Post-mortem Examination of the Nervous System: Fresh versus Fixed.

BACKGROUND: Post-mortem examination of the nervous system is a complex task that culminates in "brain cutting". It relies on expertise in neuroanatomy, clinical neurosciences, neuroimaging and experience in order to recognise the most subtle abnormalities. Like any specialist examination in medicine, it warrants formal training, a standardised approach and optimal conditions. Revelations of aberrant tissue retention practices of a select few pathologists (e.g. Goudge, Liverpool and Alder Hey inquiries) and a motivated sociopolitical climate led some Canadian jurisdictions to impose broad restrictions on tissue retention. This raised concerns that nervous system examinations for diagnosis, education and research were at risk by limiting examinations to the fresh or incompletely fixed state. Professional experience indicates that cutting an unfixed or partly fixed brain is inferior. METHODS: To add objectivity and further insight we sought the expert opinion of a group of qualified specialists. Canadian neuropathologists were surveyed for their opinion on the relative merits of examining brains in the fresh or fully fixed state. RESULTS: A total of 14 out of 46 Canadian neuropathologists responded (30%). In the pervasive opinion of respondents, cutting and sampling a brain prior to full fixation leads to a loss of diagnostic accuracy, biosafety and academic deliverables. CONCLUSIONS: Brain cutting in the fresh state is significantly impaired along multiple dimensions of relevance to a pathologist's professional roles and obligations.

Pervasive polymorphic imprinted methylation in the human placenta.

The maternal and paternal copies of the genome are both required for mammalian development, and this is primarily due to imprinted genes, those that are monoallelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilization. There are a large number of germline DMRs that have not yet been associated with imprinting, and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta and investigated the dynamics of these imprinted DMRs during development in somatic and extraembryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publicly available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. Forty-three known and 101 novel imprinted DMRs were identified in the human placenta by comparing methylation between diandric and digynic triploid conceptions in addition to female and male gametes. Seventy-two novel DMRs showed a pattern consistent with placental-specific imprinting, and this monoallelic methylation was entirely maternal in origin. Strikingly, these DMRs exhibited polymorphic imprinted methylation between placental samples. These data suggest that imprinting in human development is far more extensive and dynamic than previously reported and that the placenta preferentially maintains maternal germline-derived DNA methylation.

A review of congenital lung malformations with a simplified classification system for clinical and research use.

PURPOSE: Congenital lung abnormalities are rare malformations increasingly detected early by prenatal ultrasound. Whether management of these frequently asymptomatic lesions should be surgical or conservative is an unresolved issue. The necessary prospective studies are limited by the absence of a widely accepted practical classification system. Our aim was to develop a simple, clinically relevant system for classifying and studying congenital lung abnormalities. MATERIALS AND METHODS: We based our proposed grouping on a detailed analysis of clinical, radiological, and histological data from well-documented cases, plus an extensive review of the literature. RESULTS: The existence of hybrid lesions and common histological findings suggested a unified embryological mechanism-possibly obstruction of developing airways with distal dysplasia. Malformations could be classified by their anatomical and pathological findings; however, a system based on the prenatal ultrasound plus initial chest X-ray findings had greater clinical relevance: Group 1-Congenital solid/cystic lung malformation, Group 2-Congenital hyperlucent lobe, Group 3-Congenital small lung. CONCLUSIONS: Pathological classification is academically important but is unnecessarily complex for clinical and research use. Our simple radiological-based system allows unambiguous comparison between the results of different studies and also guides the choice of necessary investigations specific to each group.

DNA methylation profiling of placental villi from karyotypically normal miscarriage and recurrent miscarriage.

Miscarriage occurs in 15% of clinical pregnancies. Although chromosomal errors are observed in >50%, causes of karyotypically normal losses are poorly understood. DNA methylation undergoes reprogramming during development and must be appropriately set to maintain a healthy pregnancy. We hypothesize that aberrant DNA methylation may cause karyotypically normal miscarriage, particularly among women experiencing recurrent miscarriage (RM). DNA methylation in first-trimester chorionic villi was assessed in chromosomally normal miscarriages from women with RM (N = 33) or isolated miscarriage (M; N = 21) and elective terminations (TA; N = 16). Differentially methylated candidate loci were identified using the Illumina Infinium HumanMethylation27 BeadChip array. Follow-up bisulfite pyrosequencing at promoter regions showed an increase in methylation in M compared with TA at cytochrome P450, subfamily 1A, polypeptide 2 (CYP1A2; P = 0.002) and RM compared with TA at AXL receptor tyrosine kinase (P = 0.02), and a decrease in RM and M compared with TA at defensin ß 1 (DEFB1; P = 0.008). Gene ontology analysis showed an enrichment of imprinted genes (P = 9.53 × 10(-10)) and genes previously associated with RM (P = 9.51 × 10(-6)). An increase of outliers at seven imprinted loci was observed in RM (3.9%) compared with M (0%) and TA (0.9%) (P = 0.02), with increased average methylation at H19/IGF2 ICR1 in M samples (P < 0.0001). Altered DNA methylation in the placenta at specific loci, and global dysregulation in specific cases, may contribute to or be a consequence of poor placental function in karyotypically normal miscarriage.

Widespread DNA hypomethylation at gene enhancer regions in placentas associated with early-onset pre-eclampsia.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.

Protein kinase profiling in miscarriage: implications for the pathogenesis of trisomic pregnancy.

OBJECTIVE: Trisomy in pregnancy increases risks of miscarriage, fetal anomalies, and perinatal complications, with trisomy 16 the most common trisomy in human conceptions. The pathogenesis and protein expression profiles in trisomic pregnancy have not been well elucidated. Our objective was to profile protein kinase expression in trisomic and chromosomally normal miscarriage. METHODS: Placental chorionic villus cultures were set up from first-trimester miscarriages (n = 73). Protein kinases (n = 75) were profiled using a 2D Western blot in cultures from trisomic miscarriages (n = 6) (trisomy 16 or trisomy 15) and compared to chromosomally normal (euploid) miscarriages (n = 4). RESULTS: Distinct patterns of protein kinase expression were seen in the two trisomic groups, including dosage-dependent over-expression of the chromosome 16-encoded ERK1 in trisomy 16. This supports a role for chromosome-specific effects in the pathogenesis of trisomy (gene dosage hypothesis). In addition, both trisomic groups had increased inter-individual variation in protein kinase expression, which supports a role for amplified sensitivity to environmental and genetic variation in the pathogenesis of trisomy (amplified instability hypothesis). CONCLUSION: Both gene dosage effects and amplified instability operate simultaneously on the expression of protein kinases in trisomic pregnancies ending in miscarriage.

Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation.

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E-16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset of genes predicted different XCI statuses in different tissues. Using previously published expression data the effect of transcription on gene-body methylation was investigated and while X-linked introns of highly expressed genes were more methylated than the introns of lowly expressed genes, exonic methylation did not differ based on expression level. We conclude that the XCI status predicted using methylation of X-linked promoters with CpG islands was usually the same as determined by expression analysis and that 12% of X-linked genes examined show tissue-specific XCI whereby a gene has a different XCI status in at least one of the four tissues examined.

Early inflammation in the absence of overt infection in preterm neonates exposed to intensive care.

BACKGROUND: Systemic inflammation, typically attributed to sepsis, has been repeatedly linked to adverse long-term outcomes in infants born prematurely. However, it is unclear whether other factors can contribute to potentially harmful systemic inflammatory responses. OBJECTIVE: To determine the timing and extent of systemic inflammation occurring in absence of infection in preterm infants exposed to intensive care. METHODS: First, we screened for inflammation biomarkers most strongly linked to infection in a large prospective cohort of 425 newborns (gestational age 24-42 weeks). Second, we longitudinally measured levels of infection-related inflammation biomarkers up to 42 days of post-natal life in a series of 58 infants born ≤30 weeks of gestation exposed to intensive care. Ante- or post-natal infections were excluded using stringent definitions including rigorous histological placental examination. Spearman correlations were used to identify putative clinical factors potentially linked to inflammation. RESULTS: Three biomarkers were most strongly associated with neonatal sepsis (IL-6, IL-8 and G-CSF) in the first cohort. Using these markers, we found a predominant early high intensity systemic inflammation period within the first 72 h of preterm infants' extra-uterine life. Remarkably, this systemic inflammatory response was of magnitude comparable to that observed during sepsis in absence of ante- or post-natal signs of infection, and correlated with the amount of supplemental oxygen exposure (r=0.51-0.60). CONCLUSIONS: Non-infectious sources of systemic inflammation are significant in preterm infants exposed to intensive care and may contribute to intensive care-related organ injury.

Infantile cardioencephalopathy due to a COX15 gene defect: report and review.

We describe respiratory chain complex IV deficiency (cytochrome c oxidase deficiency) in a female infant with a neonatal rapidly progressive fatal course characterized by microcephaly, encephalopathy, persistent lactic acidosis, and hypertrophic cardiomyopathy. Postmortem cardiac muscle study showed marked complex IV deficiency. In contrast, complex IV activity was only slightly decreased in the skeletal muscle. Subsequent molecular investigations showed compound heterozygosity for two known pathogenic mutations in the COX15 gene. We compare the findings in our patient to those of the three previously reported cases.

Developmental origin of chorionic villus cultures from spontaneous abortion and chorionic villus sampling.

OBJECTIVE: Chorionic villus cultures from spontaneous abortions and chorionic villus sampling (CVS) are routinely used for clinical cytogenetic analysis. Although these cultures are assumed to represent the chorionic villus mesenchymal core, and therefore the inner cell mass (ICM) of the blastocyst, immunochemical studies using a true trophoblast-specific marker to definitively rule out trophoblast contamination have not been done. Therefore, we used cytokeratin-7 (CK7), a trophoblast-specific marker, to assess the developmental origin of these chorionic villus cultures. METHODS: We assessed chorionic villus cultures from CVS and spontaneous abortions for CK7 immunostaining (n = 20). RESULTS: Cultures from both CVS and spontaneous abortions showed little or no CK7 staining (≤ 1%). CONCLUSION: Chorionic villus cultures from CVS and spontaneous abortions exhibit little or no trophoblast contamination. They are therefore representative of the villus mesenchymal core and ultimately originate from the ICM of the blastocyst.

Effect of chorioamnionitis on brain development and injury in premature newborns.

OBJECTIVE: The association of chorioamnionitis and noncystic white matter injury, a common brain injury in premature newborns, remains controversial. Our objectives were to determine the association of chorioamnionitis and postnatal risk factors with white matter injury, and the effects of chorioamnionitis on early brain development, using advanced magnetic resonance imaging. METHODS: Ninety-two preterm newborns (24-32 weeks gestation) were studied at a median age of 31.9 weeks and again at 40.3 weeks gestation. Histopathological chorioamnionitis and white matter injury were scored using validated systems. Measures of brain metabolism (N-acetylaspartate/choline and lactate/choline) on magnetic resonance spectroscopy, and microstructure (average diffusivity and fractional anisotropy) on diffusion tensor imaging were calculated from predefined brain regions. RESULTS: Thirty-one (34%) newborns were exposed to histopathological chorioamnionitis, and 26 (28%) had white matter injury. Histopathological chorioamnionitis was not associated with an increased risk of white matter injury (relative risk: 1.2; p = 0.6). Newborns with postnatal infections and hypotension requiring therapy were at higher risk of white matter injury (p < 0.03). Adjusting for gestational age at scan and regions of interest, histopathological chorioamnionitis did not significantly affect brain metabolic and microstructural development (p > 0.1). In contrast, white matter injury was associated with lower N-acetylaspartate/choline (-8.9%; p = 0.009) and lower white matter fractional anisotropy (-11.9%; p = 0.01). INTERPRETATION: Histopathological chorioamnionitis does not appear to be associated with an increased risk of white matter injury on magnetic resonance imaging or with abnormalities of brain development. In contrast, postnatal infections and hypotension are associated with an increased risk of white matter injury in the premature newborn.

Assessing the role of placental trisomy in preeclampsia and intrauterine growth restriction.

OBJECTIVE: Prenatally diagnosed confined placental trisomy is associated with increased risk for intrauterine growth restriction (IUGR) and preeclampsia. However, it is unclear how often this might underlie pregnancy complications. Our objective was to evaluate the frequency and distribution of trisomic cells in placentae ascertained for IUGR and/or preeclampsia. METHOD: Comparative genomic hybridization was applied to two uncultured biopsies from each of 61 placentae referred with maternal preeclampsia and/or IUGR, 11 cases with elevated maternal serum hCG and/or AFP but no IUGR or preeclampsia, and 85 control placentae. RESULTS: Trisomy was observed in four placentae among the IUGR group (N = 43) but in no case of preeclampsia in the absence of IUGR (N = 18). Trisomy was observed in 1 of the 11 cases ascertained for abnormal maternal serum screen. Each of these five cases was mosaic and not all sampled sites showed the presence of trisomy. None of the 84 control placentas showed mosaic trisomy, although 1 case of nonmosaic 47,XXX was identified in this group. CONCLUSION: In cases in which diagnosis of the cause of IUGR may provide some benefit, testing should be performed using uncultured cells from multiple placental biopsies for the accurate diagnosis of trisomy mosaicism.

Placental weight in pregnancies with trisomy confined to the placenta.

OBJECTIVE: Mosaicism with trisomy confined to the placenta is present in ~1% of ongoing pregnancies at the time of chorionic villus sampling. Some studies have found reduced fetal growth in confined placental trisomy. The objective of this study was to assess placental weight and feto-placental weight ratio in pregnancies with trisomy confined to the placenta, and to correlate them with the level of trisomy in the three major placental lineages. METHODS: We conducted a retrospective study of 69 pregnancies with prenatally diagnosed mosaic trisomy in which the trisomic cells were confined to the placenta. Placental weight and feto-placental weight ratio were compared to those of matched controls, and placental weight was also analyzed for associations with the type and level of trisomy. Placental pathology was also reviewed. RESULTS: The pregnancies with mosaic trisomy were found to have lower placental weights than matched controls, but normal feto-placental weight ratios. Placental weight was not associated with the type or level of trisomic cells in the three placental lineages at term (chorionic plate, chorionic villus mesenchyme, and trophoblast). There were no pathognomonic findings on routine placental pathology of the trisomic placentas. CONCLUSION: Although placental weight was reduced (with normal feto-placental weight ratio) in pregnancies with trisomy confined to the placenta, the level of placental trisomy was not correlated with placental weight. Thus, trisomy may alter placental function rather than have a direct hypoplastic effect on placental growth. More in-depth studies beyond routine pathology are required to identify how trisomy affects placental function.

Schinzel-Giedion syndrome: report of splenopancreatic fusion and proposed diagnostic criteria.

We report on the 46th patient with Schinzel-Giedion syndrome (SGS) and the first observation of splenopancreatic fusion in this syndrome. In the antenatal period, a male fetus was found to have bilateral hydronephrosis. Postnatally, in keeping with a diagnosis of SGS, there were large fontanelles, ocular hypertelorism, a wide, broad forehead, midface retraction, a short, upturned nose, macroglossia, and a short neck. Other anomalies included cardiac defects, widened and dense long bone cortices, cerebral ventriculomegaly, and abnormal fundi. Splenopancreatic fusion, usually encountered in trisomy 13, was found on autopsy. Schinzel-Giedion syndrome is likely a monogenic condition for which neither the heritability pattern nor pathogenesis has yet been determined. A clinical diagnosis may be made by identifying the facial phenotype, including prominent forehead, midface retraction, and short, upturned nose, plus one of either of the two other major distinguishing features: typical skeletal abnormalities or hydronephrosis. Typical skeletal anomalies include a sclerotic skull base, wide supraoccipital-exoccipital synchondrosis, increased cortical density or thickness, and broad ribs. Other highly supportive features include neuroepithelial tumors (found in 17%), hypertrichosis, and brain abnormalities. Severe developmental delay and poor survival are constant features in reported patients.

The impact of prenatal diagnosis on neural tube defect (NTD) pregnancy versus birth incidence in British Columbia.

The birth incidence of neural tube defect (NTD) cases in British Columbia (B.C.), and elsewhere in North America, is reported to be declining. This decline is being attributed to folic acid (FA) supplementation and food fortification, but 2nd trimester prenatal screening of pregnancies for NTDs and other congenital anomalies has increased during this timeframe, as well. This descriptive, population-based study evaluates the impact of prenatal screening of NTD-affected pregnancies on (1) pregnancy outcome and (2) reporting of NTD births to the provincial Health Status Registry (B.C.H.S.R.); and it assesses (3) the use of periconceptional FA supplementation. NTD cases were ascertained from medical records of health centres providing care to families with NTD-affected pregnancies and newborns; and from NTD cases reported to the B.C.H.S.R. In 1997-1999, the B.C.H.S.R. published a NTD incidence of 0.77/1000. In this study, 151 NTD-affected pregnancies were identified, with an incidence of 1.16/1000. Partial Reporting of induced abortions in a NTD incidence 45.5% low than the actual incidence. Medical records were available for review on 144/151 pregnancies. Prenatal screening identified 86.1% (124/144) of NTD-affected pregnancies, with 72.6% (90/124) resulting in pregnancy termination, and 27.4% (34/124) continuing to term. Use of FA supplementation in the periconceptional period was recorded in 36.4% of pregnancies (39/107). Thus in B.C. the decline in the NTD incidence is due predominantly to pregnancy terminations following prenatal diagnosis, which reduces the NTD incidence by 60%, from 1.16/1000 to 0.47/1000. Continued efforts for primary and the option of secondary prevention of NTDs are recommended in order to improve newborn health in B.C. and elsewhere. These interventions need to be monitored, however, for optimal health care planning.

Profiling placental and fetal DNA methylation in human neural tube defects.

BACKGROUND: The incidence of neural tube defects (NTDs) declined by about 40 % in Canada with the introduction of a national folic acid (FA) fortification program. Despite the fact that few Canadians currently exhibit folate deficiency, NTDs are still the second most common congenital abnormality. FA fortification may have aided in reducing the incidence of NTDs by overcoming abnormal one carbon metabolism cycling, the process which provides one carbon units for methylation of DNA. We considered that NTDs persisting in a folate-replete population may also occur in the context of FA-independent compromised one carbon metabolism, and that this might manifest as abnormal DNA methylation (DNAm). Second trimester human placental chorionic villi, kidney, spinal cord, brain, and muscle were collected from 19 control, 22 spina bifida, and 15 anencephalic fetuses in British Columbia, Canada. DNA was extracted, assessed for methylenetetrahydrofolate reductase (MTHFR) genotype and for genome-wide DNAm using repetitive elements, in addition to the Illumina Infinium HumanMethylation450 (450k) array. RESULTS: No difference in repetitive element DNAm was noted between NTD status groups. Using a false discovery rate <0.05 and average group difference in DNAm ≥0.05, differentially methylated array sites were identified only in (1) the comparison of anencephaly to controls in chorionic villi (n = 4 sites) and (2) the comparison of spina bifida to controls in kidney (n = 3342 sites). CONCLUSIONS: We suggest that the distinctive DNAm of spina bifida kidneys may be consequent to the neural tube defect or reflective of a common etiology for abnormal neural tube and renal development. Though there were some small shifts in DNAm in the other tested tissues, our data do not support the long-standing hypothesis of generalized altered genome-wide DNAm in NTDs. This finding may be related to the fact that most Canadians are not folate deficient, but it importantly opens the field to the investigation of other epigenetic and non-epigenetic mechanisms in the etiology of NTDs.

Novel mutations in the TRIM37 gene in Mulibrey Nanism.

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder of unknown pathogenesis. The main clinical features are pre- and postnatal growth failure, characteristic dysmorphic craniofacial features, heart disease, and hepatomegaly. Five truncating mutations in the TRIM37 gene have previously been reported in Mulibrey nanism patients. The TRIM37 protein encodes a novel protein of unknown function. It contains a tripartite motif (TRIM, also denoted the RING-B-box-Coiled-coil or RBCC domain) and a TRAF (tumor necrosis factor-receptor associated factor) domain. TRIM37 localizes to peroxisomes classifying Mulibrey nanism as a peroxisomal disorder. Here we have characterized the genomic structure of the TRIM37 gene, which has 24 exons spanning approximately 109 kb of genomic DNA. Further, we report six novel disease-associated mutations, five of which predict a truncated protein: c.745C>T (p.Gln249X), c.1411C>T (p.Arg471X), c.2056C>T (p.Arg686X), and an 8.6 kb genomic deletion (c.1314+507_1668-207del resulting in p.Arg439fsX4). The sixth mutation (c.965G>T) is the first missense mutation (p.Gly322Val) associated with Mulibrey nanism. It affects the TRAF domain of TRIM37 and results in altered subcellular localization of the mutant TRIM37 protein, further suggesting that it is pathogenic.

Chorioamnionitis in the pathogenesis of brain injury in preterm infants.

Chorioamnionitis (or placental infection) is suspected to be a risk factor for brain injury in premature infants. The suggested association between chorioamnionitis and cystic periventricular leukomalacia and cerebral palsy is uncertain because of the variability of study designs and definitions of chorioamnionitis. Improvements in neonatal intensive care may have attenuated the impact of chorioamnionitis on brain health outcomes. Large multicenter studies using rigorous definitions of chorioamnionitis on placental pathologies and quantitative magnetic resonance techniques may offer the optimal way to clarify the complex role of chorioamnionitis in modifying brain health and long-term outcomes.

Early onset pre-eclampsia is associated with altered DNA methylation of cortisol-signalling and steroidogenic genes in the placenta.

Placental cortisol is inactivated in normotensive pregnancies, but is frequently present in pre-eclampsia associated placentae. Since glucocorticoids are strongly associated with the programming of long-term health, we assessed DNA methylation of genes involved in cortisol signalling and bioavailability, and hormonal signalling in the placenta of normotensive and hypertensive pregnancies. Candidate genes/CpG sites were selected through analysis of Illumina Infinium HumanMethylation450 BeadChip array data on control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placental samples. DNA methylation was further quantified by bisulfite pyrosequencing in a larger cohort of control (n = 111) cases, in addition to EOPET (n = 19), late onset pre-eclampsia (LOPET; n = 18) and normotensive intrauterine growth restriction (nIUGR; n = 13) cases. DNA methylation (percentage points) was increased at CpG sites within genes encoding the glucocorticoid receptor (NR3C1 exon 1D promoter; +8.46%; P<0.01) and corticotropin releasing hormone (CRH) binding protein (CRHBP intron 3; +9.14%; P<0.05), and decreased within CRH (5' UTR; -4.30%; P = 0.11) in EOPET-associated placentae, but not in LOPET nor nIUGR cases, compared to controls. Differential DNA methylation was not observed among groups at the 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene promoter. Significant hypomethylation was observed in pre-eclampsia but not nIUGR placentae for steroidogenic genes, including CYP11A1 (exon1; EOPET; -9.66%; P<0.00001, and LOPET; -5.77%; P<0.001), 3ß-hydroxy-delta-5-steroid dehydrogenase type 1 (HSD3B1 exon 2; EOPET; -12.49%; P<0.00001, and LOPET; -6.88%; P<0.001), TEA domain family member 3 (TEAD3 intron 1; EOPET; -12.56%; P<0.00001) and CYP19 (placental-specific exon 1.1 promoter; EOPET; -10.62%, P<0.0001). These data represent dysregulation of the placental epigenome in pre-eclampsia related to genes involved in maintaining the hormonal environment during pregnancy and highlights particular susceptibility in the early onset syndrome.