Mouse whole embryo culture and the analysis of neural tube defects

Whole embryo culture enables direct observation and manipulation of organogenesis stage embryos, that would otherwise be relatively inaccessible within the maternal uterus. Rat serum is the primary medium for mouse embryo culture and can sustain growth and development comparable to that in utero. To enhance the “replacement, reduction and refinement” (3Rs) of animals in research, culture experiments were performed to determine whether serum-free medium can substitute for whole rat serum, or whether rat serum can be diluted, and yet still maintain high quality development in vitro. Of two serum-free media tested, neither could sustain development comparable to that achieved in 100% rat serum. However, dilution of rat serum 1:1 with a defined medium supported growth and development with no significant differences from 100% rat serum. Hence, rat usage can be reduced by culture in medium containing diluted serum. Neural tube defects (NTDs) are severe birth defects of the brain or spinal cord. NTDs occur in mice lacking ASPP2, a p53 agonist and tumour suppressor. Embryos with a deletion of exon 3 in the ASPP2 gene, Trp53bp2, were found to have a progressive NTD phenotype that worsened with gestational age. The Trp53bp2Δ3/Δ3 embryonic neural tube phenotype involves ventral neuroepithelial overgrowth, ectopic lumen formation and re-opening of the neural tube. Cellular analysis revealed disruption in Trp53bp2Δ3/Δ3 embryos with and without a macroscopic phenotype. These histological abnormalities included disrupted proliferation, disorganised differentiation and a reduced basal to apical migration of cells prior to mitosis. Using the open-yolk sac embryo culture method, a hypothesis based on over-proliferation in Trp53bp2Δ3/Δ3 embryos was tested using the chemical inhibitor DAPT. It is proposed that deletion of ASPP2 results in an apico-basal polarity defect which increases in severity through development and results in rupture of the neural tube at variable locations along the body axis

Feasibility and design of a trial regarding the optimal mode of delivery for preterm birth:the CASSAVA multiple methods study

Background: Around 60,000 babies are born preterm (prior to 37 weeks’ gestation) each year in the UK. There is little evidence on the optimal birth mode (vaginal or caesarean section). Objective: The overall aim of the CASSAVA project was to determine if a trial to define the optimal mode of preterm birth could be carried out and, if so, determine what sort of trial could be conducted and how it could best be performed. We aimed to determine the specific groups of preterm women and babies for whom there are uncertainties about the best planned mode of birth, and if there would be willingness to recruit to, and participate in, a randomised trial to address some, but not all, of these uncertainties. This project was conducted in response to a Heath Technology Assessment programme commissioning call (17/22 ‘Mode of delivery for preterm infants’). Methods: We conducted clinician and patient surveys (n = 224 and n = 379, respectively) to identify current practice and opinion, and a consensus survey and Delphi workshop (n = 76 and n = 22 participants, respectively) to inform the design of a hypothetical clinical trial. The protocol for this clinical trial/vignette was used in telephone interviews with clinicians (n = 24) and in focus groups with potential participants (n = 13). Results: Planned sample size and data saturation was achieved for all groups except for focus groups with participants, as this had to be curtailed because of the COVID-19 pandemic and data saturation was not achieved. There was broad agreement from parents and health-care professionals that a trial is needed. The clinician survey demonstrated a variety of practice and opinion. The parent survey suggested that women and their families generally preferred vaginal birth at later gestations and caesarean section for preterm infants. The interactive workshop and Delphi consensus process confirmed the need for more evidence (hence the case for a trial) and provided rich information on what a future trial should entail. It was agreed that any trial should address the areas with most uncertainty, including the management of women at 26–32 weeks’ gestation, with either spontaneous preterm labour (cephalic presentation) or where preterm birth was medically indicated. Clear themes around the challenges inherent in conducting any trial emerged, including the concept of equipoise itself. Specific issues were as follows: different clinicians and participants would be in equipoise for each clinical scenario, effective conduct of the trial would require appropriate resources and expertise within the hospital conducting the trial, potential participants would welcome information on the trial well before the onset of labour and minority ethnic groups would require tailored approaches. Conclusion: Given the lack of evidence and the variation of practice and opinion in this area, and having listened to clinicians and potential participants, we conclude that a trial should be conducted and the outlined challenges resolved. Future work: The CASSAVA project could be used to inform the design of a randomised trial and indicates how such a trial could be carried out. Any future trial would benefit from a pilot with qualitative input and a study within a trial to inform optimal recruitment. Limitations: Certainty that a trial could be conducted can be determined only when it is attempted. Trial registration: Current Controlled Trials ISRCTN12295730. Funding: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 61. See the NIHR Journals Library website for further project information

The WHEAT pilot trial-WithHolding Enteral feeds Around packed red cell Transfusion to prevent necrotising enterocolitis in preterm neonates: a multicentre, electronic patient record (EPR), randomised controlled point-of-care pilot trial

INTRODUCTION: Necrotising enterocolitis (NEC) is a potentially devastating neonatal disease. A temporal association between red cell transfusion and NEC is well described. Observational data suggest that withholding enteral feeds around red cell transfusions may reduce the risk of NEC but this has not been tested in randomised trials; current UK practice varies. Prevention of NEC is a research priority but no appropriately powered trials have addressed this question. The use of a simplified opt-out consent model and embedding trial processes within existing electronic patient record (EPR) systems provide opportunities to increase trial efficiency and recruitment. METHODS AND ANALYSIS: We will undertake a randomised, controlled, multicentre, unblinded, pilot trial comparing two care pathways: continuing milk feeds (before, during and after red cell transfusions) and withholding milk feeds (for 4 hours before, during and for 4 hours after red cell transfusions), with infants randomly assigned with equal probability. We will use opt-out consent. A nested qualitative study will explore parent and health professional views. Infants will be eligible if born at <30+0 gestational weeks+days. Primary feasibility outcomes will be rate of recruitment, opt-out, retention, compliance, data completeness and data accuracy; clinical outcomes will include mortality and NEC. The trial will recruit in two neonatal networks in England for 9 months. Data collection will continue until all infants have reached 40+0 corrected gestational weeks or neonatal discharge. Participant identification and recruitment, randomisation and all trial data collection will be embedded within existing neonatal EPR systems (BadgerNet and BadgerEPR); outcome data will be extracted from routinely recorded data held in the National Neonatal Research Database. ETHICS AND DISSEMINATION: This study holds Research Ethics Committee approval to use an opt-out approach to consent. Results will inform future EPR-embedded and data-enabled trials and will be disseminated through conferences, publications and parent-centred information. TRIAL REGISTRATION NUMBER: ISRCTN registry ISRCTN62501859; Pre-results

Culshaw et al, dataset for BDR paper

Spreadsheet containing raw data and statistical analyses for Figures 1, 4, 5, 6 and 8

A spatio-temporal network for video semantic segmentation in surgical videos

PURPOSE: Semantic segmentation in surgical videos has applications in intra-operative guidance, post-operative analytics and surgical education. Models need to provide accurate predictions since temporally inconsistent identification of anatomy can hinder patient safety. We propose a novel architecture for modelling temporal relationships in videos to address these issues. METHODS: We developed a temporal segmentation model that includes a static encoder and a spatio-temporal decoder. The encoder processes individual frames whilst the decoder learns spatio-temporal relationships from frame sequences. The decoder can be used with any suitable encoder to improve temporal consistency. RESULTS: Model performance was evaluated on the CholecSeg8k dataset and a private dataset of robotic Partial Nephrectomy procedures. Mean Intersection over Union improved by 1.30% and 4.27% respectively for each dataset when the temporal decoder was applied. Our model also displayed improvements in temporal consistency up to 7.23%. CONCLUSIONS: This work demonstrates an advance in video segmentation of surgical scenes with potential applications in surgery with a view to improve patient outcomes. The proposed decoder can extend state-of-the-art static models, and it is shown that it can improve per-frame segmentation output and video temporal consistency

Developing a complex intervention to assess the feasibility of cue-based feeding for preterm infants in neonatal units (CuBS)

There is conflicting evidence of an association between breastfeeding and dental decay in very young children (early childhood caries (ECC)). While a recent systematic review and meta‐analysis reported that breastfeeding up to the age of 12 months reduced the risk of ECC, breastfeeding beyond 12 months was associated with a greater risk (Cui et al., 2017). The evidence however, is limited and inconsistent, with some studies finding an association only if breastfeeding continued beyond 18 or 24 months. Furthermore, a recent national study from Australia demonstrated that sustained breastfeeding (> 24 months) was not associated with ECC amongst children exposed to fluoridated water (Ha et al., 2019), indicating that the use of fluoridated water early in life moderated the relationship. The purpose of this presentation is to 1) critique the evidence related to breastfeeding practices and ECC; and 2) report the findings of an investigation into the effects of breastfeeding duration and night‐time breastfeeding on ECC amongst a cohort of Australian toddlers. The Study of Mothers’ and Infants’ Life Events affecting oral health (SMILE) is a population‐based birth cohort study conducted in Adelaide, Australia (Do et al., 2014). Data on breastfeeding practices were derived from questionnaires completed by mothers at recruitment, 3, 6, 12 and 24 months. The primary exposures were 1) duration of breastfeeding, defined as minimal (0 ‐ <1mo), breastfed for 1 ‐<6 months, breastfed for 6 ‐ < 12 months, and sustained (≥ 12months), and 2) the practice of night‐time breastfeeding at 12 months, defined as Yes or No. Standardised oral epidemiological examinations were conducted by trained and calibrated dental practitioners on children when they were 2‐3 years old (median 29.1, IQR 5.2 months). The primary outcome was ECC prevalence (presence of any decayed, missing or filled primary tooth surfaces), defined as Yes or No. Multivariable regression models generated adjusted prevalence ratios for the association between ECC and breastfeeding duration (n = 965); and between ECC and night time breastfeeding (n = 873). Confounders adjusted for in the models were free sugars intake, child age, maternal education and index of relative socioeconomic advantage and disadvantage. We found no independent association between the prevalence of caries and sustained breastfeeding (PR = 1.42, 95% CI 0.85–2.38) or the practice of breastfeeding at night‐time at 12 months (PR 1.27, 95% CI 0.80–2.01). The only variables that were significantly associated with ECC were high free sugars intakes and greater socioeconomic disadvantage. Adelaide is a city with a fluoridated mains water supply and hence the SMILE cohort were exposed to fluoridated water. More than 85% of participants reported tap water as their primary water source. We failed to find an association between either sustained breastfeeding or night‐time breastfeeding and ECC reported in other studies. The results of our study support the findings of Ha et al, (2017), that any negative association between breastfeeding duration and ECC is likely to be moderated by exposure to fluoride. Future studies investigating the association between breastfeeding and ECC should, where possible, investigate the interaction between water fluoridation and breastfeeding duration