Early prediction of preeclampsia in pregnancy with cell-free RNA.

Liquid biopsies that measure circulating cell-free RNA (cfRNA) offer an opportunity to study the development of pregnancy-related complications in a non-invasive manner and to bridge gaps in clinical care1-4. Here we used 404 blood samples from 199 pregnant mothers to identify and validate cfRNA transcriptomic changes that are associated with preeclampsia, a multi-organ syndrome that is the second largest cause of maternal death globally5. We find that changes in cfRNA gene expression between normotensive and preeclamptic mothers are marked and stable early in gestation, well before the onset of symptoms. These changes are enriched for genes specific to neuromuscular, endothelial and immune cell types and tissues that reflect key aspects of preeclampsia physiology6-9, suggest new hypotheses for disease progression and correlate with maternal organ health. This enabled the identification and independent validation of a panel of 18 genes that when measured between 5 and 16 weeks of gestation can form the basis of a liquid biopsy test that would identify mothers at risk of preeclampsia long before clinical symptoms manifest themselves. Tests based on these observations could help predict and manage who is at risk for preeclampsia-an important objective for obstetric care10,11.

RNA profiles reveal signatures of future health and disease in pregnancy.

Maternal morbidity and mortality continue to rise, and pre-eclampsia is a major driver of this burden1. Yet the ability to assess underlying pathophysiology before clinical presentation to enable identification of pregnancies at risk remains elusive. Here we demonstrate the ability of plasma cell-free RNA (cfRNA) to reveal patterns of normal pregnancy progression and determine the risk of developing pre-eclampsia months before clinical presentation. Our results centre on comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data include 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks (s.d., 4.5 weeks) before delivery. We show that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal and fetal levels and can robustly predict pre-eclampsia, with a sensitivity of 75% and a positive predictive value of 32.3% (s.d., 3%), which is superior to the state-of-the-art method2. cfRNA signatures of normal pregnancy progression and pre-eclampsia are independent of clinical factors, such as maternal age, body mass index and race, which cumulatively account for less than 1% of model variance. Further, the cfRNA signature for pre-eclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of pre-eclampsia.

Association of maternal prenatal selenium concentration and preterm birth: a multicountry meta-analysis.

BACKGROUND: Selenium (Se), an essential trace mineral, has been implicated in preterm birth (PTB). We aimed to determine the association of maternal Se concentrations during pregnancy with PTB risk and gestational duration in a large number of samples collected from diverse populations. METHODS: Gestational duration data and maternal plasma or serum samples of 9946 singleton live births were obtained from 17 geographically diverse study cohorts. Maternal Se concentrations were determined by inductively coupled plasma mass spectrometry analysis. The associations between maternal Se with PTB and gestational duration were analysed using logistic and linear regressions. The results were then combined using fixed-effect and random-effect meta-analysis. FINDINGS: In all study samples, the Se concentrations followed a normal distribution with a mean of 93.8 ng/mL (SD: 28.5 ng/mL) but varied substantially across different sites. The fixed-effect meta-analysis across the 17 cohorts showed that Se was significantly associated with PTB and gestational duration with effect size estimates of an OR=0.95 (95% CI: 0.9 to 1.00) for PTB and 0.66 days (95% CI: 0.38 to 0.94) longer gestation per 15 ng/mL increase in Se concentration. However, there was a substantial heterogeneity among study cohorts and the random-effect meta-analysis did not achieve statistical significance. The largest effect sizes were observed in UK (Liverpool) cohort, and most significant associations were observed in samples from Malawi. INTERPRETATION: While our study observed statistically significant associations between maternal Se concentration and PTB at some sites, this did not generalise across the entire cohort. Whether population-specific factors explain the heterogeneity of our findings warrants further investigation. Further evidence is needed to understand the biologic pathways, clinical efficacy and safety, before changes to antenatal nutritional recommendations for Se supplementation are considered.

External validation of postnatal gestational age estimation using newborn metabolic profiles in Matlab, Bangladesh.

This study sought to evaluate the performance of metabolic gestational age estimation models developed in Ontario, Canada in infants born in Bangladesh. Cord and heel prick blood spots were collected in Bangladesh and analyzed at a newborn screening facility in Ottawa, Canada. Algorithm-derived estimates of gestational age and preterm birth were compared to ultrasound-validated estimates. 1036 cord blood and 487 heel prick samples were collected from 1069 unique newborns. The majority of samples (93.2% of heel prick and 89.9% of cord blood) were collected from term infants. When applied to heel prick data, algorithms correctly estimated gestational age to within an average deviation of 1 week overall (root mean square error = 1.07 weeks). Metabolic gestational age estimation provides accurate population-level estimates of gestational age in this data set. Models were effective on data obtained from both heel prick and cord blood, the latter being a more feasible option in low-resource settings.

The Zambian Preterm Birth Prevention Study (ZAPPS): Cohort characteristics at enrollment.

Background:Sub-Saharan Africa bears a disproportionate burden of preterm birth and other adverse outcomes. A better understanding of the demographic, clinical, and biologic underpinnings of these adverse outcomes is urgently needed to plan interventions and inform new discovery.  Methods:The Zambian Preterm Birth Prevention Study (ZAPPS) is a prospective observational cohort established at the Women and Newborn Hospital (WNH) in Lusaka, Zambia. We recruit pregnant women from district health centers and the WNH and offer ultrasound examination to determine eligibility. Participants receive routine obstetrical care, lab testing, midtrimester cervical length measurement, and serial fetal growth monitoring. At delivery, we assess gestational age, birthweight, vital status, and sex and assign a delivery phenotype. We collect blood, urine, and vaginal swab specimens at scheduled visits and store them in an on-site biorepository. In September 2017, enrollment of the ZAPPS Phase 1 - the subject of this report - was completed. Phase 2 - which is limited to HIV-uninfected women - reopened in January 2018.  Results:Between August 2015 and September 2017, we screened 1784 women, of whom 1450 (81.2%) met inclusion criteria and were enrolled. The median age at enrollment was 27 years (IQR 23-32) and thee median gestational age was 16 weeks (IQR 13-18). Among parous women (N=866; 64%), 21% (N=182) reported a prior miscarriage, 49% (N=424) reported a prior preterm birth, and 13% (N=116) reported a prior stillbirth. The HIV seroprevalence was 24%. Discussion:We have established a large cohort of pregnant women and newborns at the WHN to characterize the determinants of adverse birth outcomes in Lusaka, Zambia. Our overarching goal is to elucidate biological mechanisms in an effort to identify new strategies for early detection and prevention of adverse outcomes. We hope that findings from this cohort will help guide future studies, clinical care, and policy.

Postnatal gestational age estimation using newborn screening blood spots: a proposed validation protocol.

BACKGROUND: Knowledge of gestational age (GA) is critical for guiding neonatal care and quantifying regional burdens of preterm birth. In settings where access to ultrasound dating is limited, postnatal estimates are frequently used despite the issues of accuracy associated with postnatal approaches. Newborn metabolic profiles are known to vary by severity of preterm birth. Recent work by our group and others has highlighted the accuracy of postnatal GA estimation algorithms derived from routinely collected newborn screening profiles. This protocol outlines the validation of a GA model originally developed in a North American cohort among international newborn cohorts. METHODS: Our primary objective is to use blood spot samples collected from infants born in Zambia and Bangladesh to evaluate our algorithm's capacity to correctly classify GA within 1, 2, 3 and 4 weeks. Secondary objectives are to 1) determine the algorithm's accuracy in small-for-gestational-age and large-for-gestational-age infants, 2) determine its ability to correctly discriminate GA of newborns across dichotomous thresholds of preterm birth (≤34 weeks, <37 weeks GA) and 3) compare the relative performance of algorithms derived from newborn screening panels including all available analytes and those restricted to analyte subsets. The study population will consist of infants born to mothers already enrolled in one of two preterm birth cohorts in Lusaka, Zambia, and Matlab, Bangladesh. Dried blood spot samples will be collected and sent for analysis in Ontario, Canada, for model validation. DISCUSSION: This study will determine the validity of a GA estimation algorithm across ethnically diverse infant populations and assess population specific variations in newborn metabolic profiles.

Prevention of preterm birth: harnessing science to address the global epidemic.

Preterm birth is a leading cause of infant morbidity and mortality worldwide, but current interventions to prevent prematurity are largely ineffective. Preterm birth is increasingly recognized as an outcome that can result from a variety of pathological processes. Despite current research efforts, the mechanisms underlying these processes remain poorly understood and are influenced by a range of biological and environmental factors. Research with modern techniques is needed to understand the mechanisms responsible for preterm labor and birth and identify targets for diagnostic and therapeutic solutions. This review evaluates the state of reproductive science relevant to understanding the causes of preterm birth, identifies potential targets for prevention, and outlines challenges and opportunities for translating research findings into effective interventions.

Serious and life-threatening pregnancy-related infections: opportunities to reduce the global burden.

Michael Gravett and colleagues review the burden of pregnancy-related infections, especially in low- and middle-income countries, and offer suggestions for a more effective intervention strategy.

Epidemiology and aetiology of maternal bacterial and viral infections in low- and middle-income countries.

BACKGROUND: Maternal morbidity and mortality in low- and middle-income countries has remained exceedingly high. However, information on bacterial and viral maternal infections, which are important contributors to poor pregnancy outcomes, is sparse and poorly characterised. This review aims to describe the epidemiology and aetiology of bacterial and viral maternal infections in low- and middle-income countries. METHODS: A systematic search of published literature was conducted and data on aetiology and epidemiology of maternal infections was extracted from relevant studies for analysis. Searches were conducted in parallel by two reviewers (using OVID) in the following databases: Medline (1950 to 2010), EMBASE (1980 to 2010) and Global Health (1973 to 2010). RESULTS: Data from 158 relevant studies was used to characterise the epidemiology of the 10 most extensively reported maternal infections with the following median prevalence rates: Treponema pallidum (2.6%), Neisseria gonorrhoeae (1.5%), Chlamydia trachomatis (5.8%), Group B Streptococcus (8.6%), bacterial vaginosis (20.9%), hepatitis B virus (4.3%), hepatitis C virus (1.4%), Cytomegalovirus (95.7% past infection), Rubella (8.9% susceptible) and Herpes simplex (20.7%). Large variations in the prevalence of these infections between countries and regions were noted. CONCLUSION: This review confirms the suspected high prevalence of maternal bacterial and viral infections and identifies particular diseases and regions requiring urgent attention in public health policy planning, setting research priorities and donor funding towards reducing maternal morbidity and mortality in low- and middle-income countries.

Epidemiology and aetiology of maternal parasitic infections in low- and middle-income countries.

BACKGROUND: There have been very few systematic reviews looking at maternal infections in the developing world, even though cutting maternal mortality by three quarters is United Nation's Millennium Development Goal number five. This systematic review has two aims. The first is to present the prevalence of parasitic infections in the developing world over the last 30 years and the second is to evaluate the quality and distribution of research in this field. METHODS: A systematic review of Medline, EMBASE and Global Health databases was undertaken using pre-determined search criteria. Three levels of quality criteria for exclusion of inadequate studies identified 115 out of initial 8580 titles. The data were extracted for 5 domains: worldwide pathogen prevalence, year of study, study setting, sample size and diagnostic test for each pathogen. RESULTS: The initial search retrieved 8580 results. From these titles, 43 studies on malaria, 12 studies on helminths, 49 studies on Toxoplasma gondii, 7 studies on Chagas disease, 5 studies on Trichomonas, 1 leishmaniasis study and 1 study on trichinellosis were extracted for analysis. High prevalence of malaria was found in Gabon (up to 57%) India (55%), Cameroon (50%), Yemen (55%), Nigeria (up to 64%) and Ghana (54%). High prevalence of hookworm infections was found in Nepal at 78.8% and high values of Ascaris lumbricoides were found in Nepal, (56.2%), Kenya (52.3%) and Gabon (45.5%). High levels of Schistosoma mansoni were found in Zimbabwe (50%) and Tanzania (63.5%). The prevalence of active Toxoplasma gondii infection was found to be highest in India (27.7%). CONCLUSION: This study highlights the large burden of maternal parasitic infections globally. It may serve as a useful starting point for health policy development and research prioritization in this area.

Emerging biomarkers for the diagnosis of severe neonatal infections applicable to low resource settings.

More than 500 000 children die each year in low resource settings due to serious neonatal infections. Better diagnostics that can be utilized in these settings to identify infected infants have the potential to significantly reduce neonatal deaths and the associated morbidity. A systematic review was performed and identified more than 250 potential new biomarkers for the diagnosis of serious neonatal infections. Eight of these biomarkers were both high-performance and high-abundance (antithrombin, inter-α inhibitor proteins, interferon-γ inducible protein-10, interleukin-1 receptor antagonist, LPS binding protein, mannose binding lectin, serum amyloid A, resistin, visfatin), and are promising for the diagnosis of serious neonatal infections in low resource settings. Future clinical trials comparing these biomarkers with more traditional biomarkers seem warranted.

The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques.

BACKGROUND: ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. RESULTS: We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses. CONCLUSION: The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.

Activated cholinergic signaling provides a target in squamous cell lung carcinoma.

The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non-small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of alpha5 and beta3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC.

M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion.

The importance of acetylcholine as a neurotransmitter in the nervous system is well established, but little is yet known about its recently described role as an autocrine and paracrine hormone in a wide variety of nonneuronal cells. Consistent with the expression of acetylcholine in normal lung, small cell lung carcinoma (SCLC) synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms. The purpose of this study was to determine if interruption of autocrine muscarinic cholinergic signaling has potential to inhibit SCLC growth. Muscarinic receptor (mAChR) agonists caused concentration-dependent increases in intracellular calcium and mitogen-activated protein kinase (MAPK) and Akt phosphorylation in SCLC cell lines. The inhibitory potency of mAChR subtype-selective antagonists and small interfering RNAs (siRNAs) on acetylcholine-increased intracellular calcium and MAPK and Akt phosphorylation was consistent with mediation by M3 mAChR (M3R). Consistent with autocrine acetylcholine secretion stimulating MAPK and Akt phosphorylation, M3R antagonists and M3R siRNAs alone also caused a decrease in basal levels of MAPK and Akt phosphorylation in SCLC cell lines. Treatment of SCLC cells with M3R antagonists inhibited cell growth both in vitro and in vivo and also decreased MAPK phosphorylation in tumors in nude mice in vivo. Immunohistochemical staining of SCLC and additional cancer types showed frequent coexpression of acetylcholine and M3R. These findings suggest that M3R antagonists may be useful adjuvants for treatment of SCLC and, potentially, other cancers.

Leveraging human genomic information to identify nonhuman primate sequences for expression array development.

BACKGROUND: Nonhuman primates (NHPs) are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays. RESULTS: We have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey) orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey), Papio hamadryas (Baboon), and Chlorocebus aethiops (African green monkey, vervet). The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species. CONCLUSION: This study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays.