Plasma S1P and Sphingosine are not Different Prior to Pre-Eclampsia in Women at High Risk of Developing the Disease.

Sphingolipids like sphingosine-1-phosphate (S1P) have been implicated in the pathophysiology of pre-eclampsia. We hypothesized that plasma S1P would be increased in women at high risk of developing pre-eclampsia who subsequently develop the disease. Low circulating placental growth factor (PlGF) is known to be associated with development of pre-eclampsia; so further, we hypothesized that increased S1P would be associated with concurrently low PlGF. This was a case-control study using stored maternal blood samples from 14 to 24 weeks of pregnancy, collected from 95 women at increased risk of pre-eclampsia. Pregnancy outcome was classified as uncomplicated, preterm pre-eclampsia (<37 weeks), or term pre-eclampsia. Plasma lipids were extracted and analyzed by ultraperformance liquid chromatography coupled to electrospray ionization MS/MS to determine concentrations of S1P and sphingosine. Median plasma S1P was 0.339 nmol/ml, and median sphingosine was 6.77 nmol/l. There were no differences in the plasma concentrations of S1P or sphingosine in women who subsequently developed pre-eclampsia, no effect of gestational age, fetal sex, ethnicity, or the presence of pre-existing hypertension. There was a correlation between S1P and sphingosine plasma concentration (P < 0.0001). There was no relationship between S1P or sphingosine with PlGF. Previous studies have suggested that plasma S1P may be a biomarker of pre-eclampsia. In our larger study, we failed to demonstrate there are women at high risk of developing the disease. We did not show a relationship with known biomarkers of the disease, suggesting that S1P is unlikely to be a useful predictor of the development of pre-eclampsia later in pregnancy.

Murine models of advanced maternal age: a systematic review and meta-analysis.

In brief: Animal models have been developed to aid understanding of the increased incidence of adverse pregnancy complications observed in women of advanced maternal age (AMA). This systematic review of murine models of AMA demonstrates consistent effects of decreased litter size and fetal weight; this supports the future use of these models to determine pathophysiological mechanisms and test therapeutic strategies to improve poor pregnancy outcomes in AMA. Abstract: Advanced maternal age (AMA; ≥35 years of age) is associated with an increased risk of adverse pregnancy outcomes. To explore causes of adverse pregnancy outcomes in AMA, and to test candidate therapies, an increasing number of murine AMA models have been developed. The aim of this study was to systematically review the literature to assess whether murine AMA models demonstrate a reproducible effect on pregnancy outcomes. PubMed, Ovid, Web of Science and Google Scholar were searched. Studies that reported on pregnancy outcomes in AMA mice and rats were included; the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool evaluated the risk of bias. Eleven mouse and six rat studies were included. AMA mice and rats had reduced litter size (standardised mean difference (SMD): -1.59, 95% confidence interval (CI): -1.84, -1.34 for mice; SMD: -1.66, 95% (CI): -2.09, -1.23 for rats) and reduced fetal weight (SMD: -0.87, 95% CI: -1.24, -0.49 for mice; SMD: -1.05, 95% CI: -1.40, -0.69 for rats). Placental weight was increased in AMA mice (SMD: 0.62, 95% CI: 0.16, 1.08). Subgroup analysis indicated that C57Bl/6 mice had less heterogeneity than other, mostly outbred, mouse strains with regards to litter size (C57 strain I2 = 68.2% vs other strain types I2 = 85.7%). The risk of bias was high, mostly due to the lack of methodological detail and unclear reporting of findings. Murine models of AMA demonstrate similar adverse pregnancy outcomes to those observed in large human epidemiological studies. The reproducible phenotypes in AMA murine models allow the exploration of mechanisms underpinning poor pregnancy outcomes and the pursuit of therapeutic interventions.

The molecular landscape of well differentiated retroperitoneal liposarcoma.

Well differentiated liposarcoma (WD-LPS) is a relatively rare tumour, with fewer than 50 cases occurring per year in the UK. These tumours are both chemotherapy- and radiotherapy-resistant and present a significant treatment challenge requiring radical surgery. Little is known of the molecular landscape of these tumours and no current targets for molecular therapy exist. We aimed to carry out a comprehensive molecular characterisation of WD-LPS via whole genome sequencing, RNA sequencing, and methylation array analysis. A recurrent mutation within exon 1 of FOXD4L3 was observed (chr9:70,918,189A>T; c.322A>T; p.Lys108Ter). Recurrent mutations were also observed in Wnt signalling, immunity, DNA repair, and hypoxia-associated genes. Recurrent amplification of HGMA2 was observed, although this was in fact part of a general amplification of the region around this gene. Recurrent gene fusions in HGMA2, SDHA, TSPAN31, and MDM2 were also observed as well as consistent rearrangements between chromosome 6 and chromosome 12. Our study has demonstrated a recurrent mutation within FOXD4L3, which shows evidence of interaction with the PAX pathway to promote tumourigenesis. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Sacroperineal Reconstruction With Inferior Gluteal Artery Perforator Flaps After Resection of Locally Advanced Primary and Recurrent Anorectal Malignancy.

ABSTRACT: Modern interdisciplinary concepts with involvement of various surgical specialties can considerably reduce perioperative morbidity after sacroperineal resection of locally advanced primary or recurrent anorectal malignancies. Resultant defects can represent a major challenge for reconstruction particularly with chemoradiotherapy. The aim is to assess the long-term outcomes of sacroperineal reconstruction using inferior gluteal artery perforator flaps.We performed a retrospective data analysis on 31 patients who were treated with inferior gluteal artery perforator flaps (n = 61) over the period 2009-2021. The demographic data, comorbidities, operative details, and outcomes with special focus on wound infection and dehiscence were recorded.The median age was 42 year (range, 25-82 years) with preponderance of males (n = 21). The follow-up period ranged from 6 to 80 months. Early minor complications included superficial wound dehiscence (3), which was managed conservatively, whereas the major (2) included deep wound collection and infection (1), which required surgical drainage, and perineal hernia, which required repair. All flaps survived completely.Inferior gluteal artery perforator flaps are safe, robust, and reliable with less donor side morbidity and positive impact on quality of life. It should be considered as a valuable tool in the reconstructive armamentarium of sacroperineal defects within a multidisciplinary setting.

Evidence of adaptation of maternofetal transport of glutamine relative to placental size in normal mice, and in those with fetal growth restriction.

KEY POINTS: Fetal growth restriction (FGR) is a major risk factor for stillbirth and has significant impact upon lifelong health. A small, poorly functioning placenta, as evidenced by reduced transport of nutrients to the baby, underpins FGR. It remains unclear how a small but normal placenta differs from the small FGR placenta in terms of ability to transfer nutrients to the fetus. Placental transport of glutamine and glutamate, key amino acids for fetal growth, was assessed in normal mice and those with FGR. Glutamine and glutamate transport was greater in the lightest versus heaviest placenta in a litter of normally grown mice. Placentas of mice with FGR had increased transport capacity in mid-pregnancy, but this adaptation was insufficient in late pregnancy. Placental adaptations, in terms of increased nutrient transport (per gram) to compensate for small size, appear to achieve appropriate fetal growth in normal pregnancy. Failure of this adaptation might contribute to FGR. ABSTRACT: Fetal growth restriction (FGR), a major risk factor for stillbirth, and neonatal and adulthood morbidity, is associated with reduced placental size and decreased placental nutrient transport. In mice, a small, normal placenta increases its nutrient transport, thus compensating for its reduced size and maintaining normal fetal growth. Whether this adaptation occurs for glutamine and glutamate, two key amino acids for placental metabolism and fetal growth, is unknown. Additionally, an assessment of placental transport of glutamine and glutamate between FGR and normal pregnancy is currently lacking. We thus tested the hypothesis that the transport of glutamine and glutamate would be increased (per gram of tissue) in a small normal placenta [C57BL6/J (wild-type, WT) mice], but that this adaptation fails in the small dysfunctional placenta in FGR [insulin-like growth factor 2 knockout (P0) mouse model of FGR]. In WT mice, comparing the lightest versus heaviest placenta in a litter, unidirectional maternofetal clearance (Kmf ) of 14 C-glutamine and 14 C-glutamate (glutamine Kmf and glutamate Kmf ) was significantly higher at embryonic day (E) 18.5, in line with increased expression of LAT1, a glutamine transporter protein. In P0 mice, glutamine Kmf and glutamate Kmf were higher (P0 versus wild-type littermates, WTL) at E15.5. At E18.5, glutamine Kmf remained elevated whereas glutamate Kmf was similar between groups. In summary, we provide evidence that glutamine Kmf and glutamate Kmf adapt according to placental size in WT mice. The placenta of the growth-restricted P0 fetus also elevates transport capacity to compensate for size at E15.5, but this adaptation is insufficient at E18.5; this may contribute to decreased fetal growth.

Whole Genome Methylation Analysis of Nondysplastic Barrett Esophagus that Progresses to Invasive Cancer.

OBJECTIVE: To investigate differences in methylation between patients with nondysplastic Barrett esophagus who progress to invasive adenocarcinoma and those who do not. BACKGROUND: Identifying patients with nondysplastic Barrett esophagus who progress to invasive adenocarcinoma remains a challenge. Previous studies have demonstrated the potential utility of epigenetic markers for identifying this group. METHODS: A whole genome methylation interrogation using the Illumina HumanMethylation 450 array of patients with nondysplastic Barrett esophagus who either develop adenocarcinoma or remain static, with validation of findings by bisulfite pyrosequencing. RESULTS: In all, 12 patients with "progressive" versus 12 with "nonprogressive" nondysplastic Barrett esophagus were analyzed via methylation array. Forty-four methylation markers were identified that may be able to discriminate between nondysplastic Barrett esophagus that either progress to adenocarcinoma or remain static. Hypomethylation of the recently identified tumor suppressor OR3A4 (probe cg09890332) validated in a separate cohort of samples (median methylation in progressors 67.8% vs 96.7% in nonprogressors; P = 0.0001, z = 3.85, Wilcoxon rank-sum test) and was associated with the progression to adenocarcinoma. There were no differences in copy number between the 2 groups, but a global trend towards hypomethylation in the progressor group was observed. CONCLUSION: Hypomethylation of OR3A4 has the ability to risk stratify the patient with nondysplastic Barrett esophagus and may form the basis of a future surveillance program.

The impact of a human IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction.

Enhancing placental insulin-like growth factor (IGF) availability appears to be an attractive strategy for improving outcomes in fetal growth restriction (FGR). Our approach was the novel use of [Leu(27)]IGF-II, a human IGF-II analog that binds the IGF-II clearance receptor IGF-IIR in fetal growth-restricted (FGR) mice. We hypothesized that the impact of [Leu(27)]IGF-II infusion in C57BL/6J (wild-type) and endothelial nitric oxide synthase knockout (eNOS(-/-); FGR) mice would be to enhance fetal growth and investigated this from mid- to late gestation; 1 mg·kg(-1)·day(-1) [Leu(27)]IGF-II was delivered via a subcutaneous miniosmotic pump from E12.5 to E18.5. Fetal and placental weights recorded at E18.5 were used to generate frequency distribution curves; fetuses <5th centile were deemed growth restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum was collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu(27)]IGF-II treatment halved the number of FGR fetuses, reduced fetal(P = 0.028) and placental weight variations (P = 0.0032), and increased the numbers of pups close to the mean fetal weight (131 vs. 112 pups within 1 SD). Mixed-model analysis confirmed litter size to be negatively correlated with fetal and placental weight and showed that [Leu(27)]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional (14C)MeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu(27)]IGF-II-treated WT animals (P < 0.01). In eNOS(-/-) mice, [Leu(27)]IGF-II reduced the number of FGR fetuses(1 vs. 5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS(-/-) litters suggests the use of this analog as a means of standardizing and rescuing fetal growth, preferentially in the smallest offspring.

In vitro assessment of mouse fetal abdominal aortic vascular function.

Fetal growth restriction (FGR) affects 3-8% of human pregnancies. Mouse models have provided important etiological data on FGR; they permit the assessment of treatment strategies on the physiological function of both mother and her developing offspring. Our study aimed to 1) develop a method to assess vascular function in fetal mice and 2) as a proof of principle ascertain whether a high dose of sildenafil citrate (SC; Viagra) administered to the pregnant dam affected fetal vascular reactivity. We developed a wire myography methodology for evaluation of fetal vascular function in vitro using the placenta-specific insulin-like growth factor II (Igf2) knockout mouse (P0; a model of FGR). Vascular function was determined in abdominal aortas isolated from P0 and wild-type (WT) fetuses at embryonic day (E) 18.5 of gestation. A subset of dams received SC 0.8 mg/ml via drinking water from E12.5; data were compared with water-only controls. Using wire myography, we found that fetal aortic rings exhibited significant agonist-induced contraction, and endothelium-dependent and endothelium-independent relaxation. Sex-specific alterations in reactivity were noted in both strains. Maternal treatment with SC significantly attenuated endothelium-dependent and endothelium-independent relaxation of fetal aortic rings. Mouse fetal abdominal aortas reproducibly respond to vasoactive agents. Study of these vessels in mouse genetic models of pregnancy complications may 1) help to delineate early signs of abnormal vascular reactivity and 2) inform whether treatments given to the mother during pregnancy may impact upon fetal vascular function.

Establishing a COVID-secure site for elective surgery during the COVID pandemic: An observational study.

INTRODUCTION: Maintaining timely and safe delivery of major elective surgery during the COVID-19 pandemic is essential to manage cancer and time-critical surgical conditions. Our NHS Trust established a COVID-secure elective site with a level 2 Post Anaesthetic Care Unit (PACU) facility. Patients requiring level 3 Intensive Care Unit admission were transferred to a non-COVID-secure site. We investigated the relationship between perioperative anaesthetic care and outcomes. MATERIALS AND METHODS: All consecutive patients undergoing major surgery at the COVID-secure site between June and November 2020 were included. Patient demographics, operative interventions and 30-day outcomes were recorded. Multivariate logistic regression was used to determine the odds ratio of outcomes according to PACU length of stay and the use of spinal or epidural anaesthesia, with age, sex, malignancy status and American Society of Anesthesiologists grade as independent co-variables. RESULTS: There were 280 patients. PACU length of stay >23h was associated with increased 30-day complications. Epidural anaesthesia was associated with PACU length of stay >23h, increased total length of stay, increase hospital transfer and 30-day complications. Two patients acquired nosocomial COVID-19 following hospital transfer. DISCUSSION: Establishing a separate COVID-secure site has facilitated delivery of major elective surgery during the COVID-19 pandemic. Choice of perioperative anaesthesia and utilisation of PACU appear likely to affect the risk of adverse outcomes.

Effect of the anti-oxidant tempol on fetal growth in a mouse model of fetal growth restriction.

Fetal growth restriction (FGR) greatly increases the risk of perinatal morbidity and mortality and is associated with increased uterine artery resistance and levels of oxidative stress. There are currently no available treatments for this condition. The hypothesis that the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) would improve uterine artery function and rescue fetal growth was tested in a mouse model of FGR, using the endothelial nitric oxide synthase knockout mouse (Nos3(-/-)). Pregnant Nos3(-/-) and control C57BL/6J mice were treated with the superoxide dismutase-mimetic Tempol (1 mmol/L) or vehicle from Gestational Day 12.5 to 18.5. Tempol treatment significantly increased pup weight (P < 0.05) and crown-rump length (P < 0.01) in C57BL/6J and Nos3(-/-) mice. Uterine artery resistance was increased in Nos3(-/-) mice (P < 0.05); Tempol significantly increased end diastolic velocity in Nos3(-/-) mice (P < 0.05). Superoxide production in uterine arteries did not differ between C57BL/6J and Nos3(-/-) mice but was significantly increased in placentas from Nos3(-/-) mice (P < 0.05). This was not reduced by Tempol treatment. Placental System A activity was reduced in Nos3(-/-) mice (P < 0.01); this was not improved by treatment with Tempol. Treatment of Nos3(-/-) mice with Tempol, however, was associated with reduced vascular density in the placental bed (P < 0.05). This study demonstrated that treatment with the antioxidant Tempol is able to improve fetal growth in a mouse model of FGR. This was associated with an increase in uterine artery blood flow velocity but not an improvement in uterine artery function or placental System A activity.

eNOS knockout mouse as a model of fetal growth restriction with an impaired uterine artery function and placental transport phenotype.

Fetal growth restriction (FGR) is the inability of a fetus to reach its genetically predetermined growth potential. In the absence of a genetic anomaly or maternal undernutrition, FGR is attributable to "placental insufficiency": inappropriate maternal/fetal blood flow, reduced nutrient transport or morphological abnormalities of the placenta (e.g., altered barrier thickness). It is not known whether these diverse factors act singly, or in combination, having additive effects that may lead to greater FGR severity. We suggest that multiplicity of such dysfunction might underlie the diverse FGR phenotypes seen in humans. Pregnant endothelial nitric oxide synthase knockout (eNOS(-/-)) dams exhibit dysregulated vascular adaptations to pregnancy, and eNOS(-/-) fetuses of such dams display FGR. We investigated the hypothesis that both altered vascular function and placental nutrient transport contribute to the FGR phenotype. eNOS(-/-) dams were hypertensive prior to and during pregnancy and at embryonic day (E) 18.5 were proteinuric. Isolated uterine artery constriction was significantly increased, and endothelium-dependent relaxation significantly reduced, compared with wild-type (WT) mice. eNOS(-/-) fetal weight and abdominal circumference were significantly reduced compared with WT. Unidirectional maternofetal (14)C-methylaminoisobutyric acid (MeAIB) clearance and sodium-dependent (14)C-MeAIB uptake into mouse placental vesicles were both significantly lower in eNOS(-/-) fetuses, indicating diminished placental nutrient transport. eNOS(-/-) mouse placentas demonstrated increased hypoxia at E17.5, with elevated superoxide compared with WT. We propose that aberrant uterine artery reactivity in eNOS(-/-) mice promotes placental hypoxia with free radical formation, reducing placental nutrient transport capacity and fetal growth. We further postulate that this mouse model demonstrates "uteroplacental hypoxia," providing a new framework for understanding the etiology of FGR in human pregnancy.

Complete response to neoadjuvant chemoradiotherapy in rectal cancer is associated with RAS/AKT mutations and high tumour mutational burden.

BACKGROUND: Pathological complete response (pathCR) in rectal cancer is beneficial, as up to 75% of patients do not experience regrowth of the primary tumour, but it is poorly understood. We hypothesised that the changes seen in the pre-treatment biopsies of pathCR but not seen in residual tumour after chemoradiotherapy were the determinants of responsiveness. METHODS: Two groups of patients with either complete response (pathCR group, N = 24) or no response (poor response group, N = 24) were retrieved. Pre-treatment biopsies of cancers from these patients underwent high read depth amplicon sequencing for a targeted panel, exome sequencing, methylation profiling and immunohistochemistry for DNA repair pathway proteins. RESULTS: Twenty four patients who underwent pathCR and twenty-four who underwent poor response underwent molecular characterisation. Patients in the pathCR group had significantly higher tumour mutational burden and neoantigen load, frequent copy number alterations but fewer structural variants and enrichment for driver mutations in the PI3K/AKT/mTOR signalling pathway. There were no significant differences in tumour heterogeneity as measured by MATH score. Methylation analysis demonstrated enrichment for hypomethyation in the PI3K/AKT/mTOR signalling pathway. DISCUSSION: The phenomenon of pathCR in rectal cancer may be related to immunovisibility caused by a high tumour mutational burden phenotype. Potential therapy resistance mechanisms involve the PI3K/AKT/mTOR signalling pathway, but tumour heterogeneity does not seem to play a role in resistance.

Clinical-grade whole-genome sequencing and 3' transcriptome analysis of colorectal cancer patients.

BACKGROUND: Clinical-grade whole-genome sequencing (cWGS) has the potential to become the standard of care within the clinic because of its breadth of coverage and lack of bias towards certain regions of the genome. Colorectal cancer presents a difficult treatment paradigm, with over 40% of patients presenting at diagnosis with metastatic disease. We hypothesised that cWGS coupled with 3' transcriptome analysis would give new insights into colorectal cancer. METHODS: Patients underwent PCR-free whole-genome sequencing and alignment and variant calling using a standardised pipeline to output SNVs, indels, SVs and CNAs. Additional insights into the mutational signatures and tumour biology were gained by the use of 3' RNA-seq. RESULTS: Fifty-four patients were studied in total. Driver analysis identified the Wnt pathway gene APC as the only consistently mutated driver in colorectal cancer. Alterations in the PI3K/mTOR pathways were seen as previously observed in CRC. Multiple private CNAs, SVs and gene fusions were unique to individual tumours. Approximately 30% of patients had a tumour mutational burden of > 10 mutations/Mb of DNA, suggesting suitability for immunotherapy. CONCLUSIONS: Clinical whole-genome sequencing offers a potential avenue for the identification of private genomic variation that may confer sensitivity to targeted agents and offer patients new options for targeted therapies.

Evaluation of a bundle of care to reduce incisional surgical site infection after gastrointestinal surgery.

BACKGROUND: Surgical site infection (SSI) is associated with morbidity, mortality and increased care costs; many SSIs are considered preventable. The aim of the present study was to test implementation of a pragmatic, evidence-based bundle designed to reduce incisional SSI after emergency laparotomy and elective major lower gastrointestinal surgery. METHOD: This was a prospective before-and-after study. Data were collected before the intervention and for two separate subsequent time periods. An evidence-based bundle of care (BOC) was implemented; the primary outcome measure was incisional SSI at 30 days. The secondary outcome measure was 30-day unplanned readmissions. The initial post-intervention group, Group 2, assessed a variable number of potential impacting factors; however, due to funding and staffing levels the second post-bundle group, Group 3, focused on the core aspects of the BOC and rates of incisional SSI and readmission. RESULTS: In total, 99 patients were included in the 'before' group; and 71 in Group 2 and 92 in Group 3, the post-intervention groups. The incisional SSI rate was 29.3% (29/99) before and 28.2% (20/71) in Group 2 (P=0.873) and 21.7% (20/92) in Group 3 (P=0.234) after the intervention. After adjustment for confounders, the care bundle was associated with a non-significant reduction in SSI (Group 2: odds ratio [OR] = 0.93, 95% confidence interval [CI] = 0.45-1.93, P=0.0843). However, it was associated with significantly reduced readmissions 18.1% (18/99) before versus 5.6% (4/71) in Group 2 (OR = 0.236, 95% CI = 0.077-0.72, P=0.012) and 8.7% (8/92) in Group 3 (OR = 0.38, 95% CI = 0.16-0.9, P=0.029). Comparing the pre-bundle group to the post-bundle groups, there was an overall significant reduction in readmissions (P=0.003). This implies a number needed to treat of 8-11 patients to prevent one readmission. Adherence to antibiotic prophylaxis with the Trust guidelines increased from 91% to 99% (1 vs. 2, P=0.047). CONCLUSION: Introduction of the bundle was associated with a reduction in the observed rate of incisional SSI from 29.3% to 21.7%; significantly fewer patients required unplanned readmission. Use of the bundle was associated with significantly improved compliance with appropriate antimicrobial prophylaxis.

Human placental uptake of glutamine and glutamate is reduced in fetal growth restriction.

Fetal growth restriction (FGR) is a significant risk factor for stillbirth, neonatal complications and adulthood morbidity. Compared with those of appropriate weight for gestational age (AGA), FGR babies have smaller placentas with reduced activity of amino acid transporter systems A and L, thought to contribute to poor fetal growth. The amino acids glutamine and glutamate are essential for normal placental function and fetal development; whether transport of these is altered in FGR is unknown. We hypothesised that FGR is associated with reduced placental glutamine and glutamate transporter activity and expression, and propose the mammalian target of rapamycin (mTOR) signaling pathway as a candidate mechanism. FGR infants [individualised birth weight ratio (IBR) < 5th centile] had lighter placentas, reduced initial rate uptake of 14C-glutamine and 14C-glutamate (per mg placental protein) but higher expression of key transporter proteins (glutamine: LAT1, LAT2, SNAT5, glutamate: EAAT1) versus AGA [IBR 20th-80th]. In further experiments, in vitro exposure to rapamycin inhibited placental glutamine and glutamate uptake (24 h, uncomplicated pregnancies) indicating a role of mTOR in regulating placental transport of these amino acids. These data support our hypothesis and suggest that abnormal glutamine and glutamate transporter activity is part of the spectrum of placental dysfunction in FGR.

A missense mutation of ErbB2 produces a novel mouse model of stillbirth associated with a cardiac abnormality but lacking abnormalities of placental structure.

BACKGROUND: In humans, stillbirth describes the death of a fetus before birth after 28 weeks gestation, and accounts for approximately 2.6 million deaths worldwide annually. In high-income countries, up to half of stillbirths have an unknown cause and are described as "unexplained stillbirths"; this lack of understanding impairs efforts to prevent stillbirth. There are also few animal models of stillbirth, but those that have been described usually have significant placental abnormalities. This study describes a novel mutant murine model of fetal death with atrial conduction block due to an ErbB2 missense mutation which is not associated with abnormal placental morphology. METHODS: Phenotypic characterisation and histological analysis of the mutant mouse model was conducted. The mRNA distribution of the early cardiomyocyte marker Nkx2-5 was assessed via in situ hybridisation. Cardiac structure was quantified and cellular morphology evaluated by electron microscopy. Immunostaining was employed to quantify placental structure and cell characteristics on matched heterozygous and homozygous mutant placental samples. RESULTS: There were no structural abnormalities observed in hearts of mutant embryos. Comparable Nkx2-5 expression was observed in hearts of mutants and controls, suggesting normal cardiac specification. Additionally, there was no significant difference in the weight, placenta dimensions, giant cell characteristics, labyrinth tissue composition, levels of apoptosis, proliferation or vascularisation between placentas of homozygous mutant mice and controls. CONCLUSION: Embryonic lethality in the ErbB2 homozygous mutant mouse cannot be attributed to placental pathology. As such, we conclude the ErbB2M802R mutant is a model of stillbirth with a non-placental cause of death. The mechanism of the atrial block resulting from ErbB2 mutation and its role in embryonic death is still unclear. Studying this mutant mouse model could identify candidate genes involved in stillbirth associated with structural or functional cardiac defects.

Validation of epigenetic markers to identify colitis associated cancer: Results of module 1 of the ENDCAP-C study.

BACKGROUND: Chronic inflammation caused by ulcerative colitis (UC) causes a pro-neoplastic drive in the inflamed colon, leading to a markedly greater risk of invasive malignancy compared to the general population. Despite surveillance protocols, 50% of cases proceed to cancer before neoplasia is detected. The Enhanced Neoplasia Detection and Cancer Prevention in Chronic Colitis (ENDCaP-C) trial is an observational multi-centre test accuracy study to ascertain the role of molecular markers in improving the detection of dysplasia. We aimed to validate previously identified biomarkers of neoplasia in a retrospective cohort and create predictive models for later validation in a prospective cohort. METHODS: A retrospective analysis using bisulphite pyrosequencing of an 11 marker panel (SFRP1, SFRP2, SRP4, SRP5, WIF1, TUBB6, SOX7, APC1A, APC2, MINT1, RUNX3) in samples from 35 patients with cancer, 78 with dysplasia and 343 without neoplasia undergoing surveillance for UC associated neoplasia across 6 medical centres. Predictive models for UC associated cancer/dysplasia were created in the setting of neoplastic and non-neoplastic mucosa. FINDINGS: For neoplastic mucosa a five marker panel (SFRP2, SFRP4, WIF1, APC1A, APC2) was accurate in detecting pre-cancerous and invasive neoplasia (AUC = 0.83; 95% CI: 0.79, 0.88), and dysplasia (AUC = 0.88; (0.84, 0.91). For non-neoplastic mucosa a four marker panel (APC1A, SFRP4, SFRP5, SOX7) had modest accuracy (AUC = 0.68; 95% CI: 0.62,0.73) in predicting associated bowel neoplasia through the methylation signature of distant non-neoplastic colonic mucosa. INTERPRETATION: This multiplex methylation marker panel is accurate in the detection of ulcerative colitis associated dysplasia and neoplasia and is currently being validated in a prospective clinical trial. FUNDING: The ENDCAP-C study was funded by the National Institute for Health Research Efficacy and Mechanism Evaluation (EME) Programme (11/100/29).

Development and functional capacity of transplanted rat metanephroi.

BACKGROUND: Transplantation of embryonic kidneys (metanephroi) offers a potential solution to the problem of kidney donor shortage. The aim of this study was to characterise the haemodynamic capacity of transplanted rat metanephroi and to determine the number and maturity of the tubules. METHODS: Metanephroi from E15 Lewis rat embryos were transplanted adjacent to the abdominal aorta of uninephrectomised adult female syngeneic Lewis rats. Twenty-one days later, a single metanephros ureter was anastomosed to the host's urinary system. Three months later animals were prepared for standard clearance measurements. RESULTS: Effective renal blood flow (149 +/- 33 microl min(-1) per g kidney weight) and glomerular filtration rate (17 +/- 9 microl min(-1) per g kidney weight), standardised to kidney weight, were significantly lower in transplanted metanephroi compared with control adult kidneys (P < 0.001); renal vascular resistance (934 +/- 209 mmHg ml min(-1) per g kidney weight) was significantly higher (P < 0.001). Nephron number in transplanted metanephroi was significantly greater than that of E21 kidneys (P < 0.01) but lower than that of postnatal day (PND) 1 kidneys (P < 0.001). Angiotensin II type 2 receptor mRNA expression, a marker of nephrogenesis, was markedly reduced in metanephroi. Aquaporins 1 and 2, epithelial Na channel and Na-K-2Cl cotransporter type 2 mRNA and protein were expressed in transplanted metanephroi; the urea transporters-A1, 2 and 3 were absent. Vascular markers (alpha-smooth muscle actin and CD31) were identified in metanephroi but their expression did not differ from that of E21 and PND 1 kidneys. CONCLUSIONS: This study shows that metanephroi continue to develop post-transplantation but only reach a stage of development equivalent to that of a normal rat kidney at birth.

The problem with using the birthweight:placental weight ratio as a measure of placental efficiency.

INTRODUCTION: The ratio of birthweight to placental weight (BW:PW) is often used as a measure of placental efficiency in humans and animals. However, ratios have properties that are known to lead to spurious results. An alternative approach is the use of residuals from regression, which reflect whether birthweight is higher or lower than expected for a given placental weight, given the population pattern. We hypothesized that biologically meaningful measures of placental efficiency would differ between placentas with and without pathology, and between adverse and normal perinatal and postnatal outcomes. METHODS: We examined associations between measures of placental efficiency (BW:PW ratio or residuals) and placental pathology, Apgar scores and infant death using National Collaborative Perinatal Project data (4645 preterm births and 28497 term births). RESULTS: BW:PW ratios and residuals were significantly lower in placentas showing pathologies including signs of large infarcts or hemorrhage, although many of these differences were small. Low BW:PW ratios and residuals were also associated with low Apgar scores and increased risk of postnatal death. Whereas residuals were lower in term placentas that appeared immature by microscopic examination, the opposite was true for BW:PW ratios. CONCLUSION: The BW:PW ratio produced an artefact whereby histologically less mature placentas at term appeared to be more "efficient" than mature placentas, illustrating a known problem with the use of ratios. For other traits, residuals generally showed differences between placentas with and without pathology that were as great as those seen with BW:PW ratios, and often showed stronger associations with adverse outcomes.