Distinct maternal metabolites are associated with obesity and glucose-insulin axis in the first trimester of pregnancy.

BACKGROUND/OBJECTIVES: Obesity in pregnancy associates with changes in the glucose-insulin axis. We hypothesized that these changes affect the maternal metabolome already in the first trimester of human pregnancy and, thus, aimed to identify these metabolites. PATIENTS/METHODS: We performed untargeted metabolomics (HPLC-MS/MS) on maternal serum (n = 181, gestational weeks 4+0-11+6). For further analysis, we included only non-smoking women as assessed by serum cotinine levels (ELISA) (n = 111). In addition to body mass index (BMI) and leptin as measures of obesity and adiposity, we metabolically phenotyped women by their fasting glucose, C-peptide and insulin sensitivity (ISHOMA index). To identify metabolites (outcome) associated with BMI, leptin, glucose, C-peptide and/or ISHOMA (exposures), we used a combination of univariable and multivariable regression analyses with multiple confounders and machine learning methods (Partial Least Squares Discriminant Analysis, Random Forest and Support Vector Machine). Additional statistical tests confirmed robustness of results. Furthermore, we performed network analyses (MoDentify package) to identify sets of correlating metabolites that are coordinately regulated by the exposures. RESULTS: We detected 2449 serum features of which 277 were annotated. After stringent analysis, 15 metabolites associated with at least one exposure (BMI, leptin, glucose, C-peptide, ISHOMA). Among these, palmitoleoyl ethanolamine (POEA), an endocannabinoid-like lipid endogenously synthesized from palmitoleic acid, and N-acetyl-L-alanine were consistently associated with C-peptide in all the analyses (95% CI: 0.10-0.34; effect size: 21%; p < 0.001; 95% CI: 0.04-0.10; effect size: 7%; p < 0.001). In network analysis, most features correlating with palmitoleoyl ethanolamide and N-acetyl-L-alanine and associated with C-peptide, were amino acids or dipeptides (n = 9, 35%), followed by lipids (n = 7, 27%). CONCLUSIONS: We conclude that the metabolome of pregnant women with overweight/obesity is already altered early in pregnancy because of associated changes of C-peptide. Changes of palmitoleoyl ethanolamide concentration in pregnant women with obesity-associated hyperinsulinemia may reflect dysfunctional endocannabinoid-like signalling.

Maternal C-Peptide and Insulin Sensitivity, but Not BMI, Associate with Fatty Acids in the First Trimester of Pregnancy.

Maternal obesity in pregnancy is a pro-inflammatory condition exposing the fetus to an adverse environment. Here, we tested associations of maternal obesity (primary exposures: BMI, leptin) and metabolic parameters (secondary exposures: glucose, C-peptide, and insulin sensitivity) with total serum concentrations of fatty acids in the first trimester of human pregnancy. This cross-sectional study included 123 non-smoking women with singleton pregnancy. In maternal serum, cotinine, leptin, and C-peptide (ELISA), glucose (hexokinase-based test) and fatty acids (gas chromatography) were quantified, and the insulin sensitivity index (ISHOMA) was calculated. Concentrations of fatty acid classes and total fatty acids did not differ between BMI or leptin categories. However, n-3 polyunsaturated fatty acids (PUFA) were decreased in the category with the highest C-peptide concentration (n-3 PUFA: CI -35.82--6.28, p < 0.006) and in the lowest ISHOMA category (n-3 PUFA: CI -36.48--5.61, p < 0.008). In a subcohort, in which fetal sex was determined (RT-qPCR of placental tissue), C-peptide was significantly associated with docosahexaenoic acid (DHA) in mothers bearing a female (n = 46), but not male (n = 37) fetus. In conclusion, pregnant women with high fasting C-peptide and low ISHOMA had decreased n-3 PUFA, and DHA was lower with higher C-peptide only in mothers bearing a female fetus.

Type 1 Diabetes Mellitus and the First Trimester Placenta: Hyperglycemia-Induced Effects on Trophoblast Proliferation, Cell Cycle Regulators, and Invasion.

Type 1 diabetes mellitus (T1DM) is associated with reduced fetal growth in early pregnancy, but a contributing role of the placenta has remained elusive. Thus, we investigated whether T1DM alters placental development in the first trimester. Using a protein array, the level of 60 cell-cycle-related proteins was determined in human first trimester placental tissue (gestational week 5-11) from control (n = 11) and T1DM pregnancies (n = 12). Primary trophoblasts (gestational week 7-12, n = 32) were incubated in the absence (control) or presence of hyperglycemia (25 mM D-glucose) and hyperosmolarity (5.5 mM D-glucose + 19.5 mM D-mannitol). We quantified the number of viable and dead trophoblasts (CASY Counter) and assessed cell cycle distribution (FACS) and trophoblast invasion using a transwell assay. T1DM was associated with a significant (p < 0.05) downregulation of Ki67 (-26%), chk1 (-25%), and p73 (-26%). The number of viable trophoblasts was reduced under hyperglycemia (-23%) and hyperosmolarity (-18%), whereas trophoblast invasion was increased only under hyperglycemia (+6%). Trophoblast cell death and cell cycle distribution remained unaffected. Collectively, our data demonstrate that hyperglycemia decreases trophoblast proliferation as a potential contributing factor to the reduced placental growth in T1DM in vivo.

Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model.

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1-10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (-28%; P < 0.05 and -24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (-61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Maternal Obesity Affects the Glucose-Insulin Axis During the First Trimester of Human Pregnancy.

Background and objective: The maternal glucose-insulin axis is central for metabolic adaptations required for a healthy pregnancy. Metabolic changes in obese mothers in early pregnancy have been scantly described. Here we characterized the glucose-insulin axis in the first trimester of human pregnancy and assessed the effect of maternal obesity and fat mass. Methods: In this cross-sectional study, maternal blood samples (N = 323) were collected during voluntary pregnancy termination (gestational age 4+0-11+6 weeks) after overnight fasting. Smokers (N = 198) were identified by self-report and serum cotinine levels (ELISA). Maternal BMI (kg/m2) and serum leptin (ELISA) were used as proxy measures of obesity and maternal fat mass, respectively. BMI was categorized into under-/normal weight (BMI < 25.0 kg/m2), overweight (BMI 25.0-29.9 kg/m2) and obese (BMI ≥ 30.0 kg/m2), and leptin in tertiles (1st tertile: leptin < 6.80 ng/ml, 2nd tertile: leptin 6.80-12.89 ng/ml, 3rd tertile: leptin > 12.89 ng/ml). ISHOMA insulin sensitivity index was calculated from glucose and C-peptide (ELISA) serum concentrations. Analyses of covariance including multiple confounders were performed to test for differences in glucose, C-peptide and ISHOMA between gestational age periods, BMI and leptin groups. C-peptide and ISHOMA were log-transformed before analyses. Results: At weeks 7-9, fasting glucose and C-peptide levels were lower (P < 0.01 and P < 0.001, respectively) and insulin sensitivity higher (P < 0.001) than at weeks 4-6. Glucose levels were not significantly different between BMI or leptin categories. In contrast, C-peptide increased by 19% (P < 0.01) between the normal weight and the overweight group and by 39% (P < 0.001) between the overweight and obese group. In the leptin groups, C-peptide increased by 25% (P < 0.001) between the 1st and 2nd leptin tertile and by 15% (P < 0.05) between the 2nd and 3rd leptin tertile. ISHOMA decreased with higher BMI and fat mass. ISHOMA decreased by 18% (P < 0.01) between the normal weight and the overweight group and by 30% (P < 0.01) between the overweight and the obese group. In the leptin groups, ISHOMA decreased by 22% (P < 0.001) between the 1st and 2nd leptin tertile and by 14% (P < 0.05) between the 2nd and 3rd leptin tertile. Conclusions: At the group level, fasting glucose, C-peptide and insulin sensitivity dynamically change in the first trimester of human pregnancy. Maternal obesity is associated with higher C-peptide and lower insulin sensitivity at all periods in the first trimester of human pregnancy, while glucose is unaltered. These findings have implications for the timing of early gestational diabetes mellitus risk screening.