Ultrasound is better tolerated than vaginal examination in and before labour

BACKGROUND: Intrapartum ultrasound has been proposed as a method of assessing labour progress but its acceptability has not been comprehensively assessed. AIMS: We evaluated the acceptability of intrapartum ultrasound in women having vaginal examination (VE) and ultrasound (US) assessment (transabdominal (TA) and transperineal (TP)) prior to delivery, with and without regional analgesia (RA). MATERIALS AND METHODS: Women at 24-42 weeks gestation were included in a prospective observational cohort study. The acceptability of digital VE and TP US were assessed pre- and post-examination using the modified validated Wijma Delivery Experience Questionnaire. Acceptability scores ranged 6-36 (6 being most and 36 being least positive) in six domains: positive-trust and relax, negative-harmful to baby, worrying, painful, intrusive. RESULTS: Of 119 women recruited, 104 completed both pre- and post-assessment questionnaires. Eighty-nine per cent of women were nulliparous with median gestation 40 + 2 weeks (25-42+1 ). Thirty-two per cent had RA before assessment, 91% in total. The combined acceptability scores of both negative and positive experiences (6 = most acceptable, 36 = least acceptable) for VE and US pre-assessment were 15 and 7 respectively (P < 0.0001: Mann-Whitney U-test). VE was associated with less positive / more negative domain scoring post-assessment 12 and 6, respectively (P < 0.0001). Although RA made no difference to the perceived experience pre-VE (P = 0.9), post-VE, women with RAs considered VEs more acceptable than those without RA (P = 0.0022). CONCLUSION(S): This is the first study to comprehensively assess the acceptability of VE and intrapartum US. US assessment prior to delivery is more acceptable than VE. RA ameliorated the negative experience of the VE post-assessment

Transcriptional implications of intragenic DNA methylation in the oestrogen receptor alpha gene in breast cancer cells and tissues

Background DNA methylation variability regions (MVRs) across the oestrogen receptor alpha (ESR1) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in ESR1 in breast cancer cell lines, tumour biopsies and exfoliated epithelial cells from expressed breast milk (EBM), to determine their significance for ESR1 transcription. Methods DNA methylation levels in eight MVRs across ESR1 were assessed by pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with DAC (1 μM), or DMSO (controls). ESR1 methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumours (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450 K (n = 36) and pyrosequencing in a further 53 donor samples. ESR1 mRNA levels were measured by qRT-PCR. Results We show that ER-positive cell lines had unmethylated ESR1 promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The ESR1 promoter was unmethylated in breast tumour biopsies with high levels of intragenic methylation, independent of ER status. However, ESR1 methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumour cell lines. Conclusion DAC treatment inhibited ESR1 transcription in cells with an unmethylated ESR1 promoter and reduced intragenic DNA methylation. Intragenic methylation levels correlated with ESR1 expression in homogenous cell populations (cell lines and exfoliated primary breast epithelial cells), but not in heterogeneous tumour biopsies, highlighting the significant differences between the in vivo tumour microenvironment and individual homogenous cell types. These findings emphasise the need for care when choosing material for epigenetic research and highlights the presence of aberrant intragenic methylation levels in tumour tissue

Investigating genome-wide transcriptional and methylomic consequences of a balanced t(1;11) translocation linked to major mental illness

Schizophrenia, bipolar disorder and major depressive disorder are devastating psychiatric conditions with a complex, overlapping genetic and environmental architecture. Previously, a family has been reported where a balanced chromosomal translocation between chromosomes 1 and 11 [t(1;11)] shows significant linkage to these disorders. This translocation transects three genes: Disrupted in schizophrenia- 1 (DISC1) on chromosome 1, a non-coding RNA, Disrupted in schizophrenia-2 (DISC2) antisense to DISC1, and a non-coding transcript, DISC1 fusion partner-1 (DISC1FP1) on chromosome 11, all of which could result in pathogenic properties in the context of the translocation. This thesis focuses on the genome-wide effects of the t(1;11) translocation, primarily examining differences in gene expression and DNA methylation, using various biological samples from the t(1;11) family. To assess the genome-wide effects of the t(1;11) translocation on methylation, DNA methylation was profiled in whole-blood from 41 family members using the Infinium HumanMethylation450 BeadChip. Significant differential methylation was observed within the translocation breakpoint regions on chromosomes 1 and 11. Downstream analysis identified additional regions of differential methylation outwith these chromosomes, while pathway analysis showed terms related to psychiatric disorders and neurodevelopment were enriched amongst differentially methylated genes, in addition to more general terms pertaining to cellular function. Using induced pluripotent stem cell (iPSC) technology, neuronal samples were developed from fibroblasts in a subset of individuals profiled for genome-wide methylation in whole blood (N = 6) with an aim to replicate the significant findings around the breakpoint regions. Here, methylation was profiled using the Infinium HumanMethylation450 BeadChip’s successor: the Infinium MethylationEPIC BeadChip. The results from the blood-based study failed to replicate in the neuronal samples, which could be attributed to low statistical power or tissue-specific factors such as methylation quantitative trait loci. The differences in methylation in the most significantly differentially methylated loci were found to be driven by a single individual, rendering further interpretation of the findings from this analysis difficult without additional samples. Cross-tissue analyses of DNA methylation were performed on blood and neuronal DNA from these six individuals, revealing little correlation between cell types. DISC1 is central to a network of interacting protein partners, including the transcription factor ATF4, and PDE4; both of which are associated with the cAMP signalling pathway. Haploinsufficiency of DISC1 due to the translocation may therefore be disruptive to cAMP-mediated gene expression. In order to identify transcriptomic effects which may be related to the t(1;11) translocation, genome-wide expression profiling was performed in lymphoblastoid cell line RNA from 13 family members. No transcripts were found to be differentially expressed at the genome-wide significant level. A post-hoc power analysis suggested that more samples would be required in order to detect genome-wide significant differential expression. However, imposing a fold-change cut-off to the data identified a number of candidate genes for follow-up analysis, including SORL1: a member of the brain-expressed Sortilin gene family. Sortilin genes have been linked to multiple psychiatric disorders including schizophrenia, bipolar disorder and Alzheimer’s disease. Follow-up analyses of Sortilin family members were performed in a Disc1 mouse model of schizophrenia, containing an amino acid substitution (L100P). Here, developmental gene expression profiling was performed with an additional aim to optimise and validate work performed by others using this mouse model. However, results from these experiments were variable between two independent batches mice tested. Additional investigation of Sortilin family genes was performed using GWAS data from human samples, using machine learning techniques to identify epistatic interactions linked to depression and brain function, revealing no statistically significant interactions. The results presented in this thesis suggest a potential mechanism for differential DNA methylation in the context of chromosomal translocations, and suggests mechanisms whereby increased risk of illness is conferred upon translocation carriers through dysregulation of transcription and DNA methylation

Gestational length assignment based on last menstrual period, first trimester crown-rump length, ovulation, and implantation timing.

PURPOSE: Understanding the natural length of human pregnancy is central to clinical care. However, variability in the reference methods to assign gestational age (GA) confound our understanding of pregnancy length. Assignation from ultrasound measurement of fetal crown-rump length (CRL) has superseded that based on last menstrual period (LMP). Our aim was to estimate gestational length based on LMP, ultrasound CRL, and implantation that were known, compared to pregnancy duration assigned by day of ovulation. METHODS: Prospective study in 143 women trying to conceive. In 71 ongoing pregnancies, gestational length was estimated from LMP, CRL at 10-14 weeks, ovulation, and implantation day. For each method of GA assignment, the distribution in observed gestational length was derived and both agreement and correlation between the methods determined. RESULTS: Median ovulation and implantation days were 16 and 27, respectively. The gestational length based on LMP, CRL, implantation, and ovulation was similar: 279, 278, 276.5 and 276.5 days, respectively. The distributions for observed gestational length were widest where GA was assigned from CRL and LMP and narrowest when assigned from implantation and ovulation day. The strongest correlation for gestational length assessment was between ovulation and implantation (r = 0.98) and weakest between CRL and LMP (r = 0.88). CONCLUSIONS: The most accurate method of predicting gestational length is ovulation day, and this agrees closely with implantation day. Prediction of gestational length from CRL and known LMP are both inferior to ovulation and implantation day. This information could have important implications on the routine assignment of gestational age

In Utero Exposures, Infant Growth, and DNA Methylation of Repetitive Elements and Developmentally Related Genes in Human Placenta

BACKGROUND: Fetal programming describes the theory linking environmental conditions during embryonic and fetal development with risk of diseases later in life. Environmental insults in utero may lead to changes in epigenetic mechanisms potentially affecting fetal development. OBJECTIVES: We examined associations between in utero exposures, infant growth, and methylation of repetitive elements and gene-associated DNA in human term placenta tissue samples. METHODS: Placental tissues and associated demographic and clinical data were obtained from subjects delivering at Women and Infants Hospital in Providence, Rhode Island (USA). Methylation levels of long interspersed nuclear element-1 (LINE-1) and the Alu element AluYb8 were determined in 380 placental samples from term deliveries using bisulfite pyrosequencing. Genomewide DNA methylation profiles were obtained in a subset of 184 samples using the Illumina Infinium HumanMethylation27 BeadArray. Multiple linear regression, model-based clustering methods, and gene set enrichment analysis examined the association between birth weight percentile, demographic variables, and repetitive element methylation and gene-associated CpG locus methylation. RESULTS: LINE-1 and AluYb8 methylation levels were found to be significantly positively associated with birth weight percentile (p = 0.01 and p \u3c 0.0001, respectively) and were found to differ significantly among infants exposed to tobacco smoke and alcohol. Increased placental AluYb8 methylation was positively associated with average methylation among CpG loci found in polycomb group target genes; developmentally related transcription factor binding sites were overrepresented for differentially methylated loci associated with both elements. CONCLUSIONS: Our results suggest that repetitive element methylation markers, most notably AluYb8 methylation, may be susceptible to epigenetic alterations resulting from the intrauterine environment and play a critical role in mediating placenta function, and may ultimately inform on the developmental basis of health and disease

Study protocol : Minimum effective low dose: anti-human thymocyte globulin (MELD-ATG): phase II, dose ranging, efficacy study of antithymocyte globulin (ATG) within 6 weeks of diagnosis of type 1 diabetes

Introduction Type 1 diabetes (T1D) is a chronic autoimmune disease, characterised by progressive destruction of the insulin-producing beta cells of the pancreas. One immunosuppressive agent that has recently shown promise in the treatment of new-onset T1D subjects aged 12-45 years is antithymocyte globulin (ATG), Thymoglobuline, encouraging further exploration in lower age groups. Methods and analysis Minimal effective low dose (MELD)-ATG is a phase 2, multicentre, randomised, double-blind, placebo-controlled, multiarm parallel-group trial in participants 5-25 years diagnosed with T1D within 3-9 weeks of planned treatment day 1. A total of 114 participants will be recruited sequentially into seven different cohorts with the first cohort of 30 participants being randomised to placebo, 2.5 mg/kg, 1.5 mg/kg, 0.5 mg/kg and 0.1 mg/kg ATG total dose in a 1:1:1:1:1 allocation ratio. The next six cohorts of 12-15 participants will be randomised to placebo, 2.5 mg/kg, and one or two selected middle ATG total doses in a 1:1:1:1 or 1:1:1 allocation ratio, as dependent on the number of middle doses, given intravenously over two consecutive days. The primary objective will be to determine the changes in stimulated C-peptide response over the first 2 hours of a mixed meal tolerance test at 12 months for 2.5 mg/kg ATG arm vs the placebo. Conditional on finding a significant difference at 2.5 mg/kg, a minimally effective dose will be sought. Secondary objectives include the determination of the effects of a particular ATG treatment dose on (1) stimulated C-peptide, (2) glycated haemoglobin, (3) daily insulin dose, (4) time in range by intermittent continuous glucose monitoring measures, (5) fasting and stimulated dry blood spot (DBS) C-peptide measurements. Ethics and dissemination MELD-ATG received first regulatory and ethical approvals in Belgium in September 2020 and from the German and UK regulators as of February 2021. The publication policy is set in the INNODIA (An innovative approach towards understanding and arresting Type 1 diabetes consortium) grant agreement (www.innodia.eu).Peer reviewe

Review of processing and analysis methods for DNA methylation array data

The promise of epigenome-wide association studies (EWAS) and cancer specific somatic changes in improving our understanding of cancer coupled with the decreasing cost and increasing coverage of DNA methylation microarrays, has brought about a surge in the use of these technologies. Here, we aim to provide both a review of issues encountered in the processing and analysis of array-based DNA methylation data, as well as to summarize advantages of recent approaches proposed for handling those issues; focusing on approaches publicly available in open-source environments such as R and Bioconductor. The processing tools and analysis flowchart described we hope will facilitate researchers to effectively use these powerful DNA methylation array-based platforms, thereby advancing our understanding of human health and disease.Keywords: Processing, Microarray, Analysis, DNA methylation, Bioconductor and R package

DNA methylation signature of passive smoke exposure is less pronounced than active smoking: the Understanding Society study

Introduction The extent of the biological impact of passive smoke exposure is unclear. We sought to investigate the association between passive smoke exposure and DNA methylation, which could serve as a biomarker of health risk. Materials and methods We derived passive smoke exposure from self-reported questionnaire data among smoking and non-smoking partners of participants enrolled in the UK Household Longitudinal Study ‘Understanding Society’ (n=769). We performed an epigenome-wide association study (EWAS) of passive smoke exposure with DNA methylation in peripheral blood measured using the Illumina Infinium Methylation EPIC array. Results No CpG sites surpassed the epigenome-wide significance threshold of p<5.97 × 10−8 in relation to partner smoking, compared with 10 CpG sites identified in relation to own smoking. However, 10 CpG sites surpassed a less stringent threshold of p<1 × 10−5 in a model of partner smoking adjusted for own smoking (model 1), 7 CpG sites in a model of partner smoking restricted to non-smokers (model 2) and 16 CpGs in a model restricted to regular smokers (model 3). In addition, there was evidence for an interaction between own smoking status and partners’ smoking status on DNA methylation levels at the majority of CpG sites identified in models 2 and 3. There was a clear lack of enrichment for previously identified smoking signals in the EWAS of passive smoke exposure compared with the EWAS of own smoking. Conclusion The DNA methylation signature associated with passive smoke exposure is much less pronounced than that of own smoking, with no positive findings for ‘expected’ signals. It is unlikely that changes to DNA methylation serve as an important mechanism underlying the health risks of passive smoke exposure