Antenatal detection of large-for-gestational-age fetuses following implementation of the Growth Assessment Protocol: secondary analysis of a randomised control trial.

OBJECTIVE: To determine whether the Growth Assessment Protocol (GAP) affects the antenatal detection of large for gestational age (LGA) or maternal and perinatal outcomes amongst LGA babies. DESIGN: Secondary analysis of a pragmatic open randomised cluster control trial comparing the GAP with standard care. SETTING: Eleven UK maternity units. POPULATION: Pregnant women and their LGA babies born at ≥36+0  weeks of gestation. METHODS: Clusters were randomly allocated to GAP implementation or standard care. Data were collected from electronic patient records. Trial arms were compared using summary statistics, with unadjusted and adjusted (two-stage cluster summary approach) differences. MAIN OUTCOME MEASURES: Rate of detection of LGA (estimated fetal weight on ultrasound scan above the 90th centile after 34+0  weeks of gestation, defined by either population or customised growth charts), maternal and perinatal outcomes (e.g. mode of birth, postpartum haemorrhage, severe perineal tears, birthweight and gestational age, neonatal unit admission, perinatal mortality, and neonatal morbidity and mortality). RESULTS: A total of 506 LGA babies were exposed to GAP and 618 babies received standard care. There were no significant differences in the rate of LGA detection (GAP 38.0% vs standard care 48.0%; adjusted effect size -4.9%; 95% CI -20.5, 10.7; p = 0.54), nor in any of the maternal or perinatal outcomes. CONCLUSIONS: The use of GAP did not change the rate of antenatal ultrasound detection of LGA when compared with standard care.

Evaluation of the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age: The DESiGN cluster randomised trial.

BACKGROUND: Antenatal detection and management of small for gestational age (SGA) is a strategy to reduce stillbirth. Large observational studies provide conflicting results on the effect of the Growth Assessment Protocol (GAP) in relation to detection of SGA and reduction of stillbirth; to the best of our knowledge, there are no reported randomised control trials. Our aim was to determine if GAP improves antenatal detection of SGA compared to standard care. METHODS AND FINDINGS: This was a pragmatic, superiority, 2-arm, parallel group, open, cluster randomised control trial. Maternity units in England were eligible to participate in the study, except if they had already implemented GAP. All women who gave birth in participating clusters (maternity units) during the year prior to randomisation and during the trial (November 2016 to February 2019) were included. Multiple pregnancies, fetal abnormalities or births before 24+1 weeks were excluded. Clusters were randomised to immediate implementation of GAP, an antenatal care package aimed at improving detection of SGA as a means to reduce the rate of stillbirth, or to standard care. Randomisation by random permutation was stratified by time of study inclusion and cluster size. Data were obtained from hospital electronic records for 12 months prerandomisation, the washout period (interval between randomisation and data collection of outcomes), and the outcome period (last 6 months of the study). The primary outcome was ultrasound detection of SGA (estimated fetal weight <10th centile using customised centiles (intervention) or Hadlock centiles (standard care)) confirmed at birth (birthweight <10th centile by both customised and population centiles). Secondary outcomes were maternal and neonatal outcomes, including induction of labour, gestational age at delivery, mode of birth, neonatal morbidity, and stillbirth/perinatal mortality. A 2-stage cluster-summary statistical approach calculated the absolute difference (intervention minus standard care arm) adjusted using the prerandomisation estimate, maternal age, ethnicity, parity, and randomisation strata. Intervention arm clusters that made no attempt to implement GAP were excluded in modified intention to treat (mITT) analysis; full ITT was also reported. Process evaluation assessed implementation fidelity, reach, dose, acceptability, and feasibility. Seven clusters were randomised to GAP and 6 to standard care. Following exclusions, there were 11,096 births exposed to the intervention (5 clusters) and 13,810 exposed to standard care (6 clusters) during the outcome period (mITT analysis). Age, height, and weight were broadly similar between arms, but there were fewer women: of white ethnicity (56.2% versus 62.7%), and in the least deprived quintile of the Index of Multiple Deprivation (7.5% versus 16.5%) in the intervention arm during the outcome period. Antenatal detection of SGA was 25.9% in the intervention and 27.7% in the standard care arm (adjusted difference 2.2%, 95% confidence interval (CI) -6.4% to 10.7%; p = 0.62). Findings were consistent in full ITT analysis. Fidelity and dose of GAP implementation were variable, while a high proportion (88.7%) of women were reached. Use of routinely collected data is both a strength (cost-efficient) and a limitation (occurrence of missing data); the modest number of clusters limits our ability to study small effect sizes. CONCLUSIONS: In this study, we observed no effect of GAP on antenatal detection of SGA compared to standard care. Given variable implementation observed, future studies should incorporate standardised implementation outcomes such as those reported here to determine generalisability of our findings. TRIAL REGISTRATION: This trial is registered with the ISRCTN registry, ISRCTN67698474.

Hypothermia is not therapeutic in a neonatal piglet model of inflammation-sensitized hypoxia-ischemia.

BACKGROUND: Perinatal inflammation combined with hypoxia-ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown. METHODS: Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1-25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured. RESULTS: Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT. CONCLUSIONS: We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI. IMPACT: Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models.

Development of standard definitions and grading for Maternal and Fetal Adverse Event Terminology.

OBJECTIVE: Adverse event (AE) monitoring is central to assessing therapeutic safety. The lack of a comprehensive framework to define and grade maternal and fetal AEs in pregnancy trials severely limits understanding risks in pregnant women. We created AE terminology to improve safety monitoring for developing pregnancy drugs, devices and interventions. METHOD: Existing severity grading for pregnant AEs and definitions/indicators of 'severe' and 'life-threatening' conditions relevant to maternal and fetal clinical trials were identified through a literature search. An international multidisciplinary group identified and filled gaps in definitions and severity grading using Medical Dictionary for Regulatory Activities (MedDRA) terms and severity grading criteria based on Common Terminology Criteria for Adverse Event (CTCAE) generic structure. The draft criteria underwent two rounds of a modified Delphi process with international fetal therapy, obstetric, neonatal, industry experts, patients and patient representatives. RESULTS: Fetal AEs were defined as being diagnosable in utero with potential to harm the fetus, and were integrated into MedDRA. AE severity was graded independently for the pregnant woman and her fetus. Maternal (n = 12) and fetal (n = 19) AE definitions and severity grading criteria were developed and ratified by consensus. CONCLUSIONS: This Maternal and Fetal AE Terminology version 1.0 allows systematic consistent AE assessment in pregnancy trials to improve safety.

Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia.

BACKGROUND: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. METHODS: Sixteen piglets were randomized: (i) LPS 2 µg/kg bolus; 1 µg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. RESULTS: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h. CONCLUSIONS: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE. IMPACT: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes.

Dimensionless squared jerk: An objective differential to assess experienced and novice probe movement in obstetric ultrasound.

OBJECTIVE: Widely accepted, validated and objective measures of ultrasound competency have not been established for clinical practice. Outcomes of training curricula are often based on arbitrary thresholds, such as the number of clinical cases completed. We aimed to define metrics against which competency could be measured. METHOD: We undertook a prospective, observational study of obstetric sonographers at a UK University Teaching Hospital. Participants were either experienced in fetal ultrasound (n = 10, >200 ultrasound examinations) or novice operators (n = 10, <25 ultrasound examinations). We recorded probe motion data during the performance of biometry on a commercially available mid-trimester phantom. RESULTS: We report that Dimensionless squared jerk, an assessment of deliberate hand movements, independent of movement duration, extent, spurious peaks and dimension differed significantly different between groups, 19.26 (SD 3.02) for experienced and 22.08 (SD 1.05, p = 0.01) for novice operators, respectively. Experienced operator performance, was associated with a shorter time to task completion of 176.46 s (SD 47.31) compared to 666.94 s (SD 490.36, p = 0.0004) for novice operators. Probe travel was also shorter for experienced operators 521.23 mm (SD 27.41) versus 2234.82 mm (SD 188.50, p = 0.007) when compared to novice operators. CONCLUSION: Our results represent progress toward an objective assessment of technical skill in obstetric ultrasound. Repeating this methodology in a clinical environment may develop insight into the generalisability of these findings into ultrasound education.

Biometric assessments of the posterior fossa by fetal MRI: A systematic review.

BACKGROUND: Posterior fossa abnormalities (PFAs) are commonly identified within routine screening and are a frequent indication for fetal magnetic resonance imaging (MRI). Although biometric measurements of the posterior fossa (PF) are established on fetal ultrasound and MRI, qualitative visual assessments are predominantly used to differentiate PFAs. OBJECTIVES: This systematic review aimed to assess 2-dimensional (2D) biometric measurements currently in use for assessing the PF on fetal MRI to delineate different PFAs. METHODS: The protocol was registered (PROSPERO ID CRD42019142162). Eligible studies included T2-weighted MRI PF measurements in fetuses with and without PFAs, including measurements of the PF, or other brain areas relevant to PFAs. RESULTS: 59 studies were included - 6859 fetuses had 62 2D PF and related measurements. These included linear, area and angular measurements, representing measures of PF size, cerebellum/vermis, brainstem, and supratentorial measurements. 11 measurements were used in 10 or more studies and at least 1200 fetuses. These dimensions were used to characterise normal for gestational age, diagnose a range of pathologies, and predict outcome. CONCLUSION: A selection of validated 2D biometric measurements of the PF on fetal MRI may be useful for identification of PFA in different clinical settings. Consistent use of these measures, both clinically and for research, is recommended.

Ascending Vaginal Infection Using Bioluminescent Bacteria Evokes Intrauterine Inflammation, Preterm Birth, and Neonatal Brain Injury in Pregnant Mice.

Preterm birth is a serious global health problem and the leading cause of infant death before 5 years of age. At least 40% of cases are associated with infection. The most common way for pathogens to access the uterine cavity is by ascending from the vagina. Bioluminescent pathogens have revolutionized the understanding of infectious diseases. We hypothesized that bioluminescent Escherichia coli can be used to track and monitor ascending vaginal infections. Two bioluminescent strains were studied: E. coli K12 MG1655-lux, a nonpathogenic laboratory strain, and E. coli K1 A192PP-lux2, a pathogenic strain capable of causing neonatal meningitis and sepsis in neonatal rats. On embryonic day 16, mice received intravaginal E. coli K12, E. coli K1, or phosphate-buffered saline followed by whole-body bioluminescent imaging. In both cases, intravaginal delivery of E. coli K12 or E. coli K1 led to bacterial ascension into the uterine cavity, but only E. coli K1 induced preterm parturition. Intravaginal administration of E. coli K1 significantly reduced the proportion of pups born alive compared with E. coli K12 and phosphate-buffered saline controls. However, in both groups of viable pups born after bacterial inoculation, there was evidence of comparable brain inflammation by postnatal day 6. This study ascribes specific mechanisms by which exposure to intrauterine bacteria leads to premature delivery and neurologic inflammation in neonates.

Extracellular signal-regulated kinase 2 has duality in function between neuronal and astrocyte expression following neonatal hypoxic-ischaemic cerebral injury.

KEY POINTS: This study identifies phosphorylated extracellular signal-regulated kinase (ERK) to be immediately diminished followed by a rapid if transient increase for up to 4 h following hypoxic-ischaemic insult (HI) in the neonatal mouse. Phosphorylated ERK up-regulation was prevented with systemic injection of the mitogen-activated protein kinase kinase (MEK) inhibitor SL327. Treatment with SL327 both pre- and post-HI gave a strong reduction in the number of dying cells and microgliosis. By utilising transgenic mouse mutations, we observe that neuronal ERK2 significantly contributes to tissue damage, while ERK1 and astrocytic ERK2 are neuroprotective. Compared to global inactivation, selective cell-specific interference with ERK activity could result in stronger neuroprotection. ABSTRACT: Hypoxia-ischaemia (HI) is a major cause of neonatal brain injury resulting in cerebral palsy, epilepsy, cognitive impairment and other neurological disabilities. The role of extracellular signal-regulated kinase (ERK) isoforms and their mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation in HI has previously been explored but remains unresolved at cellular level. This is pertinent given the growing awareness of the role of non-neuronal cells in neuroprotection. Using a modified Rice-Vannucci model of HI in the neonatal mouse we observed time- and cell-dependent ERK phosphorylation (pERK), with strongly up-regulated pERK immunoreactivity first in periventricular white matter axons within 15-45 min of HI, followed by forebrain astrocytes and neurons (1-4 h post-HI), and return to baseline by 16 h. We explored the effects of pharmacological ERK blockade through the MEK inhibitor SL327 on neonatal HI-brain damage following HI alone (30 or 60 min) or lipopolysaccharide (LPS)-sensitised HI insult (30 min). Global inhibition of ERK phosphorylation with systemically applied SL327 abolished forebrain pERK immunoreactivity, and significantly reduced cell death and associated microglial activation at 48 h post-HI. We then explored the effects of cell-specific ERK2 deletion alone or in combination with global ERK1 knockout under the same conditions of HI insult. Neuronal ERK2 deletion strongly decreased infarct size, neuronal cell death and microglial activation in grey matter following both HI alone or LPS-sensitised HI. ERK1 deletion attenuated the protective effect of neuronal ERK2 deletion. Removal of astroglial ERK2 produced a reverse response, with a 3- to 4-fold increase in microglial activation and cell death. Our data suggest a cell-specific and time-dependent role of ERK in neonatal HI, with a predominant, neurotoxic effect of neuronal ERK2, which is counteracted by neuroprotection by ERK1 and astrocytic ERK2. Overall, global pharmacological inhibition of ERK phosphorylation is strongly neuroprotective.

EVERREST prospective study: a 6-year prospective study to define the clinical and biological characteristics of pregnancies affected by severe early onset fetal growth restriction.

BACKGROUND: Fetal growth restriction (FGR) is a serious obstetric condition for which there is currently no treatment. The EVERREST Prospective Study has been designed to characterise the natural history of pregnancies affected by severe early onset FGR and establish a well phenotyped bio-bank. The findings will provide up-to-date information for clinicians and patients and inform the design and conduct of the EVERREST Clinical Trial: a phase I/IIa trial to assess the safety and efficacy of maternal vascular endothelial growth factor (VEGF) gene therapy in severe early onset FGR. Data and samples from the EVERREST Prospective Study will be used to identify ultrasound and/or biochemical markers of prognosis in pregnancies with an estimated fetal weight (EFW) <3rd centile between 20+0 and 26+6 weeks of gestation. METHODS: This is a 6 year European multicentre prospective cohort study, recruiting women with a singleton pregnancy where the EFW is <3rd centile for gestational age and <600 g at 20+0 to 26+6 weeks of gestation. Detailed data are collected on: maternal history; antenatal, peripartum, and postnatal maternal complications; health economic impact; psychological impact; neonatal condition, progress and complications; and infant growth and neurodevelopment to 2 years of corrected age in surviving infants. Standardised longitudinal ultrasound measurements are performed, including: fetal biometry; uterine artery, umbilical artery, middle cerebral artery, and ductus venosus Doppler velocimetry; and uterine artery and umbilical vein volume blood flow. Samples of maternal blood and urine, amniotic fluid (if amniocentesis performed), placenta, umbilical cord blood, and placental bed (if caesarean delivery performed) are collected for bio-banking. An initial analysis of maternal blood samples at enrolment is planned to identify biochemical markers that are predictors for fetal or neonatal death. DISCUSSION: The findings of the EVERREST Prospective Study will support the development of a novel therapy for severe early onset FGR by describing in detail the natural history of the disease and by identifying women whose pregnancies have the poorest outcomes, in whom a therapy might be most advantageous. The findings will also enable better counselling of couples with affected pregnancies, and provide a valuable resource for future research into the causes of FGR. TRIAL REGISTRATION: NCT02097667 registered 31st October 2013.

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) reduces neonatal hypoxic-ischaemic brain damage.

Hypoxic-ischaemic encephalopathy is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy and cognitive disabilities. Hypoxia-ischaemia (HI) strongly up-regulates Signal Transducer and Activator of Transcription 3 (STAT3) in the immature brain. Our aim was to establish whether STAT3 up-regulation is associated with neonatal HI-brain damage and evaluate the phosphorylated STAT3-contribution from different cell types in eliciting damage. We subjected postnatal day seven mice to unilateral carotid artery ligation followed by 60 min hypoxia. Neuronal STAT3-deletion reduced cell death, tissue loss, microglial and astroglial activation in all brain regions. Astroglia-specific STAT3-deletion also reduced cell death, tissue loss and microglial activation, although not as strongly as the deletion in neurons. Systemic pre-insult STAT3-blockade at tyrosine 705 (Y705) with JAK2-inhibitor WP1066 reduced microglial and astroglial activation to a more moderate degree, but in a pattern similar to the one produced by the cell-specific deletions. Our results suggest that STAT3 is a crucial factor in neonatal HI-brain damage and its removal in neurons or astrocytes, and, to some extent, inhibition of its phosphorylation via JAK2-blockade reduces inflammation and tissue loss. Overall, the protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal HI. Current data show that neuronal and astroglial STAT3 molecules are involved in the pathways underlying cell death, tissue loss and gliosis following neonatal hypoxia-ischaemia, but differ with respect to the target of their effect. Y705-phosphorylation contributes to hypoxic-ischaemic histopathology. Protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal hypoxia-ischaemia.

Is variation in copy number of the human beta defensin gene cluster associated with preterm birth?

BACKGROUND: Increased expression of antimicrobial peptides including human beta defensins (HBD) has been reported in the amniotic fluid and vaginal secretions of women who deliver preterm. We have previously shown that these women have increased first trimester serum HBD2. The gene encoding HBD2, DEFB4A, is part of a defensin beta (DEFB) cluster on chromosome 8 that is variable in copy number. Increased serum HBD2 is associated with increased DEFB copy number. We aimed to test the hypothesis that variation in DEFB copy number is associated with preterm birth. METHODS: In a retrospective, case-control study, genomic DNA and serum were extracted from blood collected from white European women at 11-13 weeks' gestation attending King's College Hospital between March 1, 2006, and Sept 30, 2010. DEFB copy number was determined by paralogue ratio test. Serum HBD2 concentration was measured by ELISA. Data were analysed with Pearson correlation (Excel, version 2010) and binary logistic regression (SPSS, version 20). FINDINGS: Cases were 102 women who either delivered preterm in the index pregnancy or had a history of preterm delivery. Controls were 152 women who had had at least one previous term delivery and delivered at term in the index pregnancy; they had no history of preterm birth. Modal copy number was 4 (range 2-7). Serum was available from 140 women (30 cases, 54 controls, 56 not included in the genetic association study). Median HBD2 concentration was 761·5 pg/mL (IQR 449·6-1232·0). There was no association between DEFB copy number and preterm birth, nor was there a correlation between copy number and serum HBD2 concentration. INTERPRETATION: Although variation in HBD2 protein expression in the first trimester might be useful to predict risk of preterm birth, we found no association between DEFB copy number and preterm birth. Nor did we find a correlation between DEFB copy number and serum HBD2 expression in the first trimester of pregnancy; this might be due to variation in regulatory sequences-some of which are progesterone and oestrogen sensitive-between individual copies. FUNDING: Wellcome Trust, Wellbeing of Women.

Modeling hormonal and inflammatory contributions to preterm and term labor using uterine temporal transcriptomics.

BACKGROUND: Preterm birth is now recognized as the primary cause of infant mortality worldwide. Interplay between hormonal and inflammatory signaling in the uterus modulates the onset of contractions; however, the relative contribution of each remains unclear. In this study we aimed to characterize temporal transcriptome changes in the uterus preceding term labor and preterm labor (PTL) induced by progesterone withdrawal or inflammation in the mouse and compare these findings with human data. METHODS: Myometrium was collected at multiple time points during gestation and labor from three murine models of parturition: (1) term gestation; (2) PTL induced by RU486; and (3) PTL induced by lipopolysaccharide (LPS). RNA was extracted and cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 system. Resulting RNA-Seq data were analyzed using multivariate modeling approaches as well as pathway and causal network analyses and compared against human myometrial transcriptome data. RESULTS: We identified a core set of temporal myometrial gene changes associated with term labor and PTL in the mouse induced by either inflammation or progesterone withdrawal. Progesterone withdrawal initiated labor without inflammatory gene activation, yet LPS activation of uterine inflammation was sufficient to override the repressive effects of progesterone and induce a laboring phenotype. Comparison of human and mouse uterine transcriptomic datasets revealed that human labor more closely resembles inflammation-induced PTL in the mouse. CONCLUSIONS: Labor in the mouse can be achieved through inflammatory gene activation yet these changes are not a requisite for labor itself. Human labor more closely resembles LPS-induced PTL in the mouse, supporting an essential role for inflammatory mediators in human "functional progesterone withdrawal." This improved understanding of inflammatory and progesterone influence on the uterine transcriptome has important implications for the development of PTL prevention strategies.

Peri- and Postnatal Effects of Prenatal Adenoviral VEGF Gene Therapy in Growth-Restricted Sheep.

Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period.

Specific Lipopolysaccharide Serotypes Induce Differential Maternal and Neonatal Inflammatory Responses in a Murine Model of Preterm Labor.

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models.

Activator protein 1 is a key terminal mediator of inflammation-induced preterm labor in mice.

Activation of uterine inflammatory pathways leads to preterm labor (PTL), associated with high rates of neonatal mortality and morbidity. The transcription factors nuclear factor κB (NFκB) and activator protein 1 (AP-1) regulate key proinflammatory and procontractile genes involved in normal labor and PTL. Here we show that NFκB activation normally occurs in the mouse myometrium at gestation day E18, prior to labor, whereas AP-1 and JNK activation occurs at labor onset. Where labor was induced using the progesterone receptor antagonist RU486, NFkB and AP-1/JNK activation both occurred at the time of labor (20 h compared to 60 h in DMSO-treated controls). Using an LPS (Escherichia coli: serotype O111)-induced PTL model that selectively activates AP-1 but not NFkB, we show that myometrial AP-1 activation drives production of cytokines (Il-6, Il-8, and Il-1ß), metalloproteinases (Mmp3 and Mmp10), and procontractile proteins (Cox-2 and Cx43) resulting in PTL after 7 h. Protein levels of CX43 and IL-1ß, and IL-1ß cleavage, were increased following LPS-induced activation of AP-1. Inhibition of JNK by SP600125 (30 mg/kg) delayed PTL by 6 h (7.5 vs. 13.5 h P<0.05). Our data reveal that NFκB activation is not a functional requirement for infection/inflammation-induced preterm labor and that AP-1 activation is sufficient to drive inflammatory pathways that cause PTL.

Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates.

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca(+2) -regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma. Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

Ultrasound Plane Pose Regression: Assessing Generalized Pose Coordinates in the Fetal Brain.

In obstetric ultrasound (US) scanning, the learner's ability to mentally build a three-dimensional (3D) map of the fetus from a two-dimensional (2D) US image represents a significant challenge in skill acquisition. We aim to build a US plane localization system for 3D visualization, training, and guidance without integrating additional sensors. This work builds on top of our previous work, which predicts the six-dimensional (6D) pose of arbitrarily oriented US planes slicing the fetal brain with respect to a normalized reference frame using a convolutional neural network (CNN) regression network. Here, we analyze in detail the assumptions of the normalized fetal brain reference frame and quantify its accuracy with respect to the acquisition of transventricular (TV) standard plane (SP) for fetal biometry. We investigate the impact of registration quality in the training and testing data and its subsequent effect on trained models. Finally, we introduce data augmentations and larger training sets that improve the results of our previous work, achieving median errors of 2.97 mm and 6.63° for translation and rotation, respectively.

Probe motion during mid-trimester fetal anomaly scan in the clinical setting: A prospective observational study.

INTRODUCTION: This study aims to investigate probe motion during full mid-trimester anomaly scans. METHODS: We undertook a prospective, observational study of obstetric sonographers at a UK University Teaching Hospital. We collected prospectively full-length video recordings of routine second-trimester anomaly scans synchronized with probe trajectory tracking data during the scan. Videos were reviewed and trajectories analyzed using duration, path metrics (path length, velocity, acceleration, jerk, and volume) and angular metrics (spectral arc, angular area, angular velocity, angular acceleration, and angular jerk). These trajectories were then compared according to the participant level of expertise, fetal presentation, and patient BMI. RESULTS: A total of 17 anomaly scans were recorded. The average velocity of the probe was 12.9 ± 3.4 mm/s for the consultants versus 24.6 ± 5.7 mm/s for the fellows (p = 0.02), the average acceleration 170.4 ± 26.3 mm/s2 versus 328.9 ± 62.7 mm/s2 (p = 0.02), and the average jerk 7491.7 ± 1056.1 mm/s3 versus 14944.1 ± 3146.3 mm/s3 (p = 0.02), the working volume 9.106 ± 4.106 mm3 versus 29.106 ± 11.106 mm3 (p = 0.03), respectively. The angular metrics were not significantly different according to the participant level of expertise, the fetal presentation, or to patients BMI. CONCLUSION: Some differences in the probe path metrics (velocity, acceleration, jerk and working volume) were noticed according to operator's level.