Medical Research Council Grant Research Articles

Epidemiology of Alcohol Misuse and Illicit Drug Use Among Young People Aged 15-24 Years in Fishing Communities in Uganda

Background: Alcohol use and illicit drug use are global problems that increase substantially during adolescence, and heighten the risk of HIV infection. We determined the prevalence of and risk factors for alcohol misuse and illicit drug use among young Ugandans in fishing communities, a recognised “key population” for HIV infection. Methods: We conducted a cross-sectional survey among young people (15-24 years) in fishing communities in Koome, Uganda, in December 2017-July 2018. Using Audio Assisted Self-Interviewing, we collected data on sociodemographic characteristics and alcohol use, including Alcohol Use Disorders Identification Test (AUDIT) and timeline-follow-back-calendar (TLFB). Blood samples were analysed for HIV, HSV2, and phosphatidyl ethanol (PEth 16:0/18:1). Urine samples were analysed for illicit drugs. Findings: Among 1281 participants (52.7% male, mean age 20 years), 659 (51.4%) reported ever drinking alcohol, 248 (19.4%) had 12-month-AUDIT≥8, and 261 (20.5%) had whole blood PEth 16:0/18:1 concentration ≥20ng/mL indicating significant consumption. In multivariable analyses, PEth 16:0/18:1≥20ng/mL, AUDIT≥8 and binge drinking (≥6 standard drinks per drinking occasion in the previous month from TLFB) were all strongly associated with older age, low education, smoking, and HSV2. Illicit drug use prevalence was 4.4% and was associated with older age, low education, being single, and smoking. Interpretation: Levels of alcohol misuse were high among young people in fishing communities and associated with HSV2, a proxy for risky sexual behaviour. Alcohol and illicit drug harm reduction services and HIV prevention programs in Uganda should prioritise young people in fishing communities. Funding Statement: This award is jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement, and by the MRC Tropical Epidemiology Group (TEG). This work was funded in part by IAVI and made possible by the support of many donors, including: the Bill & Melinda Gates Foundation, the Ministry of Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan in partnership with The World Bank, the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency for Development Cooperation (NORAD), the United Kingdom Department for International Development (DFID), and the United States Agency for International Development (USAID). Declaration of Interests: E Webb and H Weiss received salary funding from MRC Grant Reference MR/K012126/1. Other authors have no conflict of interest to declare. Ethic Approval Statement: We obtained ethical approval from the Uganda Virus Research Institute research and ethics committee, the London School of Hygiene and Tropical Medicine ethics committee, and the Uganda National Council for Science and Technology. Any participants requiring medical treatment were referred to nearby government health centres where free treatment was offered.

Probing human sperm metabolism using 13C-magnetic resonance spectroscopy.

STUDY QUESTIONCan 13C-Magnetic Resonance Spectroscopy (MRS) of selected metabolites provide useful information about human sperm metabolism and how glycolysis or oxidative phosphorylation are used by different sperm populations?SUMMARY ANSWERSperm populations, prepared by density gradient centrifugation (DGC) and incubated with either 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate, showed consistent evidence of metabolism generating principally lactate and more intermittently bicarbonate, and significantly more lactate was produced from 13Cu-glucose by vital or motile sperm recovered from the 40/80% interface compared to those from the pellet, which could not be accounted for by differences in the non-sperm cells present.WHAT IS KNOWN ALREADYPrevious studies have focused on CO2 or other specific metabolite production by human sperm and there remains considerable debate about whether glycolysis and/or oxidative phosphorylation is the more important pathway for ATP production in sperm.STUDY DESIGN, SIZE, DURATIONSperm populations were prepared by DGC and subjected to 13C-MRS to answer the following questions. (i) Is it possible to detect human sperm metabolism of 13C substrates implicated in energy generation? (ii) What are the kinetics of such reactions? (iii) Do different sperm populations (e.g. ‘80%’ pellet sperm and ‘40%’ interface sperm) utilise substrates in the same way? Semen samples from 97 men were used in these experiments; 52 were used in parallel for aims (i) and (ii) and 45 were used for aim (iii).PARTICIPANTS/MATERIALS, SETTING, METHODSSperm populations were prepared from ejaculates of healthy men using a Percoll/Phosphate Buffered Saline (PBS) DGC and then incubated with a range of 13C-labelled substrates (13Cu-glucose, 13Cu-fructose, 13C1-pyruvate, 13C1-butyrate, 13C3-lactate, 13C2,4-D-3-hydroxybutyrate, 13C5-l-glutamate, 13C1,2-glycine or 13Cu-galactose) along with penicillin/streptomycin antibiotic at 37°C for 4 h, 24 h or over 48 h for an estimated rate constant. Sperm concentration, vitality and motility were measured and, for a subset of experiments, non-sperm cell concentration was determined. A 9.4 T magnetic resonance spectrometer was used to acquire 1D 13C, inverse gated 1H decoupled, MRS spectra. Spectrum processing was carried out using spectrometer software and Matlab scripts to determine peak integrals for each spectrum.MAIN RESULTS AND THE ROLE OF CHANCE 13Cu-glucose, 13Cu-fructose and 13C1-pyruvate were consistently converted into lactate and, to a lesser extent, bicarbonate. There was a significant correlation between sperm concentration and lactate peak size for 13Cu-glucose and 13Cu-fructose, which was not observed for 13C1-pyruvate. The lactate peak did not correlate with the non-sperm cell concentration up to 6.9 × 106/ml. The concentration of 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate (1.8, 3.6, 7.2 or 14.4 mM) had no influence on the size of the observed lactate peak over a 4 h incubation. The rate of conversion of 13C1-pyruvate to lactate was approximately three times faster than for 13Cu-glucose or 13Cu-fructose which were not significantly different from each other. After incubating for 4 h, the utilisation of 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate by sperm from the ‘40%’ interface of the DGC was no different from those from the pellet when normalised to total sperm concentration. However, after normalising by either the vital or motile sperm concentration, there was a significant increase in conversion of 13Cu-glucose to lactate by ‘40%’ interface sperm compared to pellet sperm (Vital = 3.3 ± 0.30 × 106 vs 2.0 ± 0.21 × 106; P = 0.0049; Motile = 7.0 ± 0.75 × 106 vs 4.8 ± 0.13 × 106; P = 0.0032. Mann–Whitney test P < 0.0055 taken as statistically significant). No significant differences were observed for 13Cu-fructose or 13C1-pyruvate.LARGE SCALE DATANot applicable.LIMITATIONS, REASONS FOR CAUTIONOnly 13C labelled metabolites that accumulate to a sufficiently high concentration can be observed by 13C MRS. For this reason, intermediary molecules in the metabolic chain are difficult to observe without trapping the molecule at a particular step using inhibitors. Non-sperm cell concentration was typical of the general population and no link was found between these cells and the magnitude of the 13C-lactate peak. However, it is possible that higher concentrations than the maximum observed (6.9 × 106/ml) may contribute to exogenous substrate metabolism in other experiments.WIDER IMPLICATIONS OF THE FINDINGS 13C-MRS can provide information on the underlying metabolism of multiple pathways in live sperm. Dysfunction in sperm metabolism, as a result of either impaired enzymes of lack of metabolisable substrate, could be detected in sperm by a non-destructive assay, potentially offering new treatment options to improve overall sperm quality and outcomes for reproduction.STUDY FUNDING AND COMPETING INTERESTSThis work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Hyperandrogenism and Insulin Resistance-Induced Fetal Loss: Evidence for Placental Mitochondrial Abnormalities and Elevated ROS Production in PCOS-Like Pregnant Rats

Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanism of PCOS-induced fetal loss during pregnancy is unclear. We used pregnant rats treated with 5α-dihydrotestosterone (DHT) and insulin to investigate the effects of hyperandrogenism and insulin resistance on fetal survival and to determine the molecular link between PCOS conditions and placental dysfunction during pregnancy. We show that pregnant rats chronically treated with a combination of DHT and insulin exhibited endocrine aberrations such as hyperandrogenism and insulin resistance that mirror pregnant PCOS patients. Compared to normal pregnant rats, PCOS-like pregnant rats had greater fetal loss and subsequently decreased litter sizes. This negative effect was accompanied by impaired trophoblast differentiation, abnormal glycogen accumulation, and decreased angiogenesis in the placenta. We report that over-production of reactive oxygen species (ROS) in the placenta, mitochondrial dysfunction, and disturbed SOD1 and Keap1/Nrf2 antioxidant responses constitute important contributors to fetal loss in PCOS-like pregnant rats. Furthermore, dose-dependent treatment with the antioxidant N-acetylcysteine (NAC) improved fetal survival in PCOS-like pregnant rats dependent on Keap1/Nrf2 and NFκB signaling molecules. However, similar treatment resulted in fetal loss in normal pregnant rats, most likely due to PCOS-like endocrine abnormality induced by NAC. Our work suggests that the deleterious effects of hyperandrogenism and insulin resistance on fetal survival are related to a constellation of mitochondrial-ROS-SOD1/Nrf2 changes in the placenta. Our findings also suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy. Funding: This work was supported by the Swedish Medical Research Council (grant number 10380), the Swedish federal government under the LUA/ALF agreement (grant number ALFGBG-147791), the Jane and Dan Olsson’s Foundation, and the Adlerbert Research Foundation to HB and LRS as well as the National Natural Science Foundation of China (grant number 81774136), the Project of Young Innovation Talents in Heilongjiang Provincial University (grant number UNPYSCT-2015121), the Project of Innovation Talents (Young Reserve Talents) in Harbin city (grant number 2015RAQYJ089), and the Scientific Research Foundation of the Heilongjiang Province for Returned Chinese Scholars to YZ. ANSP is supported by a Royal Society Dorothy Hodgkin Research Fellowship. Declaration of Interest: The authors declare that no competing interests exist. Ethical Approval: The study was approved and authorized by the Animal Care and Use Committee of the Heilongjiang University of Chinese Medicine, China (HUCM 2015-0112), and followed the National Institutes of Health guidelines on the care and use of animals.

A Signature of Gene Expression in Peripheral Blood that Enables Earlier Detection of Acute Rejection in Kidney Transplant Recipients

Background Acute rejection (AR) is often indicated by an increase in creatinine levels, after a considerable graft damage has already occurred. There is, therefore, a need for early identification of AR. Monitoring of RNA gene signatures in peripheral blood and urine samples offers the opportunity for surveillance of the recipient immune system and earlier detection of acute rejection. Samples We examined RT-qPCR expression of 22 literature-based candidate genes in 335 longitudinal whole-blood samples from 27 stable kidney transplant recipients (KTRs) from the KALIBRE study (Kidney Allograft Immune Biomarkers of Rejection Episodes) and 237 samples from 27 KTRs with a biopsy-proven cellular rejection during the first post-transplant year. Creatinine in stable KTRs reached no higher than 20% above the reference range and had less than 15% standard deviation. Methods To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene expression as the residuals of linear mixed-effects models with stable patients. To select a parsimonious AR signature, we used penalised logistic regression with a pre-biopsy sample in rejectors and the median gene expression for each stable patient. As a test set we used the longitudinal data. We were also able to examine longitudinal expression of genes as well as effect of different anti-rejection therapies. We subsequently tested the performance of our signature in a second cohort. Results A 7-gene signature of AR showed area under the curve (AUC) 0.95 (95% confidence interval 0.91-1.00), sensitivity 0.85, specificity 0.93, positive and negative predictive values 0.92 and 0.86 and cross-validated AUC 0.85 (0.75-0.95). The estimated probability of rejection increased as early as six weeks prior to the AR biopsy and decreased after administration of treatment. Notably, gene expression in stable KTRs, as well as in rejectors, showed a pronounced variability, with 22% AR-positives longitudinal samples.Conclusion Molecular marker alterations in blood emerge well ahead of the time of clinically overt AR. Monitoring of a gene-expression signature of AR in peripheral blood could unravel AR-related pro-inflammatory activity and provide information on subclinical processes and further justification for a biopsy in cases of sustained kidney dysfunction with no marked change in creatinine. Medical Research Council (MRC) (grants G0801537/ID: 88245 and MRC Centre for Transplantation, – MRC grant no. MR/J006742/1). MHF - HEALTH-F5– 2010–260687: The ONE Study. SC, PM and MPH-F received funding from FP7-HEALTH-2012-INNOVATION-1 project number 305147: BIO-DrIM. National Institute for Health Research Biomedical Research Centre at Guy’s and St Thomas’ and King’s College London. NIHR Health Informatics Collaborative.

Agreement between diagnoses of otitis media by audiologists and otolaryngologists in Aboriginal Australian children.

To determine the degree of agreement of diagnoses by audiologists and otolaryngologists of otitis media (OM) in Aboriginal children. Cross-sectional study of agreement between diagnoses. Study of Environment on Aboriginal Resilience and Child Health (SEARCH), a prospective cohort study of Aboriginal children attending four Aboriginal Community Controlled Health Services in New South Wales (three metropolitan, one regional) during 2008-2012. 1310 of 1669 SEARCH participants (78.5%; mean age, 7.0 years; SD, 4.4 years) were assessed and received a diagnosis from one of five experienced audiologists. Test results (but not case histories) were forwarded to one of three otolaryngologists for blinded independent assessment. Agreement of OM diagnoses by audiologists and otolaryngologists at ear and child levels; correctness of audiologist diagnoses (otolaryngologist diagnosis as reference). Paired diagnoses by audiologists and otolaryngologists were available for 863 children at the child level and 1775 ears (989 children) at the ear level. Otolaryngologists diagnosed OM in 251 children (29.1%), including 11 (1.3%) with tympanic membrane perforation, and in 396 ears (22.3%), including 12 (0.7%) with perforation. Agreement between audiologists and otolaryngologists for OM at the ear level was 92.2% (κ = 0.78; 95% CI, 0.74-0.82), and at the child level 91.7% (κ = 0.81; 95% CI, 0.77-0.85). No otolaryngologist-diagnosed perforation was missed by audiologists. Among 1000 children triaged by an audiologist, there would be 45 false positives and 30 false negatives when compared with assessments by an otolaryngologist, with no missed perforations. There was substantial agreement between audiologists' and otolaryngologists' diagnoses of OM in a high prevalence population of Aboriginal children. In settings with limited access to otolaryngologists, audiologists may appropriately triage children and select those requiring specialist review.

Lung function trajectories throughout childhood in survivors of very preterm birth: a longitudinal cohort study

Data on longitudinal respiratory follow-up after preterm birth in the surfactant era are scarce and of increasing importance, with concerns that preterm survivors are destined for early onset chronic obstructive airway disease. We aimed to comprehensively assess lung function longitudinally from early childhood to mid-childhood in very preterm children (≤32 weeks gestation), and to explore factors negatively impacting on lung function trajectories. Preterm children (with and without bronchopulmonary dysplasia) and healthy term children as controls were studied. All preterm participants were born at 32 weeks' gestation or earlier at King Edward Memorial Hospital, Perth, WA, Australia, between 1997 and 2003. Bronchopulmonary dysplasia was defined as at least 28 days of supplemental oxygen requirement as assessed at 36 weeks' post-menstrual age. Spirometry, oscillatory mechanics, gas exchange, lung volumes, and respiratory symptoms were assessed at three visits, two in early childhood (4-8 years) and one in mid-childhood (9-12 years). CT of the chest was done in preterm children in mid-childhood. Respiratory symptoms were documented via questionnaire at each visit. Data were analysed longitudinally using linear mixed models. 200 very preterm children (126 with bronchopulmonary dysplasia and 74 without bronchopulmonary dysplasia) and 67 healthy term control children attended 458 visits between age 4 and 12 years. Chest CT was done on 133 preterm children at a mean age of 10·9 (SD 0·6) years. Preterm children, with and without bronchopulmonary dysplasia, had declines in spirometry z-scores over time compared with controls: forced expiratory volume in 1 s (FEV1), forced expiratory flow at 25-75% of the pulmonary volume, and FEV1/forced vital capacity all declined by at least 0·1 z-score per year in children with bronchopulmonary dysplasia (all p<0·001). Respiratory mechanics and gas exchange also deteriorated over time in children with bronchopulmonary dysplasia (relative to term controls, respiratory system reactance at 8 Hz decreased by -0·05 z-score per year [95% CI -0·08 to -0·01; p=0·006] and diffusing capacity of the lungs for carbon monoxide decreased by -0·03 z-score per year [95% CI -0·06 to -0·01; p=0·048]). Preterm children with bronchial wall thickening on chest CT (suggestive of inflammation) had bigger decreases in spirometry outcomes through childhood. For example, children with bronchial wall thickening on chest CT had an FEV1 z-score decline of -0·61 (95% CI -1·03 to-0·19; p=0·005) more than those without. Similarly, children exposed to tobacco smoke, those with earlier gestation, or those requiring more neonatal supplemental oxygen declined at a faster rate. Lung function trajectories are impaired in survivors of very preterm birth. Survivors with bronchopulmonary dysplasia, ongoing respiratory symptoms, or CT changes reflecting inflammation have the poorest trajectories and might be at increased risk of lung disease in later life. Close targeted pulmonary follow-up of these individuals is necessary. National Health and Medical Research Council grants APP634519, APP1073301 (to SJS), APP1077691 (to JJP), and APP1025550 (to GLH), Princess Margret Hospital Foundation, and Raine Medical Foundation.

Interleukin-34 is present at the fetal-maternal interface and induces immunoregulatory macrophages of a decidual phenotype in vitro.

Is the newly discovered cytokine interleukin (IL)-34 expressed at the human fetal-maternal interface in order to influence polarization of monocytes into macrophages of a decidual immunoregulatory phenotype? IL-34 was found to be present at the fetal-maternal interface, in both fetal placenta and maternal decidua, and it was able to polarize monocytes into macrophages of a decidual phenotype. IL-34 was shown to bind to the same receptor as macrophage-colony stimulating factor (M-CSF), which has an important immunomodulatory role at the fetal-maternal interface, for example by polarizing decidual macrophages to an M2-like regulatory phenotype. IL-34 is known to regulate macrophage subsets, such as microglia and Langerhans cells, but its presence at the fetal-maternal interface is unknown. The presence of IL-34 at the fetal-maternal interface was evaluated by immunohistochemistry (IHC) and ELISA in placental and decidual tissues as well as in isolated trophoblast cells and decidual stromal cells obtained from first trimester elective surgical terminations of pregnancy (n = 49). IL-34 expression was also assessed in third trimester placental biopsies from women with (n = 21) or without (n = 15) pre-eclampsia. The effect of IL-34 on macrophage polarization was evaluated in an in vitro model of blood monocytes obtained from healthy volunteers (n = 14). In this model, granulocyte macrophage-colony stimulating factor (GM-CSF) serves as a growth factor for M1-like polarization, and M-CSF as a growth factor for M2-like polarization. First trimester placental and decidual tissues were obtained from elective pregnancy terminations. Placental biopsies were obtained from women with pre-eclampsia and matched controls in the delivery ward. Polarization of macrophages in vitro was determined by flow-cytometric phenotyping and secretion of cytokines and chemokines in cell-free supernatants by multiplex bead assay. Our study shows that IL-34 is produced at the fetal-maternal interface by both placental cyto- and syncytiotrophoblasts and decidual stromal cells. We also show that IL-34, in vitro, is able to polarize blood monocytes into macrophages with a phenotype (CD14highCD163+CD209+) and cytokine secretion pattern similar to that of decidual macrophages. The IL-34-induced phenotype was similar, but not identical to the phenotype induced by M-CSF, and both IL-34- and M-CSF-induced macrophages were significantly different (P < 0.05-0.0001 depending on marker) from GM-CSF-polarized M1-like macrophages. Our findings suggest that IL-34 is involved in the establishment of the tolerant milieu found at the fetal-maternal interface by skewing polarization of macrophages into a regulatory phenotype. Although it is clear that IL-34 is present at the fetal-maternal interface and polarizes macrophages in vitro, its precise role in vivo remains to be established. The recently discovered cytokine IL-34 is present at the fetal-maternal interface and has immunomodulatory properties with regard to induction of decidual macrophages, which are important for a healthy pregnancy. Knowledge of growth factors related to macrophage polarization can potentially be translated to treatment of pregnancy complications involving dysregulation of this process. This study was funded by grants from the Medical Research Council (Grant K2013-61X-22310-01-04), the Research Council of South-East Sweden (FORSS), and the County Council of Östergötland, Sweden. No author has any conflicts of interest to declare.

Correction: Long-term intra-individual reproducibility of heart rate dynamics during exercise and recovery in the UK Biobank cohort.

[This corrects the article DOI: 10.1371/journal.pone.0183732.].

N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays

Is there a specific surface marker that identifies human endometrial epithelial progenitor cells with adult stem cell activity using in vitro assays? N-cadherin isolates clonogenic, self-renewing human endometrial epithelial progenitor cells with high proliferative potential that differentiate into cytokeratin+ gland-like structures in vitro and identifies their location in some cells of gland profiles predominantly in basalis endometrium adjacent to the myometrium. Human endometrium contains a small population of clonogenic, self-renewing epithelial cells with high proliferative potential that differentiate into large gland-like structures, but their identity and location is unknown. Stage-specific embryonic antigen-1 (SSEA-1) distinguishes the epithelium of basalis from functionalis and is a marker of human post-menopausal (Post-M) endometrial epithelium. Prospective observational study of endometrial epithelial cells obtained from hysterectomy samples taken from 50 pre-menopausal (Pre-M) and 24 Post-M women, of which 4 were from women who had taken daily estradiol valerate 2 mg/day for 8 weeks prior. Gene profiling was used to identify differentially expressed surface markers between fresh EpCAM (Epithelial Cell Adhesion Molecule)-magnetic bead-selected basalis-like epithelial cells from Post-M endometrium compared with predominantly functionalis epithelial cells from Pre-M endometrium and validated by qRT-PCR. In vitro clonogenicity and self-renewal assays were used to assess the stem/progenitor cell properties of magnetic bead-sorted N-cadherin+ and N-cadherin- epithelial cells. The cellular identity, location and phenotype of N-cadherin+ cells was assessed by dual colour immunofluorescence and confocal microscopy for cytokeratin, proliferative status (Ki-67), ERα, SSEA-1, SOX9 and epithelial mesenchymal transition (EMT) markers on full thickness human endometrium. CDH2 (N-cadherin gene) was one of 11 surface molecules highly expressed in Post-M compared to Pre-M endometrial epithelial cells. N-cadherin+ cells comprise a median 16.7% (n = 8) and 20.2% (n = 5) of Pre-M endometrial epithelial cells by flow cytometry and magnetic bead sorting, respectively. N-cadherin+ epithelial cells from Pre-M endometrium were more clonogenic than N-cadherin- cells (n = 12, P = 0.003), underwent more population doublings (n = 7), showed greater capacity for serial cloning (n = 7) and differentiated into cytokeratin+ gland-like organoids. N-cadherin immunolocalised to the lateral and apical membrane of epithelial cells in the bases of glands in the basalis of Pre-M endometrium and Post-M gland profiles, co-expressing cytokeratin, ERα but not SSEA-1 or SOX9, which localized on gland profiles proximal to N-cadherin+ cells. N-cadherin+ cells were quiescent (Ki-67-) in the basalis and in Post-M endometrial glands and co-localized with EMT markers vimentin and E-cadherin. The raw and processed data files from the gene microarray have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus data set with accession number GSE35221. This is a descriptive study in human endometrium only using in vitro stem cell assays. The differential ability of N-cadherin+ and N-cadherin-cells to generate endometrial glands in vivo was not determined. A small number of uterine tissues analysed contained adenomyosis for which N-cadherin has been implicated in epithelial-EMT. A new marker enriching for human endometrial epithelial progenitor cells identifies a different and potentially more primitive cell population than SSEA-1, suggesting a potential hierarchy of epithelial differentiation in the basalis. Using N-cadherin as a marker, the molecular and cellular characteristics of epithelial progenitor cells and their role in endometrial proliferative disorders including endometriosis, adenomyosis and thin dysfunctional endometrium can be investigated. This research was supported by Cancer Council Victoria grant 491079 (C.E.G.) and Australian National Health and Medical Research Council grants 1021127 (C.E.G.), 1085435 (C.E.G., J.A.D.), 145780 and 288713 (C.N.S.), RD Wright Career Development Award 465121 (C.E.G.), Senior Research Fellowship 1042298 (C.E.G.), the Victorian Government's Operational Infrastructure Support and an Australian Postgraduate Award (HPTN), and China Council Scholarship (L.X.). The authors have nothing to declare.

Long-term intra-individual reproducibility of heart rate dynamics during exercise and recovery in the UK Biobank cohort.

BackgroundThe heart rate (HR) response to exercise provides useful information about the autonomic function and has prognostic value, but its reproducibility over a long period of time, a critical requirement for using it as a clinical biomarker, is undetermined.AimTo determine the intra-individual reproducibility of HR dynamics during sub-maximum exercise and one minute recovery.Methods1187 individuals from the Cardio physical fitness assessment test of the UK Biobank repeated a standard exercise stress test twice (recall time 34.2 ± 2.8 months) and were prospectively studied.Results821 individuals complied with inclusion criteria for reproducibility analysis, including peak workload differences between assessments ≤10 W. Intra-individual correlation between HR profile during the first and the second assessment was very high and higher than inter-individual correlation (0.92±0.08 vs 0.87±0.11, p<0.01). Intra-individual correlation of indices describing HR dynamics was: ρ = 0.81 for maximum HR during exercise; ρ = 0.71 for minimum HR during recovery; ρ = 0.70 for HR changes during both exercise and recovery; Intra-individual correlation was higher for these indices of HR dynamics than for resting HR (ρ = 0.64). Bland-Altman plots demonstrated good agreement between HR indices estimated during the first and second assessment. A small but consistent bias was registered for all repeated measurements. The intra-individual consistency of abnormal values was about 60–70%.ConclusionsThe HR dynamics during exercise and recovery are reproducible over a period of 3 years, with moderate to strong intra-individual reproducibility of abnormal values.

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTIONCan 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa?SUMMARY ANSWERPercoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface.WHAT IS KNOWN ALREADYCurrent methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm.STUDY DESIGN, SAMPLES/MATERIALS, METHODSSperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet (‘80%’ sperm) and the 40/80 interface (‘40%’ sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of ‘40%’ and ‘80%’ sperm.MAIN RESULTS AND THE ROLE OF CHANCEDGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65–0.67, lactate/lipid ROC AUC = 0.86–0.87.LIMITATIONS, REASONS FOR CAUTIONOnly 3–4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the ‘40%’ and ‘80%’ sperm.WIDER IMPLICATIONS OF THE FINDINGS 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility.STUDY FUNDING AND COMPETING INTEREST(S)This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Effect of N-Wasp knockout on tumour burden and survival in murine models of intestinal tumorigenesis and its potential as a prognostic biomarker in human colorectal cancer

BackgroundAPC loss is a common initiating event in colorectal cancer but additional mutations are required for progression along the adenoma–carcinoma pathway. The Neural Wiskott–Aldrich syndrome protein (N-WASP) is crucial for invasion in a variety of cancers. Its role in colorectal cancer has not been investigated; however, it is implicated in Wnt signalling (the pathway affected when APC function is lost). The aim of this project was to investigate the role of N-WASP in intestinal tumorigenesis and its potential as a prognostic biomarker. MethodsThe effect of N-Wasp knockout (N-Waspfl/fl) on survival and tumour burden was investigated using two established murine models of intestinal tumorigenesis (Apcfl/+ and Apcfl/+ KrasG12D/+). Immunohistochemistry for N-WASP was performed on two human tissue microarrays—namely, colorectal cancers with linked clinicopathological data and long-term follow-up, and early cancers diagnosed via bowel screening. N-WASP expression was assessed by weighted histoscore, and correlation with survival was tested. FindingsN-Wasp knockout decreased survival in the Apcfl/+ model (median survival 143 days [IQR 127·3–167·0] vs 269 [228·8–304·5], p<0·0001) and developed more small intestinal tumours (mean 39·7 tumours [SD 14·24] vs 8 [4·34], p<0·0001). Interestingly, although mean tumour size was decreased in Apcfl/+ N-Waspfl/fl mice compared with Apcfl/+ mice (0·538 mm2 [SD 0·238] vs 1·547 [0·609], p=0·0317), there was no difference in mean size of the largest tumour. There was no effect of N-Wasp knockout on survival or small intestinal tumour burden in the Apcfl/+ KrasG12D/+ model. However, they developed fewer colonic tumours (mean 16·8 [SD 10·2] vs 35·4 [11·5], p=0·0018). In the human tissue microarray studies, there was no difference in overall or cancer-specific survival between tumours with high and low N-WASP expression. InterpretationWe found that loss of N-Wasp promotes early tumorigenesis in Apc-deficient intestinal epithelium in mice. N-WASP stabilises epithelial cell–cell junctions and might be a regulator of Wnt signalling. Further research is required to ascertain whether either of these roles explains the effects seen here and whether it promotes invasion and metastasis in more advanced intestinal cancers, as seen in other tissues. FundingFunded by a Medical Research Council grant via the Scottish Clinical Pharmacology and Pathology Programme (SCP3).

Non-growing follicle density is increased following adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy in the adult human ovary.

Do the chemotherapeutic regimens of ABVD (adriamycin, bleomycin, vinblastine and dacarbazine) or OEPA-COPDAC (combined vincristine, etoposide, prednisone, doxorubicin (OEPA) and cyclophosphamide, vincristine, prednisone, dacarbazine (COPDAC)) used to treat Hodgkin lymphoma (HL), affect the density, morphology and in vitro developmental potential of human ovarian follicles? Ovarian tissue from women treated with ABVD contained a higher density of non-growing follicles (NGFs) per cubic millimetre and increased numbers of multiovular follicles but showed reduced in vitro growth compared with patients with lymphoma who had not received chemotherapy, patients treated with OEPA-COPDAC, age-matched healthy women and age-related model-predicted values. Chemotherapy regimens can cause a loss of follicles within the ovary, which depends on the drugs given. Early stage HL is commonly treated by ABVD, a non-alkylating regimen that apparently has ovarian sparing qualities; thus it is important to investigate the histological appearance and distribution of follicles within ABVD-treated ovarian tissue. Thirteen ovarian biopsies were obtained from HL patients (six adolescents and seven adults) and one biopsy from a non-HL patient. Two HL patients and the non-HL patient had received no treatment prior to biopsy collection. The remaining 11 HL patients received one of two regimens: ABVD or OEPA-COPDAC. Tissue was analysed histologically and compared to biopsies from healthy women, and in a subgroup of patients, tissue was cultured for 6 days in vitro. Ovarian biopsies were obtained from patients undergoing ovarian cryopreservation for fertility preservation and from healthy women at the time of Caesarian section ('obstetric tissue'). Follicle number and maturity were evaluated in sections of ovarian cortical tissue, and compared to an age-related model of mean follicle density and to age-matched contemporaneous biopsies. The developmental potential of follicles was investigated after 6 days of tissue culture. A total of 6877 follicles were analysed. ABVD-treated tissue contained a higher density of NGFs per cubic millimetre (230 ± 17) (mean ± SEM) than untreated (110 ± 54), OEPA-COPDAC-treated (50 ± 27) and obstetric (20 ± 4) tissue (P < 0.01), with follicle density 9-21 SD higher than predicted by an age-related model. Biovular and binucleated NGFs occurred frequently in ABVD-treated and in adolescent-untreated tissue but were not observed in OEPA-COPDAC-treated or obstetric tissue, although OEPA-COPDAC-treated tissue contained a high proportion of morphologically abnormal oocytes (52% versus 23% in untreated, 22% in ABVD-treated and 25% in obstetric tissue; P < 0.001). Activation of follicle growth in vitro occurred in all groups, but in ABVD-treated samples there was very limited development to the secondary stage, whereas in untreated samples from lymphoma patients growth was similar to that observed in obstetric tissue (untreated; P < 0.01 versus ABVD-treated, NS versus obstetric). N/A LIMITATIONS, REASONS FOR CAUTION: Although a large number of follicles were analysed, these data were derived from a small number of biopsies. The mechanisms underpinning these observations have yet to be determined and it is unclear how they relate to future fertility. This study confirms that the number of NGFs is not depleted following ABVD treatment, consistent with clinical data that female fertility is preserved. Our findings demonstrate that immature follicle density can increase as well as decrease following at least one chemotherapy treatment. This is the first report of morphological and follicle developmental similarities between ABVD-treated tissue and the immature human ovary. Further experiments will investigate the basis for the marked increase in follicle density in ABVD-treated tissue. Funded by UK Medical Research Council Grants G0901839 and MR/L00299X/1. The authors have no competing interests.

Understanding the relative valuation of research impact: a best–worst scaling experiment of the general public and biomedical and health researchers

Objectives(1) To test the use of best–worst scaling (BWS) experiments in valuing different types of biomedical and health research impact, and (2) to explore how different types of research impact...

Maternal first trimester serum levels of free-beta human chorionic gonadotrophin and male genital anomalies.

Are maternal first trimester levels of serum free-beta hCG associated with the development of hypospadias or undescended testis (UDT) in boys? Overall, first trimester maternal levels of serum free-beta hCG are not associated with hypospadias or UDT. However, elevated levels were found in severe phenotypes (proximal hypospadias and bilateral UDT) suggesting an altered pathway of hormonal release in early pregnancy. Human chorionic gonadotrophin peaks in first trimester of pregnancy stimulating fetal testosterone production, which is key to normal male genital development. Endocrine-disrupting insults early in pregnancy have been associated with increased risk of common genital anomalies in males such as hypospadias and UDT. One plausible etiological pathway is altered release of hCG. We conducted a record-linkage study of two separate populations of women attending first trimester aneuploidy screening in two Australian states, New South Wales (NSW) and Western Australia (WA), in 2006-2009 and 2001-2003, respectively. Included were women who gave birth to a singleton live born male infant. There were 12 099 boys from NSW and 10 518 from WA included, of whom 90 and 77 had hypospadias; and 107 and 109 UDT, respectively. Serum levels of free-beta hCG were ascertained from laboratory databases and combined with relevant birth outcomes and congenital anomalies via record linkage of laboratory, birth, congenital anomalies and hospital data. Median and quartile levels of gestational age specific free-beta hCG multiple of the median (MoM) were compared between affected and unaffected boys. Logistic regression was used to evaluate the association between levels of free-beta hCG MoM and hypospadias or UDT, stratified by suspected placental dysfunction and co-existing anomalies. Where relevant, pooled analysis was conducted. There was no difference in median hCG levels amongst women with an infant with hypospadias (NSW = 0.88 MoM, P = 0.83; WA = 0.84 MoM, P = 0.76) or UDT (NSW = 0.89 MoM, P = 0.54; WA = 0.95 MoM, P = 0.95), compared with women with an unaffected boy (NSW = 0.92 MoM; WA = 0.88 MoM). Low (<25th centile) or high (>75th centile) hCG levels were not associated with hypospadias or UDT, nor when stratifying by suspected placental dysfunction and co-existing anomalies. However, there was a tendency towards high levels for severe types, although confidence intervals were wide. When combining NSW and WA results, high hCG MoM levels (>75th centile) were associated with increased risk of proximal hypospadias (odds ratio (OR) 4.34; 95% CI: 1.08-17.4) and bilateral UDT (OR 2.86; 95% CI: 1.02-8.03). There were only small numbers of proximal hypospadias and bilateral UDT in both cohorts and although we conducted pooled analyses, results reported on these should be interpreted with caution. Gestational age by ultrasound may have been inaccurately estimated in small and large for gestational age fetuses affecting hCG MoM calculation in those pregnancies. Despite the reliability of our datasets in identifying adverse pregnancy outcomes, we did not have pathology information to confirm tissue lesions in the placenta and therefore our composite outcome should be considered as a proxy for placental dysfunction. This is one of the largest population-based studies examining the association between maternal first trimester serum levels of free-beta hCG and genital anomalies-hypospadias and UDT; and the first to compare specific phenotypes by severity. Overall, our findings does not support the hypothesis that alteration in maternal hCG levels is associated with the development of male genital anomalies; however, high hCG free-beta levels found in severe types suggest different underlying etiology involving higher production and secretion of hCG. These findings require further exploration and replication. This work was funded by the National Health and Medical Research Council (NHMRC) grant APP1047263. N.N. is supported by a NHMRC Career Development Fellowship APP1067066. C.B. was supported by a NHMRC Principal Research Fellowship #634341. The funding agencies had no role in the design, analysis, interpretation or reporting of the findings. There are no competing interests. Not applicable.

Cholesterol‐Mediated Degradation of 7‐Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

Cholesterol is essential for normal embryogenesis, but in excess, it can be detrimental to human health. To achieve balanced cholesterol levels, enzymes involved in its synthesis possess many layers of regulation. Here, we investigated the post‐translational regulation of 7‐dehydrocholesterol reductase (DHCR7), a terminal enzyme of cholesterol synthesis, converting 7‐dehydrocholesterol to cholesterol. In the absence of functional DHCR7, accumulation of 7‐dehydrocholesterol and a lack of cholesterol production leads to the devastating developmental disorder, Smith‐Lemli‐Opitz Syndrome (SLOS). We found that statin treatment can ameliorate the low DHCR7 expression observed with common SLOS mutants. Furthermore, we show that DHCR7 protein is rapidly turned over and this degradation is induced by the cholesterol product itself, resulting in decreased DHCR7 protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7‐dehydrocholesterol, which in turn leads to an increased production of vitamin D. Thus, DHCR7 is an important enzymatic switch between cholesterol and vitamin D synthesis.Support or Funding InformationNational Health and Medical Research Council Grant (1060515), UNSW Goldstar Award, UNSW Career Advancement Fund

The natural history of Chlamydia trachomatis infection in women: a multi-parameter evidence synthesis.

The evidence base supporting the National Chlamydia Screening Programme, initiated in 2003, has been questioned repeatedly, with little consensus on modelling assumptions, parameter values or evidence sources to be used in cost-effectiveness analyses. The purpose of this project was to assemble all available evidence on the prevalence and incidence of Chlamydia trachomatis (CT) in the UK and its sequelae, pelvic inflammatory disease (PID), ectopic pregnancy (EP) and tubal factor infertility (TFI) to review the evidence base in its entirety, assess its consistency and, if possible, arrive at a coherent set of estimates consistent with all the evidence. Evidence was identified using 'high-yield' strategies. Bayesian Multi-Parameter Evidence Synthesis models were constructed for separate subparts of the clinical and population epidemiology of CT. Where possible, different types of data sources were statistically combined to derive coherent estimates. Where evidence was inconsistent, evidence sources were re-interpreted and new estimates derived on a post-hoc basis. An internally coherent set of estimates was generated, consistent with a multifaceted evidence base, fertility surveys and routine UK statistics on PID and EP. Among the key findings were that the risk of PID (symptomatic or asymptomatic) following an untreated CT infection is 17.1% [95% credible interval (CrI) 6% to 29%] and the risk of salpingitis is 7.3% (95% CrI 2.2% to 14.0%). In women aged 16-24 years, screened at annual intervals, at best, 61% (95% CrI 55% to 67%) of CT-related PID and 22% (95% CrI 7% to 43%) of all PID could be directly prevented. For women aged 16-44 years, the proportions of PID, EP and TFI that are attributable to CT are estimated to be 20% (95% CrI 6% to 38%), 4.9% (95% CrI 1.2% to 12%) and 29% (95% CrI 9% to 56%), respectively. The prevalence of TFI in the UK in women at the end of their reproductive lives is 1.1%: this is consistent with all PID carrying a relatively high risk of reproductive damage, whether diagnosed or not. Every 1000 CT infections in women aged 16-44 years, on average, gives rise to approximately 171 episodes of PID and 73 of salpingitis, 2.0 EPs and 5.1 women with TFI at age 44 years. The study establishes a set of interpretations of the major studies and study designs, under which a coherent set of estimates can be generated. CT is a significant cause of PID and TFI. CT screening is of benefit to the individual, but detection and treatment of incident infection may be more beneficial. Women with lower abdominal pain need better advice on when to seek early medical attention to avoid risk of reproductive damage. The study provides new insights into the reproductive risks of PID and the role of CT. Further research is required on the proportions of PID, EP and TFI attributable to CT to confirm predictions made in this report, and to improve the precision of key estimates. The cost-effectiveness of screening should be re-evaluated using the findings of this report. The Medical Research Council grant G0801947.

Absolute or relative effects? Arm-based synthesis of trial data.

Absolute or relative effects? Arm-based synthesis of trial data.

P08.24 Alternative evidence sources present a consistent picture of the population excess fraction (pef) of pelvic inflammatory disease caused bychlamydia trachomatisinfection in england

Introduction Pelvic Inflammatory Disease (PID) is a leading cause of both Tubal Factor Infertility (TFI) and Ectopic pregnancy. Chlamydia trachomatis (CT) is an important risk factor for the development of PID but it is also caused by other infections. The extent of the role of CT in the aetiology of PID is unclear and knowledge of its role is critical for assessing the efficacy and cost-effectiveness of CT screening. Methods We define the population attributable fraction as the population Excess fraction (PEF) ‘the estimated proportion of the disease that would be removed by eliminating the exposure from the population’. We consider seven separate methods of estimating age-group specific PEFs using a variety of data-sources. Estimates are based on evidence from routine data sources, survey’s, case-control studies and randomised controlled trials. Estimation is carried out using a Bayesian approach. This method ensures that all of the uncertainty in the data and estimates for all parameters is fully propagated into the estimates of PEF. Results There is a high degree of consistency between these estimates of PEF. We estimate that in women aged 16–44 around 20% of PID is caused by CT in England. However, this could be as low as 5% or as high as 40%. There is a steep decline in PEF with age with the PEF dropping by a factor of around 5-fold between younger and older women. Conclusion There is good evidence that CT plays an important role in the development of PID, especially in younger women. Future studies of the relationship between CT and PID should focus more on the relationship between risk and age. Disclosure of interest statement This study was funded by the Medical Research Council grant G0801947. No pharmaceutical grants were received in the development of this study. PH has received funding from Cepheid directly and indirectly for lecturing on point of care testing and undertaking research on the cost effectiveness of their CT/NG assay. Has also received payment from Atlas Genetics for an article in the Parliamentary Review on the benefits of point of care technology in improving the cost effectiveness of sexual health services. Has also received an honorarium from Hologic for an education talk on STI diagnostics.

O15.5 The natural history ofchlamydia trachomatisinfection in women: a multi-parameter evidence synthesis

Introduction The National Chlamydia Screening Programme (NCSP) was initiated in England in 2003. However, the evidence base supporting it has been questioned repeatedly, with little consensus on modelling assumptions, parameter values, or evidence sources to be used in cost-effectiveness analyses. Methods We reviewed all the available evidence on the Chlamydia trachomatis (CT) in the UK and its sequelae. Evidence was identified using “high yield” strategies. Bayesian Multi-Parameter Evidence Synthesis models were constructed for separate sub-parts of the clinical and population epidemiology of CT. Where possible different types of data were statistically combined to derive coherent estimates. Where evidence was inconsistent, evidence sources were re-interpreted and new estimates derived on a post hoc basis. Results An internally coherent set of estimates was generated, consistent with a multi-faceted evidence base. Among the key findings were: the risk of pelvic inflammatory disease (PID), both symptomatic and asymptomatic, following an untreated CT infection is 17% (6,29), and the risk of salpingitis is 7% (2,14). In women aged 16–24 screened at annual intervals, at best 61% (55,67) of CT-related PID and 22% (7,43) of all PID could be prevented. For women aged 16–44 in the UK, the proportions of PID, ectopic pregnancy and tubal factor infertility (TFI) that are attributable to CT are estimated to be 20% (6,38), 5% (1,12), and 29% (9,56) respectively. Conclusion The study establishes a set of interpretations of the major studies and study designs, under which a coherent set of estimates can be generated for UK decision-makers. CT is a significant cause of PID and TFI. CT screening is of benefit to the individual, but detection and treatment of incident infection may be more beneficial. Further research is required to confirm predictions, and to improve the precision of key estimates. The cost-effectiveness of screening should be re-evaluated using these estimates. Disclosure of interest statement This study was funded by the Medical Research Council grant G0801947. No pharmaceutical grants were received in the development of this study. PH has received funding from Cepheid directly and indirectly for lecturing on point of care testing and undertaking research on the cost effectiveness of their CT/NG assay. Has also received payment from Atlas Genetics for an article in the Parliamentary Review on the benefits of point of care technology in improving the cost effectiveness of sexual health services. Has also received an honorarium from Hologic for an education talk on STI diagnostics.