Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

Pregnancy Termination and Postnatal Major Congenital Heart Defect Prevalence After Introduction of Prenatal Cardiac Screening.

Importance: Prenatal cardiac screening of the first and second trimesters has had a major impact on postnatal prevalence of congenital heart defects (CHDs), rates of termination of pregnancy (TOP), and outcomes among children born alive with CHDs. Objective: To examine the prenatal and postnatal incidence of major CHDs (ie, necessitating intervention within the first year of life), detection rate trends, rates of TOP, and the association of cardiac screening with postnatal outcomes. Design, Settings, and Participants: In this cross-sectional study, 3827 fetuses with antenatally diagnosed major CHDs in the Czech Republic (population 10.7 million) between 1991 and 2021 were prospectively evaluated with known outcomes and associated comorbidities. Prenatal and postnatal prevalence of CHD in an unselected population was assessed by comparison with a retrospective analysis of all children born alive with major CHDs in the same period (5454 children), using national data registry. Data analysis was conducted from January 1991 to December 2021. Main Outcomes and Measures: Prenatal detection and postnatal prevalence of major CHDs and rate of TOPs in a setting with a centralized health care system over 31 years. Results: A total of 3 300 068 children were born alive during the study period. Major CHD was diagnosed in 3827 fetuses, of whom 1646 (43.0%) were born, 2069 (54.1%) resulted in TOP, and 112 (2.9%) died prenatally. The prenatal detection rate increased from 6.2% in 1991 to 82.8% in 2021 (P < .001). Termination of pregnancy decreased from 70% in 1991 to 43% (P < .001) in 2021. Of 627 fetuses diagnosed in the first trimester (introduced in 2007), 460 were terminated (73.3%). Since 2007, of 2066 fetuses diagnosed in the second trimester, 880 (42.6%) were terminated, resulting in an odds ratio of 3.6 (95% CI, 2.8-4.6; P < .001) for TOP in the first trimester compared with the second trimester. Postnatal prevalence of major CHDs declined from 0.21% to 0.14% (P < .001). The total incidence (combining prenatal detection of terminated fetuses with postnatal prevalence) of major CHD remained at 0.23% during the study period. Conclusions and Relevance: In this cross-sectional study, the total incidence of major CHD did not change significantly during the 31-year study period. The prenatal detection of major CHD approached 83% in the current era. Postnatal prevalence of major CHD decreased significantly due to early TOPs and intrauterine deaths. The introduction of first trimester screening resulted in a higher termination rate in the first trimester but did not revert the overall decreasing trend of termination for CHDs in general.

Genome-wide miRNA profiling in plasma of pregnant women with down syndrome fetuses.

Down syndrome (DS) is one of the most common causes of intellectual disability and new approaches allowing its rapid and effective prenatal detection are being explored. In this study, we investigated the diagnostic potential of plasma microRNAs (miRNAs). This study builds upon our previous study in DS placentas, where seven miRNAs were found to be significantly up-regulated. A total of 70 first-trimester plasma samples from pregnant women were included in the present study (35 samples with DS fetuses; 35 with euploid fetuses). Genome-wide miRNA profiling was performed in the pilot study using Affymetrix GeneChip™ miRNA 4.1 Array Strips (18 samples). Selected miRNAs were then analysed in the validation study using quantitative reverse transcription PCR (RT-qPCR; 52 samples). Based on the current pilot study results (12 miRNAs), our previous research on chorionic villi samples (7 miRNAs) and the literature (4 miRNAs), a group of 23 miRNAs was selected for the validation study. Although the results of the pilot study were promising, the validation study using the more sensitive RT-qPCR technique and a larger group of samples revealed no significant differences in miRNA profiles between the compared groups. Our results suggest that testing of the first-trimester plasma miRNAs is probably not suitable for non-invasive prenatal testing (NIPT). Different results could be theoretically achieved at later gestational ages; however, such a result probably would have limited use in clinical practice.

Differentially expressed miRNAs in trisomy 21 placentas.

OBJECTIVE: Molecular pathogenesis of Down syndrome (DS) is still incompletely understood. Epigenetic mechanisms, including miRNAs gene expression regulation, belong to potential influencing factors. The aims of this study were to compare miRNAs expressions in placentas with normal and trisomic karyotype and to associate differentially expressed miRNAs with concrete biological pathways. METHODS: A total of 80 CVS samples - 41 with trisomy 21 and 39 with normal karyotype - were included in our study. Results obtained in the pilot study using real-time PCR technology and TaqMan Human miRNA Array Cards were subsequently validated on different samples using individual TaqMan miRNA Assays. RESULTS: Seven miRNAs were verified as upregulated in DS placentas (miR-99a, miR-542-5p, miR-10b, miR-125b, miR-615, let-7c and miR-654); three of these miRNAs are located on chromosome 21 (miR-99a, miR-125b and let-7c). Many essential biological processes, transcriptional regulation or apoptosis, were identified as being potentially influenced by altered miRNA levels. Moreover, miRNAs overexpressed in DS placenta apparently regulate genes involved in placenta development (GJA1, CDH11, EGF, ERVW-1, ERVFRD-1, LEP or INHA). CONCLUSION: These findings suggest the possible participation of miRNAs in Down syndrome impaired placentation and connected pregnancy pathologies. © 2016 John Wiley & Sons, Ltd.

Comparison of right ventricular measurements and SFRV in fetuses with and without tricuspid regurgitation at 11+0 and 13+6 weeks' gestation.

OBJECTIVES: To compare right ventricular dimensions and systolic shortening fraction of the right ventricle (SFRV) in fetuses with tricuspid regurgitation (TR [+]) to those without tricuspid regurgitation (TR [-]). METHODS: Unselected patients presenting for first trimester screening between 11 + 0 and 13 + 6 weeks' gestation were examined for the presence or absence of fetal tricuspid regurgitation using a standard approach. Only euploid fetuses without structural anomalies were included in the study. The heart was examined with the aid of M-mode using a previously described method. The right ventricular diastolic diameter (RVDD) and right ventricular systolic diameter (RVSD) were measured on stored M-mode images and the SFRV was calculated using the following formula [(RVDD-RVSD)/RVDD] × 100. RESULTS: A total of 69 fetuses (n = 44 (TR [-]); n = 25 (TR [+])) were examined. The two groups were similar in maternal age, gestational age and nuchal translucency (NT) measurements. The SFRV was noted not to change with gestational age and there was no statistical difference between the two groups. Both the RVDD and the RVSD increased with gestational age. The calculated delta RVDD was statistically larger in the TR [+] group (mean: 0.29, CI 95%: 0.054-0.532) than the TR [-] group (mean: 0.013, CI 95%: -0.128 to 0.154) (p < 0.05). This was not true for the delta RVSD: TR [+] (mean: 0.17, CI 95%: 0.015-0.325) versus TR [-] group (mean: 0.035, CI 95%: -0.061 to 0.131). However, there was a trend towards larger RVSD in the TR [+] group (p = 0.13). CONCLUSIONS: The presence of TR appears to be associated with an increased RVDD in normal fetuses between 11 + 0 and 13 + 6 weeks' gestation.

Shortening fraction of the right ventricle: a comparison between euploid and trisomy 21 fetuses at week 11 to week 13 and 6 days of gestation.

OBJECTIVES: This study was designed to compare the first trimester shortening fraction of the right ventricle (SFRV) values between euploid fetuses and fetuses with trisomy 21. METHODS: SFRV was measured in 58 first trimester fetuses between September 2008 and February 2010. The stored M-mode images were used to obtain the right ventricular diastolic diameter (RVDD) and right ventricular systolic diameter (RVSD) measurements offline. RESULTS: The SFRV values were found to be significantly greater in the 9 fetuses with trisomy 21 as compared to the group of 49 euploid fetuses (mean: 48.6 mm; range: 36-56.25 mm vs mean: 34.11 mm; range: 22.73-43.48 mm) (p < 0.0001). The RVDD measurements were also found to be significantly greater in the fetuses with trisomy 21 than in the euploid fetuses (mean: 3.08 mm; range: 2.2-4.7 mm vs mean: 2.54 mm; range: 1.9-3.6 mm) (p = 0.03).There was no difference in the RVSD measurements between the two groups [mean: 1.56 mm; range: 1.2-2.3 mm (trisomy 21 fetuses) vs mean: 1.67 mm; range: 1.3-2.4 mm (euploid fetuses)] (p = 0.17). CONCLUSIONS: The SFRV values in fetuses with trisomy 21 appear to be significantly greater than in the euploid fetuses. The RVDD also appears to be greater in fetuses with trisomy 21 than in the euploid fetuses.

Left ventricle shortening fraction: a comparison between euploid and trisomy 21 fetuses in the first trimester.

OBJECTIVES: Measurement of the shortening fraction of the left ventricle (SFLV) is an objective way to assess systolic performance. The aim of the study was to compare first trimester SFLV values in euploid fetuses to those in fetuses with trisomy 21. METHODS: We measured SFLV in 56 fetuses from 11 weeks to 13 weeks 6 days. The left ventricular diastolic diameter (LVDD) and left ventricular systolic diameter (LVSD) were measured offline, and SFLV was calculated. The data were analyzed using Mann-Whitney U test. RESULTS: We found a significant difference in the SFLV measurements between the group of 49 euploid fetuses and the 7 fetuses with trisomy 21 [38.00 (95% CI: 33.72-42.27) vs 49.93 (95% CI: 43.72-56.13)] (p < 0.05). There was also a significant difference in the nuchal translucency measurements between the two groups: 1.78 mm (95% CI: 1.08-2.48 mm) in the euploid population versus 5.06 mm (95% CI: 3.61-6.71 mm) in the fetuses with trisomy 21 (p < 0.05). There were no significant differences between the group of euploid fetuses and the group of trisomy 21 fetuses in the following parameters: CRL (chorionic villus sampling), LVDD and LVSD. CONCLUSIONS: SFLV is a well-defined, simple measurement of systolic function of the fetal myocardium. SFLV values in fetuses with trisomy 21 appear to be significantly higher than in euploid fetuses.

Amniovacucentesis vs standard syringe technique for amniocentesis: experience with 1219 cases.

OBJECTIVE: The aim of the study was to compare amniovacucentesis to the usual syringe use for amniotic fluid aspiration. STUDY DESIGN: We compared 2 groups of procedures: 1117 amniocenteses performed with the usual syringe technique and 1219 amniovacucenteses. RESULTS: The numbers of needle insertions, unsuccessful amniocyte cultures, and miscarriage up to 21 days after the procedure were statistically not significant (P>.01) comparing the 2 techniques. CONCLUSION: The vacuum tube serves as an automated aspiration tool alternative. The major subjective differences between the 2 methods are the operator's comfort and dexterity during sampling and the absence of an extra manipulation of the amniotic fluid after aspiration.