Fetal metabolic adaptations to cardiovascular stress in twin-twin transfusion syndrome

Abstract

Monochorionic-diamniotic twin pregnancies are susceptible to unique complications arising from a single placenta shared by two fetuses. Twin-twin transfusion syndrome (TTTS) is a constellation of disturbances caused by unequal blood flow within the shared placenta giving rise to a major hemodynamic imbalance between the twins. Here, we applied TTTS as a model to uncover fetal metabolic adaptations to cardiovascular stress. We compared untargeted metabolomic analyses of amniotic fluid samples from severe TTTS cases vs. singleton controls. Amniotic fluid metabolites demonstrated alterations in fatty acid, glucose, and steroid hormone metabolism in TTTS. Among TTTS cases, unsupervised principal component analysis revealed two distinct clusters of disease defined by levels of glucose metabolites, amino acids, urea, and redox status. Our results suggest that the human fetal heart can adapt to hemodynamic stress by modulating its glucose metabolism and identify potential differences in the ability of individual fetuses to respond to cardiovascular stress.

Keywords: Biology of human development; Cardiovascular medicine; Developmental anatomy; Health sciences.

Graphical abstract

Figure 1

Untargeted amniotic fluid metabolomics identifies metabolic changes in TTTS fetuses with cardiovascular stress (A) Overview of study groups. (B) Stage III TTTS is defined by abnormal Doppler studies of the umbilical artery, ductus venosus, or umbilical vein. Representative images of normal and abnormal Doppler patterns from TTTS cases included in the study are shown. IVC, inferior vena cava. (C) Dilution of amniotic fluid protein in TTTS recipients due to polyhydramnios. Mean (SD), two-tailed unpaired t test, ∗∗∗p ≤ 0.001. (D) Principal component analysis (PCA) of amniotic fluid metabolomics data using top 10% most variable metabolites. (E) Volcano plot showing differential metabolites in TTTS vs. control, including enrichment of fatty acids, arachidonic acid metabolites, and steroid hormones. Fold change threshold ≥2, FDR ≤0.001. See also Table S2 and Figure S3.

Figure 2

Distinct clusters of TTTS are defined by glycolytic metabolites, redox stress, and altered nitrogen metabolism (A) PCA of metabolite profiles using top 10% most variable metabolites within TTTS samples. (B) Volcano plot of differential metabolites between TTTS cluster 2 and TTTS cluster 1 showing accumulation of amino acids, carnitines, 8-epi-PGF_2α, ATP and NAD+ metabolites. Fold-change threshold ≥2, FDR ≤0.001. See also Table S4. (C) Schematic overview of glycolysis and fatty acid oxidation pathways. (D–F) Normalized mass spectrometry peak intensities for glucose and glycolytic metabolites: glucose, lactate, and alanine. See also Figures S2A and S2B. (G) Reduced:oxidized glutathione (GSH:GSSG) suggest increased oxidative stress and redox imbalance in TTTS cluster 2 compared with TTTS cluster 1 and control. See also Figures S2C–S2G. (H–J) Normalized mass spectrometry peak intensities for urea cycle intermediates: argininosuccinate, urea, and ornithine. See also Figures S2H–S2J. One-way ANOVA with Tukey’s multiple comparisons test, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 3

Perinatal outcomes by TTTS cluster (A–C) Similar frequency of perinatal death, early preterm birth (prior to 34 weeks of gestation), and preterm premature rupture of membranes between clusters. See also Table S5. (D and E) Similar median gestational age at birth and interval between laser surgery and birth (latency in days). See also Table S5. (F) Amniotic fluid protein levels stratified by cluster (data also shown in Figure 1C). (G) Normalized amniotic fluid NT-proBNP concentration determined by ELISA. (H) Model of fetal metabolic adaptation to cardiac stress. Data are % or median (IQR). Fisher’s exact test or Mann-Whitney test.