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Abnormal fetal cerebral and vascular development in hypoplastic left heart syndrome

Abstract Objective To assess the cerebral and vascular development in fetuses with hypoplastic left heart syndrome (HLHS). Methods Pregnant women carrying fetuses diagnosed with HLHS who decided to interrupt their pregnancies were included in our study. Aortic size and blood flow were assessed based from fetal echocardiography. Immunohistochemical staining was performed in brain sections obtained from pathology in fetuses with HLHS and control fetuses without heart disease. Results Twenty‐seven midgestation fetal HLHS were included (gestational age, 23.3 ± 3.4 weeks). Head circumference z scores were lower in HLHS fetuses. Middle cerebral artery pulsatility index, a measure of cerebrovascular resistance, was inversely correlated with the ascending aortic z score (P < 0.05). Fetuses with HLHS had lower capillary density in the germinal matrix and their capillaries were larger compared with control fetuses with (P < 0.05). The expression of neuronal differentiation marker, FGFR1, and oligodendrocyte precursor, O4, were lower in HLHS brains compared with controls (P < 0.05). Conclusion Our study identified abnormalities of vascular flow and structural brain abnormalities in fetal HLHS associated with impaired neuronal and oligodendrocyte differentiation, as well as cerebral growth impairment, early in gestation. These findings may be related in part to early vascular abnormalities.


| INTRODUCTION
Neurodevelopmental abnormalities (low motor scores, low IQ scores) are common in children with HLHS. 1 Improved survival following surgery has uncovered abnormalities in neurodevelopmental outcomes that manifest in late infancy and early childhood. Although initially thought to be secondary to postnatal and perioperative insult, neuroradiological studies have shown abnormal cerebral structure in utero in association with delayed brain growth in neonates with HLHS. [2][3][4][5][6] Studies have also detected cranial ultrasound abnormalities (cerebral edema, ventriculomegaly) 7 and MRI abnormalities 8

in infants with
HLHS prior to cardiac surgery. Neonates with HLHS have been reported to have smaller head circumference emphasizing that prenatal factors may contribute to the cerebral growth impairment in HLHS. 9 In fetuses with aortic atresia, brain perfusion is dependent on ret- is not known. The aim of our study was to assess cerebral and vascular growth in fetuses with HLHS compared with normal controls.
While structural brain abnormalities and abnormalities in neurodevelopment have been reported in HLHS, our study describes abnormal vascular development that may account for abnormal neuronal and white matter differentiation in the developing fetus with HLHS.

| Statistical analysis
Echocardiographic data obtained from HLHS fetuses was analyzed by converting measurements into z scores using published normative data from a large population of healthy fetuses. 11 Doppler indices were compared between fetuses with and without antegrade aortic flow. Capillary density, capillary area, intensity of CD133 fluorescence, O4 expression, and proportion of VEGF or FGFR1 positive cells in four brain layers (germinal matrix, intermediate layer, subcortex, and cortex) were compared between HLHS and control brains. Student t test or one-way analysis of variance (ANOVA) with post hoc Bonferroni testing was used to determine differences between groups. A P value of <0.05 was considered significant.

| RESULTS
Twenty-seven midgestational fetuses with HLHS (mean gestational age, 23.3 ± 3.4 weeks) were evaluated for cardiac and brain findings at autopsy. Fetal echocardiograms were available in 16 HLHS fetuses What is known about this topic?
• Structural brain abnormalities and abnormalities in neurodevelopment have been reported in HLHS.
What does this study add?
• Our study describes abnormal vascular development that may account for abnormal neuronal and white matter differentiation in the developing fetus with HLHS. Chromosomal abnormalities were detected in five fetuses, 45 XO (n = 2), trisomy 18 (n = 2), and trisomy 21 (n = 1). Mean body weight z score was −0.17 ± 1.02. Mean head circumference z score was −0.57 ± 0.76. Although within the normal range, the lower z scores for head circumference suggests that cerebral growth impairment begins in early gestation, ie, second trimester. Central nervous system findings showed that 6 out of 27 fetuses with HLHS demonstrated structural brain abnormalities, and only one had severe HLHS and only one had chromosomal abnormalities (Table 1). There was no association of structural brain abnormalities with severity of HLHS or concomitant cardiac lesions.
Echocardiographic data were available in a subset of 16 HLHS fetuses. Ascending aortic size was smaller in 11 fetuses with retrograde aortic flow (0.17 ± 0.04 cm; z score, −5.53) compared with five fetuses with antegrade aortic blood flow (0.33 ± 0.07 cm; z score, −0.72) (P = 0.003). MCA pulsatility index, a measure of cerebral vascular resistance, in five fetuses with severe HLHS was 1.71 ± 0.7 with an inverse correlation between the MCA pulsatility index and the ascending aortic z score (r 2 = 0.89, P = 0.015); ie, resistance was higher in those with smaller aortas.
Brain sections from five fetuses with HLHS and from five gesta-

| DISCUSSION
A high proportion of neonates born with complex congenital heart diseases have neurodevelopmental problems prior to surgery raising concern about in utero cerebral injury. [12][13][14] In our study, structural brain abnormalities were seen predominantly in moderate rather than severe HLHS suggesting that the neuronal and vascular maturation abnormalities seen on histopathology in severe HLHS in our study were independent of the structural brain abnormalities.
The hypothesis is that in utero hypoxia or altered cerebrovascular, hemodynamics leads to delayed brain growth and maturation. [15][16][17][18][19] This is a particular concern in HLHS fetuses in whom cerebral blood flow and oxygen delivery is often via retrograde flow through the ductus arteriosus due to aortic stenosis or atresia. Fetal auto FIGURE 2 Flow chart of study population. Cardiac and brain anatomy and morphology were obtained from 27 fetuses with hypoplastic left heart syndrome (HLHS) at autopsy including body weight, head circumference, and ascending aortic diameter. Echocardiograms were available from 16 fetal HLHS cases. Immunohistology was focused on fetal brains with severe HLHS (n = 5) and compared with age-matched control fetuses with normal hearts (n = 5) regulatory mechanisms are expected to counteract reduced cerebral oxygen delivery by decreasing cerebrovascular resistance and improving cerebral flow to allow brain growth to occur, ie, brain sparing effect. 20 However, in our study, head circumference in the second trimester was lower in HLHS compared with controls. A smaller ascending aorta was associated with a higher MCA pulsatility index, ie, higher cerebral vascular resistance suggesting that vascular autoregulation may be impaired. This may be related to an immature or underdeveloped cerebral vascular bed, which may contribute to elevated cerebrovascular resistance and prevent a brain sparing effect in HLHS. These findings are consistent with other published reports that suggest impaired brain growth 9,16,18,21,22 including smaller head circumference in midgestation and third trimester in fetal HLHS, 9,16,21 delay in cerebral development and volume growth in the third trimester on fetal MRI, 23 and abnormal cerebral blood flow characteristics in midgestational fetuses with evolving HLHS. 16  The potential mechanism of this impaired autoregulation has not been well studied. We therefore explored fetal brain and vascular pathology to understand the pathogenesis of this dysregulation. In our study, brain capillaries were significantly larger in HLHS vs controls, but the capillary density was reduced in the germinal matrix and the cortical layers of HLHS, and VEGF, a key angiogenic factor necessary for new vessel growth, was not increased. Failure to upregulate VEGF in HLHS as well as in other cyanotic CHD has been reported by us previously 26,27 ; however, the vascular abnormalities identified by us provide a potential mechanism for the inability of the fetal brain in HLHS to activate compensatory pathways of hypoxia and ischemia. This was further reflected in reduced expression of CD133, an early stem cell marker regulated by hypoxia and mitochondrial dysfunction, 28 in the brains of HLHS fetuses. Whether the failure to upregulate hypoxic and angiogenic response in fetal brains is a feature of fetal immaturity or is secondary to an intrinsic or genetic impairment [29][30][31][32] in these pathways remains to be explored.
An important finding was that vascular changes in HLHS fetal brains were associated with markers of impaired neuronal differentiation as seen by reduced FGFR1 expression, as well as reduced oligodendrocyte progenitors, which are important for myelination. This may explain in part the susceptibility to white matter injury observed in HLHS infants undergoing cardiac surgery and highlights the need for neuroprotective strategies especially in those with severe HLHS.

| CONCLUSION
Despite the small sample size, our study uncovers novel findings that may explain the mechanism of failed cerebrovascular autoregulation in fetal HLHS and its impact on grey and white matter development that starts prenatally. Further studies are needed to determine whether early fetal intervention to augment antegrade aortic blood flow can improve vascular development and cerebral growth.

ACKNOWLEDGMENTS
We would like to acknowledge Megan Thompson, Mount Sinai Hospital, for her assistance with retrieval of pathology samples from fetal autopsies for our study.

CONFLICTS OF INTEREST
None declared