Early fetal hypoxia leads to growth restriction and myocardial thinning

Abstract

Hypoxia is necessary for fetal development; however, excess hypoxia is detrimental. Hypoxia has been extensively studied in the near-term fetus, but less is known about earlier fetal effects. The purpose of this study was to determine the window of vulnerability to severe hypoxia, what organ system(s) is most sensitive, and why hypoxic fetuses die. We induced hypoxia by reducing maternal-inspired O2 from 21% to 8%, which decreased fetal tissue oxygenation assessed by pimonidazole binding. The mouse fetus was most vulnerable in midgestation: 24 h of hypoxia killed 89% of embryonic day 13.5 (E13.5) fetuses, but only 5% of E11.5 and 51% of E17.5 fetuses. Sublethal hypoxia at E12.5 caused growth restriction, reducing fetal weight by 26% and protein by 45%. Hypoxia induced HIF-1 target genes, including vascular endothelial growth factor (Vegf), erythropoietin, glucose transporter-1 and insulin-like growth factor binding protein-1 (Igfbp-1), which has been implicated in human intrauterine growth restriction (IUGR). Hypoxia severely compromised the cardiovascular system. Signs of heart failure, including loss of yolk sac circulation, hemorrhage, and edema, were caused by 18-24 h of hypoxia. Hypoxia induced ventricular dilation and myocardial hypoplasia, decreasing ventricular tissue by 50% and proliferation by 21% in vivo and by 40% in isolated cultured hearts. Epicardial detachment was the first sign of hypoxic damage in the heart, although expression of epicardially derived mitogens, such as FGF2, FGF9, and Wnt9b was not reduced. We propose that hypoxia compromises the fetus through myocardial hypoplasia and reduced heart rate.

Fig. 1.

Fetal tissue Po₂ reflects maternal oxygenation. Representative images of embryonic day 12.5 (E12.5) fetuses labeled with a hypoxia-reactive dye, pimonidazole (120 μg/g), and exposed for 3 h to hyperoxia (A; hyperoxia, 95% O₂), normoxia (B; normoxia, 21% O₂), or hypoxia (C; hypoxia, 8% O₂) are shown. As maternal Fi_O2 decreased, dye binding increased, indicating reduced fetal tissue Po₂. These data indicate that fetal Po₂ can be manipulated by changing maternal oxygenation. All pimonidazole sections were stained at the same time. H, heart; L, liver; VB, vertebral bodies; DRG, dorsal root ganglia; SC spinal cord. Scale bar = 600 μm. The experiment was repeated on at least 3 litters per condition with at least 1 fetus examined per litter.

Fig. 2.

Sensitivity to hypoxia peaks at E13.5 and is proportional to the length of time in hypoxia. A: pregnant mice were placed in 8% oxygen for 24 h ending on the gestational day indicated in the figure. Fetuses were dissected and determined to be dead or alive by the absence or presence of a heart beat. ^Data vs. E11.5, P < 0.05. *Data vs. E13.5, P < 0.05. Number of alive/total fetuses for each age group was 39/41 for E11.5, 55/77 for E12.5, 8/71 for E13.5, 14/52 for E15.5, and 23/47 for E17.5. Four litters were used at E11.5 and E17.5; 7 litters were used at E13.5, 6 litters at E12.5, and 5 litters at E15.5. B: pregnant mice were placed in 8% oxygen for varying lengths of time prior to E13.5 so that hypoxia was complete at E13.5. All fetuses were then dissected and determined to be dead or alive. ^Data vs. 0 h of hypoxia, P < 0.05. *Data vs. 24 h of hypoxia, P < 0.05. Number of alive/total fetuses was 46/46 for 0 h, 32/35 for 12 h, 19/33 for 18 h, and 8/70 for 24 h. In B, 3 litters were used at 12 and 18 h; 4 litters were used at 0 h and 7 at 24 h. For both A and B, N = a litter, with all fetuses in a litter counted.

Fig. 3.

Hypoxia causes growth restriction. Pregnant dams were subjected to 8% O₂ for varying lengths of time prior to E12.5, so that hypoxia was complete at E12.5. A: hypoxia caused a time-dependent reduction in fetal weight among live fetuses. *Data vs. 0 h of hypoxia (i.e., normoxic E12.5 fetuses), P < 0.05. N = mean fetal weight of 5 litters at 0 h, 6 litters at 12 h, and 4 litters at 24 h, using at least 3 fetuses per litter. B: hypoxia reduces fetal protein content. *Data vs. 0 h of hypoxia, P < 0.05. N = mean fetal protein content of 3 litters at 0 h, 3 litters at 12 h, and 3 litters at 24 h, using at least 3 fetuses from each litter.

Fig. 4.

Maternal hypoxia causes signs of congestive heart failure. Representative images of normoxic (A and B), live hypoxic (C and D), and dead hypoxic (E and F) E12.5 fetuses. Hypoxic litters were housed in 8% O₂ for 24 h. A, C, and E: acutely dissected fetuses with yolk sac membranes intact. Arrows point to yolk sac blood vessels. P, placenta. H, fetal head. Scale bars in A, C, and E = 2.5 mm. B, D, and F: close-up of hearts demonstrates cardiac congestion with hypoxia. A, atrium; V, ventricle; L, liver. Scale bars in B, D, and F = 1 mm. Hue and saturation were adjusted in A to account for different lighting conditions than were used in B and C. These observations were made in at least 20 litters in each condition.

Fig. 5.

Hypoxia causes ventricular thinning and dilation. A–C: representative images of hematoxylin and eosin-stained transverse sections from hearts of live normoxic fetuses at E11.5 (A) and E12.5 (B), and from live hypoxic E12.5 (C) fetuses (8% O₂ for 18 h ending at E12.5). As the heart matures, the compact myocardium (orange circles) thickens and the epicardium (black arrow heads) adheres to the myocardium. After 18 h of hypoxia, the heart displays myocardial and septal thinning and a detached epicardium. Scale bars = 200 μm. D: ventricular tissue area reported as a fraction of normoxic tissue area. E: mean ventricular wall thickness. F: hypoxia leads to ventricular dilation, as measured by the fraction of total ventricular area occupied by ventricular lumen. The stage of contraction was not normalized by the fixation process. In D–F, 0 h of hypoxia refers to E12.5 fetuses that did not experience hypoxia (normoxic). *Data vs. 0 h, P < 0.05. N = 3, 3, and 4 fetuses (each from a different litter) for 0, 12, and 18 h of hypoxia, respectively, in both D and E. N = 5, 4, and 3 fetuses (each from a different litter) for 0, 12, and 18 h of hypoxia, respectively, in F.

Fig. 6.

The epicardium is particularly affected by hypoxia. A: representative image of hematoxylin and eosin-stained sagittal section of normoxic E12.5 heart. B: pimonidazole binding of an adjacent section reveals areas of constitutive hypoxia in the heart of a normoxic fetus. C: pimonidazole binding increases after 3 h of maternal hypoxia ending at E12.5 (8% O₂). Arrows in B and C point to pimonidazole binding in the outflow tract (OFT) myocardium. Pimonidazole binding in epicardium (circled) in normoxic (D) and hypoxic (E) E12.5 fetuses shows that hypoxia preferentially increased dye binding in epicardial cells and to a lesser extent in endocardial cells (green arrow). The red arrow points to red blood cells (RBC). Hematoxylin and eosin-stained sections of ventricular wall of normoxic (F) and hypoxic (G) E12.5 fetuses show hypoxia-induced detachment of the epicardium (black arrows) from the myocardium. Scale bars in A–C = 300 μm, D = 50 μm, E–G = 25 μm. These observations were made in at least 3 litters.

Fig. 7.

Hypoxia leads to reduced proliferation in vivo and in vitro in the myocardium, but not in brain or spinal cord. Representative images of in vivo (+)-5-bromo-2′-deoxyuridine (BrdU) labeling in ventricular myocardium of E12.5 fetuses after 0 h (A) or 24 h in 8% O₂ (i.e., from E11.5 to E12.5; B). Note the myocardial thinning in B. IVS, interventricular septum. Scale bars = 50 μm. C: hypoxia reduced the fraction of proliferating cells as determined by BrdU incorporation in the heart (left), but had no effect in the brain (middle) or spinal cord (right). *Data vs. 0 h in the same tissue type, P < 0.05. For heart, n = 7 fetuses at 0 h, 8 at 12 h, and 6 at 24 h. For spinal cord, n = 3 fetuses for each time point. For brain, n = 4 for 0 h and 5 for 24 h. One fetus was used per litter. D: in vitro tritiated thymidine incorporation into isolated hearts determined after 24 h under 21% O₂ (normoxia) or 2% O₂ (hypoxic) conditions in the presence of 10% or 0.5% fetal clonal serum. Hearts were explanted at E11.5, divided between conditions, and cultured for 21 h before being labeled with thymidine for 3 h. N = number of hearts: 21% O₂-10% serum = 18; 21% O₂-0.5% serum = 22; 2% O₂-10% serum = 19; 2% O₂-0.5% serum = 22. Average incorporation per heart ranged from 1,040 cpm to 2,510 cpm depending on the culture conditions. *Data vs. 21% O₂ in the same serum concentration, P < 0.05. Incorporation at 21% O₂ was set to 100%.