cord blood VLDL-P (total number, large and small particles), cholesterol and triglyceride content in VLDL, IDL and HDL-triglyceride content.
LDL-cholesterol concentrations were negatively associated with birth-weight in the GDM group, while no association was observed in the control group. Cord blood insulin was strongly and positively associated with small and large LDL-P and LDL-cholesterol content in the control group, while a negative relationship was observed with VLDL-P and VLDL-triglyceride content in the GDM offspring.
In both groups, neonatal adiposity was negatively correlated with cord blood VLDL-P, VLDL-and IDLtriglyceride content, and positively associated with cord blood LDL-cholesterol content and LDL-P, being these associations stronger in the control group.

Cord blood 1 H-NMR-lipoprotein pro le is associated with obesity at two years
To assess whether cord blood lipoprotein pro le could be used as a biomarker for offspring outcomes, we explored its potential association with obesity at 2 years of life in a subset of participants. One hundred and three children were available in the follow-up study. No differences in sex, GDM or birth-weight categories distribution, birth-weight, maternal pre-pregnancy BMI, GWG or cord blood lipid pro le were observed between the children lost to follow-up and those that remained in the study. Of the 103 children studied, 78 were normal weight and 25 were obese. Obese children were born to women with higher BMI, had higher birth-weight and were more exposed to GDM during the intrauterine life compared with normal weight children. Additionally, they showed a higher number of cord blood small (352 ±29 vs 326±51 nmol/L; P = 0.019) and large LDL-P (98±11 vs 91±13 nmol/L; P = 0.022). No other differences were observed between the two groups. To further assess the independence of these associations, we performed logistic regression analysis. We found that small LDL-P were associated with infant obesity at two years after adjusting for potential confounders (Table 7), whereas large LDL-P showed a trend (P = 0.058). Statistical Analysis: Logistic regression analysis. *Adjusted for gestational diabetes mellitus, gestational age at delivery, birth-weight, sex, pre-gestational BMI and gestational weight gain. LDL-P: LDL particles. CI: con dence interval.

Discussion
Both GDM and abnormal growth patterns have been associated with long-term adverse outcomes on offspring and changes in the lipoprotein composition have been proposed as potential markers of cardiovascular diseases later in life. In this study, taking advantage of a thorough lipoprotein pro ling by using 1 H-NMR, we show for the rst time that GDM modi es the umbilical cord blood lipoprotein pro le in AGA neonates. In particular, GDM alters IDL-lipoproteins, triglyceride content in LDL, and medium-size VLDL-P and LDL-P in those children. By contrast, GDM offspring belonging to the LGA and SGA groups have a lipoprotein pro le more similar to controls. Besides, we found that cord blood small LDL-P, known to be associated with atherosclerosis development, have a predictive value for later obesity in the offspring.
Both under and overnutrition in utero affects the lipoprotein pro le of neonates. 4-7 SGA neonates are reported to exhibit higher cord blood triglyceride concentrations, (6,9,27)  abnormal hepatic lipase activation also increasing IDL half-life. This scenario is similar to the dyslipidemia associated with diabetes and insulin-resistant states, where an increased generation of IDL, small and dense LDL particles, and triglyceride-enriched HDL particles is observed, (30) and which has been related to an increased atherogenic risk. These ndings lead us to hypothesize that postnatal insulin resistance, which has been described in offspring of GDM women, may be programmed in utero and would be present even in AGA neonates, suggesting that a good glycemic control during pregnancy is not enough to prevent long term complications, as has been previously reported. (32,33) When analysing the lipoprotein pro le according to birth-weight categories ( Table 4), most of the differences observed in the whole group were replicated both, in GDM and controls separately (Tables 5   and 6). These ndings may support an effect of fetal growth accretion instead of the glucose status. In this context, it must be remarked that the evidence of cord blood lipoprotein as biomarkers of later Previous studies exploring the potential relationship between prenatal lipid metabolism and adverse metabolic outcomes in offspring have generated inconsistent results. (37)(38)(39)(40)(41)(42) Following other reports, (43,44) we con rm that GDM, pre-pregnancy BMI, and GWG during pregnancy are all associated with offspring obesity in early life. Furthermore, we found that small LDL-P in cord blood were associated with early obesity, even after controlling for confounding factors. These ndings support the notion that disturbances in the lipoprotein metabolism at birth may have lasting effects independently of birth weight or maternal metabolic status.
There is evidence that an altered fetal lipoprotein pro le is associated with aorta intima thickness in SGA and LGA neonates, (8,27) indicating a potentially increased atherosclerotic risk, already at birth. We are aware that our results cannot establish a direct link between the 1 H-NMR-assessed lipoprotein pro le, observed in GDM-AGA newborns, with an increased atherogenic risk but, however, it offers new clues to understand the high metabolic and cardiovascular risk in the offspring of GDM pregnant women. (45) Long-term studies are guaranteed to con rm whether cord blood 1 H-NMR-based lipoprotein pro ling can be implemented as a useful biomarker of later metabolic diseases beyond two years of age.
One of the main limitations in observational studies is the inability to attribute causation between the observed associations. However, we have considered many confounding variables to mitigate bias in the analysis. Thus, the main prenatal factors were addressed, and the groups were comparable for maternal BMI and birth-weight categories. Otherwise, to reach a su cient sample size in the three birth-weight categories, the SGA and LGA groups were overrepresented, and further population-based studies are needed to determine the role of lipoprotein composition and subfractions in the pathogenesis of metabolic diseases in offspring.
The strengths of this study include a longitudinal birth cohort with almost complete maternal data that establish a temporal relationship between the outcome and the exposure to GDM. The novelty of the lipoprotein assessment, which allows us to identify different fetal metabolic behaviors, is also a big asset in the experimental methods.