Behavioural And Molecular Consequences Of Postnatal Stress In A Mouse Model Of Fetal Alcohol Spectrum Disorder

Fetal alcohol spectrum disorders (FASD) are caused by prenatal alcohol exposure (PAE) and affect 1‑5% of the North American population. Children born with FASD often face maternal separation throughout childhood. How this early life stress (ELS) affects the severity of FASD-related deficits is poorly understood. Using a mouse model, this dissertation establishes that behavioural deficits accumulate following prenatal alcohol exposure and early life stress, assessed using tests for activity, anxiety-like behaviour as well as learning and memory. Hippocampal gene expression was evaluated using RNA-seq followed by clustering of expression profiles through weighted gene co-expression network analysis (WGCNA). A set of transcripts are associated with anxiety-like behaviour (r = 0.79, p = 0.002) and treatment (r = 0.68, p = 0.015). Genes in this module are overrepresented by transcriptional regulation and neurodevelopment genes. One member of this module, Polr2a, is downregulated by the combination of treatments. Hippocampal promoter DNA methylation was assessed using methylated DNA immunoprecipitation sequencing (MeDIP-Seq). Methylation at different genes is affected by each treatment independently, and a unique set of genes are affected by the combination of treatments. PAE leads to altered promoter DNA methylation at genes important for transcriptional regulation and ELS leads to changes at genes important for histone methylation and immune response. The combination of treatments results in DNA methylation changes at genes important for neuronal migration and immune response. The results from the same samples show that genes with altered expression and promoter methylation are critical in brain development and function. Also, there is minimal complementarity between changes in promoter DNA methylation and gene expression. Mechanisms beyond promoter DNA methylation are likely involved in lasting gene expression changes leading to behavioural deficits seen in FASD. Although further research is required to elucidate the mechanism, the results included may be valuable towards early and reliable diagnosis, together with the development of novel strategies for the amelioration of FASD-related deficits

Exploring the Complexity of Intellectual Disability in Fetal Alcohol Spectrum Disorders

Brain development in mammals is long lasting. It begins early during embryonic growth and is finalized in early adulthood. This progression represents a delicate choreography of molecular, cellular, and physiological processes initiated and directed by the fetal genotype in close interaction with environment. Not surprisingly, most aberrations in brain functioning including intellectual disability (ID) are attributed to either gene(s), or environment or the interaction of the two. The ensuing complexity has made the assessment of this choreography, ever challenging. A model to assess this complexity has used a mouse model (C57BL/6J or B6) that is subjected to prenatal alcohol exposure. The resulting pups show learning and memory deficits similar to patients with fetal alcohol spectrum disorder (FASD), which is associated with life-long changes in gene expression. Interestingly, this change in gene expression underlies epigenetic processes including DNA methylation and miRNAs. This paradigm is applicable to ethanol exposure at different developmental times (binge at trimesters 1, 2, and 3 as well as continuous preference drinking (70%) of 10% alcohol by B6 females during pregnancy). The exposure leads to life-long changes in neural epigenetic marks, gene expression, and a variety of defects in neurodevelopment and CNS function. We argue that this cascade may be reversed postnatally via drugs, chemicals, and environment including maternal care. Such conclusions are supported by two sets of results. First, antipsychotic drugs that are used to treat ID including psychosis function via changes in DNA methylation, a major epigenetic mark. Second, post-natal environment may improve (with enriched environments) or worsen (with negative and maternal separation stress) the cognitive ability of pups that were prenatally exposed to ethanol as well as their matched controls. In this review, we will discuss operational epigenetic mechanisms involved in the development of intellectual ability/disability in response to alcohol during prenatal or post-natal development. In doing so, we will explore the potential of epigenetic manipulation in the treatment of FASD and related disorders implicated in ID

Management of fetal malposition in the second stage of labor: a propensity score analysis.

OBJECTIVE: We sought to determine the factors associated with selection of rotational instrumental vs cesarean delivery to manage persistent fetal malposition, and to assess differences in adverse neonatal and maternal outcomes following delivery by rotational instruments vs cesarean delivery. STUDY DESIGN: We conducted a retrospective cohort study over a 5-year period in a tertiary United Kingdom obstetrics center. In all, 868 women with vertex-presenting, single, liveborn infants at term with persistent malposition in the second stage of labor were included. Propensity score stratification was used to control for selection bias: the possibility that obstetricians may systematically select more difficult cases for cesarean delivery. Linear and logistic regression models were used to compare maternal and neonatal outcomes for delivery by rotational forceps or ventouse vs cesarean delivery, adjusting for propensity scores. RESULTS: Increased likelihood of rotational instrumental delivery was associated with lower maternal age (odds ratio [OR], 0.95; P < .01), lower body mass index (OR, 0.94; P < .001), lower birthweight (OR, 0.95; P < .01), no evidence of fetal compromise at the time of delivery (OR, 0.31; P < .001), delivery during the daytime (OR, 1.45; P < .05), and delivery by a more experienced obstetrician (OR, 7.21; P < .001). Following propensity score stratification, there was no difference by delivery method in the rates of delayed neonatal respiration, reported critical incidents, or low fetal arterial pH. Maternal blood loss was higher in the cesarean group (295.8 ± 48 mL, P < .001). CONCLUSION: Rotational instrumental delivery is often regarded as unsafe. However, we find that neonatal outcomes are no worse once selection bias is accounted for, and that the likelihood of severe obstetric hemorrhage is reduced. More widespread training of obstetricians in rotational instrumental delivery should be considered, particularly in light of rising cesarean delivery rates.During data analysis, A.R.A. was supported by an NICHD Predoctoral Fellowship under grant number F31HD079182 and by grant R24HD042849, awarded to the Population Research Center at The University of Texas at Austin. She is currently supported by grant R24HD047879 for Population Research at Princeton University. J.G.S. is partially funded by a CAREER grant from the National Science Foundation (DMS-1255187).This is the accepted version. It will be embargoed until 12 months after the final version is published by Elsevier. The final version is available from Elsevier at http://www.sciencedirect.com/science/article/pii/S000293781401078

Methylation of the imprinted GNAS1 gene in cell-free plasma DNA : equal steady-state quantities of methylated and unmethylated DNA in plasma

Background Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of tumour suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes. Methods We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing. Results Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers. Conclusions Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults

Long-Term Genomic and Epigenomic Dysregulation as a Consequence of Prenatal Alcohol Exposure: A Model for Fetal Alcohol Spectrum Disorders

There is abundant evidence that prenatal alcohol exposure leads to a range of behavioral and cognitive impairments, categorized under the term fetal alcohol spectrum disorders (FASDs). These disorders are pervasive in Western cultures and represent the most common preventable source of neurodevelopmental disabilities. The genetic and epigenetic etiology of these phenotypes, including those factors that may maintain these phenotypes throughout the lifetime of an affected individual, has become a recent topic of investigation. This review integrates recent data that has progressed our understanding FASD as a continuum of molecular events, beginning with cellular stress response and ending with a long-term footprint of epigenetic dysregulation across the genome. It reports on data from multiple ethanol-treatment paradigms in mouse models that identify changes in gene expression that occur with respect to neurodevelopmental timing of exposure and ethanol dose. These studies have identified patterns of genomic alteration that are dependent on the biological processes occurring at the time of ethanol exposure. This review also adds to evidence that epigenetic processes such as DNA methylation, histone modifications, and non-coding RNA regulation may underlie long-term changes to gene expression patterns. These may be initiated by ethanol-induced alterations to DNA and histone methylation, particularly in imprinted regions of the genome, affecting transcription which is further fine-tuned by altered microRNA expression. These processes are likely complex, genome-wide, and interrelated. The proposed model suggests a potential for intervention, given that epigenetic changes are malleable and may be altered by postnatal environment. This review accentuates the value of mouse models in deciphering the molecular etiology of FASD, including those processes that may provide a target for the ammelioration of this common yet entirely preventable disorder

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP+/+), heterozygous (ANP+/-) and knockout (ANP−/-) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP−/− versus ANP+/+ mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR

Systematic Identification of Placental Epigenetic Signatures for the Noninvasive Prenatal Detection of Edwards Syndrome

Background: Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively. Principal Findings: We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPAAPCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P = 0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%. Conclusions: Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is redominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18. © Tsui et al.published_or_final_versio

Impact of DNA methylation on trophoblast function

The influence of epigenetics is evident in many fields of medicine today. This is also true in placentology, where versatile epigenetic mechanisms that regulate expression of genes have shown to have important influence on trophoblast implantation and placentation. Such gene regulation can be established in different ways and on different molecular levels, the most common being the DNA methylation. DNA methylation has been shown today as an important predictive component in assessing clinical prognosis of certain malignant tumors; in addition, it opens up new possibilities for non-invasive prenatal diagnosis utilizing cell-free fetal DNA methods. By using a well known demethylating agent 5-azacytidine in pregnant rat model, we have been able to change gene expression and, consequently, the processes of trophoblast differentiation and placental development. In this review, we describe how changes in gene methylation effect trophoblast development and placentation and offer our perspective on use of trophoblast epigenetic research for better understanding of not only placenta development but cancer cell growth and invasion as well