Chinthika Piyasena

Association between preterm brain injury and exposure to chorioamnionitis during fetal life.

Preterm infants are susceptible to inflammation-induced white matter injury but the exposures that lead to this are uncertain. Histologic chorioamnionitis (HCA) reflects intrauterine inflammation, can trigger a fetal inflammatory response, and is closely associated with premature birth. In a cohort of 90 preterm infants with detailed placental histology and neonatal brain magnetic resonance imaging (MRI) data at term equivalent age, we used Tract-based Spatial Statistics (TBSS) to perform voxel-wise statistical comparison of fractional anisotropy (FA) data and computational morphometry analysis to compute the volumes of whole brain, tissue compartments and cerebrospinal fluid, to test the hypothesis that HCA is an independent antenatal risk factor for preterm brain injury. Twenty-six (29%) infants had HCA and this was associated with decreased FA in the genu, cingulum cingulate gyri, centrum semiovale, inferior longitudinal fasciculi, limbs of the internal capsule, external capsule and cerebellum (p < 0.05, corrected), independent of degree of prematurity, bronchopulmonary dysplasia and postnatal sepsis. This suggests that diffuse white matter injury begins in utero for a significant proportion of preterm infants, which focuses attention on the development of methods for detecting fetuses and placentas at risk as a means of reducing preterm brain injury.

Epigenomic profiling of preterm infants reveals DNA methylation differences at sites associated with neural function.

DNA methylation (DNAm) plays a determining role in neural cell fate and provides a molecular link between early-life stress and neuropsychiatric disease. Preterm birth is a profound environmental stressor that is closely associated with alterations in connectivity of neural systems and long-term neuropsychiatric impairment. The aims of this study were to examine the relationship between preterm birth and DNAm, and to investigate factors that contribute to variance in DNAm. DNA was collected from preterm infants (birth<33 weeks gestation) and healthy controls (birth>37 weeks), and a genome-wide analysis of DNAm was performed; diffusion magnetic resonance imaging (dMRI) data were acquired from the preterm group. The major fasciculi were segmented, and fractional anisotropy, mean diffusivity and tract shape were calculated. Principal components (PC) analysis was used to investigate the contribution of MRI features and clinical variables to variance in DNAm. Differential methylation was found within 25 gene bodies and 58 promoters of protein-coding genes in preterm infants compared with controls; 10 of these have neural functions. Differences detected in the array were validated with pyrosequencing. Ninety-five percent of the variance in DNAm in preterm infants was explained by 23 PCs; corticospinal tract shape associated with 6th PC, and gender and early nutritional exposure associated with the 7th PC. Preterm birth is associated with alterations in the methylome at sites that influence neural development and function. Differential methylation analysis has identified several promising candidate genes for understanding the genetic/epigenetic basis of preterm brain injury.

Tract shape modeling detects changes associated with preterm birth and neuroprotective treatment effects

Preterm birth is associated with altered connectivity of neural circuits. We developed a tract segmentation method that provides measures of tract shape and integrity (probabilistic neighborhood tractography, PNT) from diffusion MRI (dMRI) data to test the hypotheses: 1) preterm birth is associated with alterations in tract topology (R), and tract-averaged mean diffusivity (〈D〉) and fractional anisotropy (FA); 2) neural systems are separable based on tract-averaged dMRI parameters; and 3) PNT can detect neuroprotective treatment effects. dMRI data were collected from 87 preterm infants (mean gestational age 29+1 weeks, range 23+2 –34+6) at term equivalent age and 24 controls (mean gestational age 39+6 weeks). PNT was used to segment eight major fasciculi, characterize topology, and extract tract-averaged 〈D〉 and FA. Tract topology was altered by preterm birth in all tracts except the splenium (p < 0.05, false discovery rate [FDR] corrected). After adjustment for age at scan, tract-averaged 〈D〉 was increased in the genu and splenium, right corticospinal tract (CST) and the left and right inferior longitudinal fasciculi (ILF) in preterm infants compared with controls (p < 0.05, FDR), while tract-averaged FA was decreased in the splenium and left ILF (p < 0.05, FDR). Specific fasciculi were separable based on tract-averaged 〈D〉 and FA values. There was a modest decrease in tract-averaged 〈D〉 in the splenium of preterm infants who had been exposed to antenatal MgSO4 for neuroprotection (p = 0.002). Tract topology is a biomarker of preterm brain injury. The data provide proof of concept that tract-averaged dMRI parameters have utility for evaluating tissue effects of perinatal neuroprotective strategies.

Corrigendum: Dynamic changes in DNA methylation occur during the first year of life in preterm infants [Front Endocrinol, 7, (2016) (158)] doi: 10.3389/fendo.2016.00158

[This corrects the article on p. 158 in vol. 7, PMID: 28018293.].

Diffusion MRI parameters of corpus callosum and corticospinal tract in neonates: Comparison between region-of-interest and whole tract averaged measurements

Purpose Measures of white matter (WM) microstructure inferred from diffusion magnetic resonance imaging (dMRI) are useful for studying brain development. There is uncertainty about agreement between FA and MD values obtained from region-of-interest (ROI) versus whole tract approaches. We investigated agreement between dMRI measures using ROI and Probabilistic Neighbourhood Tractography (PNT) in genu of corpus callosum (gCC) and corticospinal tracts (CST). Materials and Methods 81 neonates underwent 64 direction DTI at term equivalent age. FA and MD values were extracted from a 8 mm3 ROI placed within the gCC, right and left posterior limbs of internal capsule. PNT was used to segment gCC and CSTs to calculate whole tract-averaged FA and MD. Agreement between values obtained by each method was compared using Bland–Altman statistics and Pearsons correlation. Results Across the 3 tracts the mean difference in FA measured by PNT and ROI ranged between 0.13 and 0.17, and the 95% limits of agreement did not include the possibility of no difference. For MD, the mean difference in values obtained from PNT and ROI ranged between 0.101 and 0.184 mm2/s × 10−3 mm2/s: the mean difference in gCC was 0.101 × 10−3 mm2/s with 95% limits of agreement that included the possibility of no difference, but there was significant disagreement in MD values measured in the CSTs. Conclusion Agreement between dMRI measures of neonatal WM microstructure calculated from ROI and whole tract averaged methods is weak. ROI approaches may not provide sufficient representation of tract microstructure at the level of neural systems in newborns.

Alterations in glucose concentrations affect DNA methylation at Lrg1 in an ex vivo rat cortical slice model of preterm brain injury

Preterm birth affects 5–18% of all babies and is associated with neurodevelopmental impairment and increased neuropsychiatric disease risk. Although preterm birth associates with differential DNA methylation at neurodevelopmental genes in buccal DNA, including leucine‐rich alpha‐2‐glycoprotein 1 (LRG1), it is not known whether these differences also occur in the brain, or whether they persist. Thus, there is a need for animal models or in vitro systems in which to undertake longitudinal and mechanistic studies. We used a combination of in vivo rat studies and ex vivo experiments in rat cortical slices to explore their utility in modelling the human preterm brain. We identified temporal changes in DNA methylation at LRG1 in human buccal DNA over the first year of life and found persistent differences in LRG1 methylation between preterm and term infants at 1 year. These developmental changes also occurred in rat brains in vivo, alongside changes in global DNA hydroxymethylation and expression of the ten‐eleven translocation (Tet1) enzyme, and were reproducible in ex vivo rat cortical slices. On the basis of the observation that neonatal glucose homeostasis can modify neurodevelopmental outcome, we studied whether glucose concentration affects Lrg1 methylation using cortical slices. Culture of slices in lower glucose concentration was associated with lower Lrg1 methylation, lower global 5hmC and Tet1 expression. Our results suggest that ex vivo organotypic cultures may be useful in the study of biological and environmental influences on the epigenome and that perturbations during early life including glucose concentration can affect methylation at specific genes implicated in neurodevelopment.

PC.12 Gender-specific effects on the development of white matter tracts in preterm babies

Introduction Diffusion tensor imaging (DTI) may provide insights into cerebral changes associated with preterm birth by supplying biomarkers of white matter microstructure in tracts of interest (TOI). We used a tractography-based segmentation method, probabilistic neighbourhood tractography (PNT),1,2 to study gender-specific effects in developing white matter tracts in preterm infants. Methods 49 preterm infants underwent DTI (3T MRI) comprising 11 T2- and 64 diffusion-weighted (b = 750s/mm2) single-shot spin echo EPI volumes with 2 mm isotropic voxels at term-equivalent age. 10 TOIs were identified using PNT (http://www.tractor-mri.org.uk).2 Using a neighbourhood of seed voxels, the seed point that produced the best matching tract to the reference (MNI standard space) was determined. The resulting tractography masks were applied to each subject’s mean diffusivity (D) and fractional anisotropy (FA) maps, which permitted tract-averaged measures for each TOI. Results Tract-averaged FA values of the left cingulum cingulate gyri (CCG) were significantly increased in male infants (p = 0.05), while (D) of the left CCG were significantly increased in female infants (p < 0.04). When the biomarkers were corrected for postmenstrual age at birth and scan, track averaged (D) of the left CCG was significantly increased (p < 0.03) in female infants (1551 ± 285 µm2/s) compared to male infants (1397 ± 858 µm2/s). Abstract PC.12 Figure 1 Maps of left CCG in a preterm infant (a. axial; b. coronal; c. sagittal). Discussion Quantitative DTI measures can be determined in TOIs in preterm brains using PNT. These values are comparable to other studies using tractography methods, and demonstrate increased diffusivities and reduced FA in CCG of female preterm infants compared with male equivalents. References Clayden JD, et al. IEEE Trans Med Imaging 2007;26:1555–1561 Clayden JD, et al. J Stat Software 2011;44(8):1–18

355 Fetal Growth is Associated with Altered Expression of Imprinted Genes in the Placenta

Background and Aims Both low and high birthweight is associated with adverse health outcomes throughout life. Altered expression of imprinted genes which regulate fetal and placental growth may be one mechanism linking the environment and later disease risk. We have studied the expression of candidate imprinted genes in placenta with respect to anthropometric parameters at birth. Methods 58 term placentas (27 male) were obtained from the Edinburgh Reproductive Tissue BioBank. Pregnancies complicated by congenital abnormalities or diabetes were excluded. Gene expression was analysed using real-time PCR. Results Median birthweight was 3900g (interquartile range: 2949–4340g). Insulin-like growth factor 2 (IGF2) mRNA levels correlated positively with standard deviation scores for birthweight (Spearman’s rho=0.335, p=0.005), head circumference (Spearman’s rho=0.424, p=0.001) and length (Spearman’s rho=0.259, p=0.041). Growth factor receptor-bound protein 10 (GRB10) mRNA levels correlated negatively with birthweight standard deviation score (Spearman’s rho= –0.221, p=0.048). The expression of two other imprinted genes, PHLDA2 and ZIM2 showed no relation to size at birth. Conclusion Both IGF2 and GRB10 are imprinted in the placenta and impact on fetal and placental growth. IGF2 is paternally imprinted and increased expression is implicated in overgrowth disorders; in contrast, GRB10 is maternally imprinted in trophoblasts and disruption in mice leads to overgrowth. Additionally, GRB10 has recently been identified as having a role in insulin signaling. As genomic imprinting is under epigenetic regulation, these targets are strong candidates for exploration of environmentally influenced non-Mendelian effects on fetal size and developmental programming.