Genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women.

BACKGROUND: Common fetal aneuploidies include Down syndrome (trisomy 21 or T21), Edward syndrome (trisomy 18 or T18), Patau syndrome (trisomy 13 or T13), Turner syndrome (45,X), Klinefelter syndrome (47,XXY), Triple X syndrome (47,XXX) and 47,XYY syndrome (47,XYY). Prenatal screening for fetal aneuploidies is standard care in many countries, but current biochemical and ultrasound tests have high false negative and false positive rates. The discovery of fetal circulating cell-free DNA (ccfDNA) in maternal blood offers the potential for genomics-based non-invasive prenatal testing (gNIPT) as a more accurate screening method. Two approaches used for gNIPT are massively parallel shotgun sequencing (MPSS) and targeted massively parallel sequencing (TMPS). OBJECTIVES: To evaluate and compare the diagnostic accuracy of MPSS and TMPS for gNIPT as a first-tier test in unselected populations of pregnant women undergoing aneuploidy screening or as a second-tier test in pregnant women considered to be high risk after first-tier screening for common fetal aneuploidies. The gNIPT results were confirmed by a reference standard such as fetal karyotype or neonatal clinical examination. SEARCH METHODS: We searched 13 databases (including MEDLINE, Embase and Web of Science) from 1 January 2007 to 12 July 2016 without any language, search filter or publication type restrictions. We also screened reference lists of relevant full-text articles, websites of private prenatal diagnosis companies and conference abstracts. SELECTION CRITERIA: Studies could include pregnant women of any age, ethnicity and gestational age with singleton or multifetal pregnancy. The women must have had a screening test for fetal aneuploidy by MPSS or TMPS and a reference standard such as fetal karyotype or medical records from birth. DATA COLLECTION AND ANALYSIS: Two review authors independently carried out study selection, data extraction and quality assessment (using the QUADAS-2 tool). Where possible, hierarchical models or simpler alternatives were used for meta-analysis. MAIN RESULTS: Sixty-five studies of 86,139 pregnant women (3141 aneuploids and 82,998 euploids) were included. No study was judged to be at low risk of bias across the four domains of the QUADAS-2 tool but applicability concerns were generally low. Of the 65 studies, 42 enrolled pregnant women at high risk, five recruited an unselected population and 18 recruited cohorts with a mix of prior risk of fetal aneuploidy. Among the 65 studies, 44 evaluated MPSS and 21 evaluated TMPS; of these, five studies also compared gNIPT with a traditional screening test (biochemical, ultrasound or both). Forty-six out of 65 studies (71%) reported gNIPT assay failure rate, which ranged between 0% and 25% for MPSS, and between 0.8% and 7.5% for TMPS.In the population of unselected pregnant women, MPSS was evaluated by only one study; the study assessed T21, T18 and T13. TMPS was assessed for T21 in four studies involving unselected cohorts; three of the studies also assessed T18 and 13. In pooled analyses (88 T21 cases, 22 T18 cases, eight T13 cases and 20,649 unaffected pregnancies (non T21, T18 and T13)), the clinical sensitivity (95% confidence interval (CI)) of TMPS was 99.2% (78.2% to 100%), 90.9% (70.0% to 97.7%) and 65.1% (9.16% to 97.2%) for T21, T18 and T13, respectively. The corresponding clinical specificity was above 99.9% for T21, T18 and T13.In high-risk populations, MPSS was assessed for T21, T18, T13 and 45,X in 30, 28, 20 and 12 studies, respectively. In pooled analyses (1048 T21 cases, 332 T18 cases, 128 T13 cases and 15,797 unaffected pregnancies), the clinical sensitivity (95% confidence interval (CI)) of MPSS was 99.7% (98.0% to 100%), 97.8% (92.5% to 99.4%), 95.8% (86.1% to 98.9%) and 91.7% (78.3% to 97.1%) for T21, T18, T13 and 45,X, respectively. The corresponding clinical specificities (95% CI) were 99.9% (99.8% to 100%), 99.9% (99.8% to 100%), 99.8% (99.8% to 99.9%) and 99.6% (98.9% to 99.8%). In this risk group, TMPS was assessed for T21, T18, T13 and 45,X in six, five, two and four studies. In pooled analyses (246 T21 cases, 112 T18 cases, 20 T13 cases and 4282 unaffected pregnancies), the clinical sensitivity (95% CI) of TMPS was 99.2% (96.8% to 99.8%), 98.2% (93.1% to 99.6%), 100% (83.9% to 100%) and 92.4% (84.1% to 96.5%) for T21, T18, T13 and 45,X respectively. The clinical specificities were above 100% for T21, T18 and T13 and 99.8% (98.3% to 100%) for 45,X. Indirect comparisons of MPSS and TMPS for T21, T18 and 45,X showed no statistical difference in clinical sensitivity, clinical specificity or both. Due to limited data, comparative meta-analysis of MPSS and TMPS was not possible for T13.We were unable to perform meta-analyses of gNIPT for 47,XXX, 47,XXY and 47,XYY because there were very few or no studies in one or more risk groups. AUTHORS' CONCLUSIONS: These results show that MPSS and TMPS perform similarly in terms of clinical sensitivity and specificity for the detection of fetal T31, T18, T13 and sex chromosome aneuploidy (SCA). However, no study compared the two approaches head-to-head in the same cohort of patients. The accuracy of gNIPT as a prenatal screening test has been mainly evaluated as a second-tier screening test to identify pregnancies at very low risk of fetal aneuploidies (T21, T18 and T13), thus avoiding invasive procedures. Genomics-based non-invasive prenatal testing methods appear to be sensitive and highly specific for detection of fetal trisomies 21, 18 and 13 in high-risk populations. There is paucity of data on the accuracy of gNIPT as a first-tier aneuploidy screening test in a population of unselected pregnant women. With respect to the replacement of invasive tests, the performance of gNIPT observed in this review is not sufficient to replace current invasive diagnostic tests.We conclude that given the current data on the performance of gNIPT, invasive fetal karyotyping is still the required diagnostic approach to confirm the presence of a chromosomal abnormality prior to making irreversible decisions relative to the pregnancy outcome. However, most of the gNIPT studies were prone to bias, especially in terms of the selection of participants.

Effects of tempol on endothelial and vascular dysfunctions and insulin resistance induced by a high-fat high-sucrose diet in the rat.

We investigated the effects of treatment with tempol (an antioxidant) on vascular and metabolic dysfunction induced by a high-fat high-sucrose (HFHS) diet. Rats were randomized to receive an HFHS or chow diet with or without tempol treatment (1.5 mmol·(kg body mass)(-1)·day(-1)) for 4 weeks. Blood pressure, heart rate, and blood flow were measured in the rats by using intravascular catheters and Doppler flow probes. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic-hyperinsulinemic clamp. In-vitro studies were performed to evaluate vascular reactivity and endothelial and inducible nitric oxide synthase (eNOS; iNOS) expression in vascular and muscle tissues. Endothelin, nitrotyrosine, and NAD(P)H oxidase expressions were determined in vascular tissues, and glucose transport activity and glucose transporter 4 (GLUT4) expression were examined in muscles. Tempol treatment was found to prevent alterations in insulin sensitivity, glucose transport activity, GLUT4 expression, and vascular reactivity, and to prevent increases in plasma insulin, blood pressure, and heart rate noted in the untreated HFHS-fed rats. These were associated with increased levels of eNOS expression in vascular and muscle tissues, but reductions in nitrotyrosine, endothelin, NAD(P)H oxidase, and iNOS expressions. Therefore, oxidative stress induced by a relatively short-term HFHS diet could contribute to the early development of vascular and metabolic abnormalities in rats.

Validation of a New Protocol to Collect and Isolate Plasma from Pregnant Women for Noninvasive Prenatal Testing (NIPT).

BACKGROUND: Most laboratories use specialized tubes (e.g., Streck) to recover circulating cell-free DNA (ccfDNA) for noninvasive prenatal testing (NIPT). We validated a low cost, simple procedure for collecting NIPT samples in remote laboratories that avoids highspeed centrifugation. EDTA gel blood sampling tube allows simple separation of plasma from blood cells. Decanted plasma is filtered to remove cell debris. The procedure can be performed within a few minutes after the blood centrifugation step, and ccfDNA-grade plasma can be frozen for transportation. METHODS: We recruited 51 pregnant women and collected blood in one EDTA-gel Greiner tube and two Streck tubes. All tubes were centrifuged at 1600 g x 10 min within 6 h of sample collection. Plasma from EDTA tubes was poured into a syringe cylinder and filtered through a 0.45 µm Millipore filter. Plasma from Streck tubes was recovered with a pipette and one was filtered as above while the second was centrifuged at 16 000 g. The ccfDNA was isolated and NGS sequencing libraries were prepared and sequenced on an Illumina system. Fetal fractions were estimated using SeqFF. This study had a power of 79% to detect a decrease of 1% in fetal fractions with the new method. RESULTS: We did not observe any significant difference between the three procedures for the fetal fraction nor for the quality or quantity of libraries produced. CONCLUSION: EDTA-gel tubes with filtration provide high quality plasma for ccfDNA analysis and can be sent frozen to the NIPT laboratory. This is economical and it frees the laboratory of time-consuming steps.

Fatty acid-induced changes in vascular reactivity in healthy adult rats.

Dietary fatty acids (FAs) are known to modulate endothelial dysfunction, which is the first stage of atherosclerosis. However, their exact role in this initial phase is still unclear. The effects of isolated or combined (by 2) purified FAs from the main FA families were studied on the vascular response of isolated thoracic aorta in healthy rats to get a better understanding of the mechanisms of action of dietary FAs in regulating vascular endothelial function. Cumulative contraction curves to phenylephrine and relaxation curves to carbachol and then to sodium nitroprusside were obtained in the absence or presence of the FAs studied allowing endothelium-dependent and endothelium-independent ability of the smooth muscle to relax to be assessed in each experimental group. The endothelium-dependent vasodilator response to carbachol was lowered by eicosapentaenoic acid, whereas it was not altered either by docosahexaenoic acid alone or by combined eicosapentaenoic acid-docosahexaenoic acid, oleic acid, or stearic acid, and it was increased by linoleic acid (LA). A decreased phenylephrine-induced contraction was observed after incubation with arachidonic acid and with stearic acid. On the other hand, the endothelium-dependent relaxation was reduced by the addition of combined LA-arachidonic acid and LA-oleic acid. In conclusion, these data point out the differential effects of different types of FAs and of FAs alone vs combined on vascular reactivity. The complex nature of these effects could be partially linked to metabolic specificities of endothelial cells and to interactions between some FAs.

Endothelial and vascular dysfunctions and insulin resistance in rats fed a high-fat, high-sucrose diet.

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis.

Effects of high-sucrose feeding on insulin resistance and hemodynamic responses to insulin in spontaneously hypertensive rats.

This study was designed to investigate the effects of a sucrose diet on vascular and metabolic actions of insulin in spontaneously hypertensive rats (SHR). Male SHR were randomized to receive a sucrose or regular chow diet for 4 wk. Age-matched, chow-fed Wistar-Kyoto (WKY) rats were used as normotensive control. In a first series of experiments, the three groups of rats had pulsed Doppler flow probes and intravascular catheters implanted to determine blood pressure, heart rate, and blood flows. Insulin sensitivity was assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine glucose transport activity in isolated muscles and to determine endothelial nitric oxide synthase (eNOS) protein expression in muscles and endothelin content in vascular tissues. Sucrose feeding was shown to markedly enhance the pressor response to insulin and its hindquarter vasoconstrictor effect when compared with chow-fed SHR. A reduction in eNOS protein content in muscle, but no change in vascular endothelin-1 protein, was noted in sucrose-fed SHR when compared with WKY rats, but these changes were not different from those noted in chow-fed SHR. Similar reductions in insulin-stimulated glucose transport were observed in soleus muscles from both groups of SHR when compared with WKY rats. In extensor digitorum longus muscles, a significant reduction in insulin-stimulated glucose transport was only seen in sucrose-fed rats when compared with the other two groups. Environmental factors, that is, high intake of simple sugars, could possibly potentiate the genetic predisposition in SHR to endothelial dysfunction and insulin resistance.