Systematic Identification of Spontaneous Preterm Birth-Associated RNA Transcripts in Maternal Plasma

<div><h3>Background</h3><p>Spontaneous preterm birth (SPB, before 37 gestational weeks) is a major cause of perinatal mortality and morbidity, but its pathogenesis remains unclear. Studies on SPB have been hampered by the limited availability of markers for SPB in predelivery clinical samples that can be easily compared with gestational age-matched normal controls. We hypothesize that SPB involves aberrant placental RNA expression, and that such RNA transcripts can be detected in predelivery maternal plasma samples, which can be compared with gestational age-matched controls.</p> <h3>Principal Findings</h3><p>Using gene expression microarray to profile essentially all human genes, we observed that 426 probe signals were changed by >2.9-fold in the SPB placentas, compared with the spontaneous term birth (STB) placentas. Among the genes represented by those probes, we observed an over-representation of functions in RNA stabilization, extracellular matrix binding, and acute inflammatory response. Using RT-quantitative PCR, we observed differences in the RNA concentrations of certain genes only between the SPB and STB placentas, but not between the STB and term elective cesarean delivery placentas. Notably, 36 RNA transcripts were observed at placental microarray signals higher than a threshold, which indicated the possibility of their detection in maternal plasma. Among them, the <em>IL1RL1</em> mRNA was tested in plasma samples taken from 37 women. It was detected in 6 of 10 (60%) plasma samples collected during the presentation of preterm labor (≤32.9 weeks) in women eventually giving SPB, but was detected in only 1 of 27 (4%) samples collected during matched gestational weeks from women with no preterm labor (Fisher exact test, p = 0.00056).</p> <h3>Conclusion</h3><p>We have identified 36 SPB-associated RNA transcripts, which are possibly detectable in maternal plasma. We have illustrated that the <em>IL1RL1</em> mRNA was more frequently detected in predelivery maternal plasma samples collected from women resulting in SPB than the gestational-age matched controls.</p> </div

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

RNA transcripts that were up-regulated in the SPB placentas, relative to the STB placentas, according to the microarray experiment (Mann-Whitney test, adjusted by the Benjamini and Hochberg method, adjusted p<0.05).

a<p>The RNA transcript interrogated by each probeset is shown as the gene symbol and name approved by the HUGO Gene Nomenclature Committee at the European Bioinformatics Institute (<a href="http://www.genenames.org" target="_blank">http://www.genenames.org</a>). Certain probesets interrogating the same gene may be listed as different GenBank accession numbers. This may reflect the different isoforms being interrogated. Other details on the probesets are freely accessible at <a href="http://www.netaffx.com" target="_blank">http://www.netaffx.com</a>.</p>b<p>Only 15 transcripts with the greatest fold-change values are shown. The remaining 225 transcripts are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s006" target="_blank">Table S5</a>.</p>c<p>Up-regulated transcripts with higher expression levels than the <i>PLAC4</i> mRNA in the placenta, according to the microarray data.</p><p>SPB, spontaneous preterm birth; STB, spontaneous term birth.</p

Gene ontology (GO) terms over-represented in the list of genes that were down-regulated in the SPB placentas, compared with the STB placentas.

<p>Terms with the highest fold of over-representation from each category are shown. The remaining terms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s009" target="_blank">Table S8</a>.</p

Gene ontology (GO) terms over-represented in the list of genes that were up-regulated in the SPB placentas, compared with the STB placentas.

<p>Terms with the highest fold of over-representation from each category are shown. The remaining terms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s008" target="_blank">Table S7</a>.</p

RNA transcripts that were down-regulated in the SPB placentas, relative to the STB placentas, according to the microarray experiment (Mann-Whitney test, adjusted by the Benjamini and Hochberg method, adjusted p<0.05).

a<p>If a probeset interrogates a RNA transcript with no gene symbol (“—”) approved by the HUGO Gene Nomenclature Committee at the European Bioinformatics Institute (<a href="http://www.genenames.org" target="_blank">http://www.genenames.org</a>), it is shown as a “transcribed locus”. Other details on the probesets are freely accessible at <a href="http://www.netaffx.com" target="_blank">http://www.netaffx.com</a>.</p>b<p>Only 15 transcripts with the greatest fold-change values are shown. The remaining 171 transcripts are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s007" target="_blank">Table S6</a>.</p><p>SPB, spontaneous preterm birth; STB, spontaneous term birth.</p

Characteristics of participants in the analysis of maternal plasma samples.

a<p>Mann-Whitney rank sum test for continuous variables. Fisher exact test for nominal variables.</p><p>SPB, spontaneous preterm birth.</p

Systematic identification of SPB-associated placental transcripts in maternal plasma.

<p>Shown in the Venn diagram are the number of microarray probesets representing transcripts which are highly expressed (pale yellow) and aberrantly expressed (red) in the SPB placentas. Transcripts fulfilling both criteria (orange) are potentially detectable in maternal plasma samples obtained from women eventually resulting in SPB. These probesets represent 36 transcripts and are shown in the box, along with the fold-change up-regulated (up arrow) or down-regulated (down arrow) in the SPB placentas, compared with the STB placentas. For transcript represented by more than one probesets, its average fold-change is shown. Transcripts with no official gene symbols are shown in their GenBank accession numbers. Transcripts chosen for RT-qPCR are shown in bold. SPB, spontaneous preterm birth. STB, spontaneous term birth.</p