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Objective: To assess the frequency of atypical chromosomal and submicroscopic anomalies, as well as fetal structural abnormalities, observed on first-trimester ultrasound scan in fetuses with nuchal translucency (NT) thickness > 99th centile, in order to evaluate the suitability of using standard cell-free DNA (cfDNA) testing as the sole screening test in these pregnancies.Methods: This was a retrospective cohort study of 226 fetuses with NT > 99th centile at 11-14 weeks' gestation, between January 2013 and December 2017, in a clinical setting in which greater than 95% of pregnant women receive first-trimester combined screening. All patients underwent genetic testing by means of quantitative fluorescence polymerase chain reaction and chromosomal microarray analysis, mainly in chorionic villus samples. We assessed the theoretical yield of two cfDNA testing models, targeted cfDNA (chromosomes 21, 18 and 13) and extended cfDNA (chromosomes 21, 18, 13 and sex chromosomes), and compared it with that of cytogenetic testing and ultrasound assessment in the first and second or third trimesters.Results: In the 226 fetuses analyzed, cytogenetic testing revealed 84 (37%) anomalies, including 68 typical aneuploidies (involving chromosomes 13, 18 or 21), six sex chromosome aneuploidies (four cases of monosomy X and two of trisomy X), three clinically relevant atypical chromosomal anomalies (one trisomy 22, one trisomy 21 mosaicism and one unbalanced translocation), five submicroscopic pathogenic variants and two cases with Noonan syndrome. Targeted and extended cfDNA testing would miss at least 12% (10/84) and 19% (16/84), respectively, of genetic anomalies, accounting for 4.4% and 7.1% of the fetuses with an increased NT, respectively. Finally, of the 142 fetuses with no identified genetic anomaly, a major fetal malformation was observed in 15 (10.6%) fetuses at the early anomaly scan, and in 19 (13.4%) in the second or third trimester.Conclusions: cfDNA does not appear to be the appropriate genetic test in fetuses with NT > 99th centile, given that it would miss 12-19% of genetic anomalies in this group. Additionally, first-trimester ultrasound will identify a major structural abnormality in 11% of the fetuses with NT > 99th centile and no genetic anomaly. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Objectives: An association between isolated, increased nuchal translucency thickness (NT) and pathogenic findings on chromosomal microarray analysis (CMA) has been reported. A recent meta-analysis reported that most studies use a NT cut-off value of 3.5 mm. However, considering NT distribution and the commonly accepted 5% false-positive rate in maternal serum screening, NT cut-off levels should be reconsidered. The aim of this study was to assess different NT cut-off levels as indication for CMA and to determine whether CMA should be recommended for mildly increased NT of 3.0-3.4 mm.Methods: This was a retrospective, multicenter study of singleton pregnancies with CMA results and either normal NT and no other finding or with increased NT as the only medical indication for CMA at the time of an invasive procedure (increased NT was considered an isolated finding in cases of advanced maternal age). Women with normal fetal NT who underwent CMA did so at their own request. A single laboratory performed all genetic analyses. Comparative genomic hybridization microarray analysis or single nucleotide polymorphism array technology was used for CMA. If combined first-trimester screening (NT and biochemistry) indicated increased risk for common aneuploidies, the case was excluded. NT was used to divide cases into three groups (≤ 2.9 mm, 3.0-3.4 mm and ≥ 3.5 mm) and their CMA results were compared.Results: CMA results were recorded in 1588 pregnancies, among which 770 fetuses had either normal NT with no other finding or isolated increased NT. Of these, 462 had NT ≤ 2.9 mm, 170 had NT of 3.0-3.4 mm and 138 had NT ≥ 3.5 mm. Pathogenic copy number variants were found in 1.7%, 6.5% and 13.8% of cases, respectively.Conclusion: Our results suggest that CMA should be recommended when fetuses have isolated, mildly increased NT (3.0-3.4 mm). Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Cell-free fetal DNA testing is being used in parallel or in contingency screening as part of the first trimester screen. The test has high sensitivity and specificity for the trisomies 21, 18 and 13. The test also offers the option of assessing sex chromosome aneuploidies (SCA) which are recognised to be the next most common group of aneuploidies in the live birth population. Companies that offer the sex chromosome assessment report an accuracy rate of above 99% and a significant number of high-risk results have been detected in a multi-site Australian ultrasound practice. A high proportion of these women underwent prenatal testing to further assess the sex chromosomes. This study reports the results of these invasive investigations and results show that many of the high-risk SCA results appear to be false positives. This study reports the clinical experience of cell-free fetal DNA (cfDNA) testing with regard to sex chromosome aneuploidies in singleton pregnancies for a multi-site Sydney specialist O&G Ultrasound practice. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the clinical accuracy of the IONA® test for aneuploidy screening.Methods: This was a multicenter blinded study in which plasma samples from pregnant women at increased risk of trisomy 21 underwent cell-free DNA analysis utilizing the IONA test. For each sample, the IONA software generated a likelihood ratio and a maternal age-adjusted probability risk score for trisomies 21, 18 and 13. All results from the IONA test were compared against accepted diagnostic karyotyping.Results: A total of 442 maternal samples were obtained, of which 437 had test results available for analysis and assessment of clinical accuracy. The IONA test had a detection rate of 100% for trisomies 21 (n = 43; 95% CI, 87.98-100%), 18 (n = 10; 95% CI, 58.72-100%) and 13 (n = 5; 95% CI, 35.88-100%) with cut-offs applied to likelihood ratio (cut-off > 1 considered high risk for trisomy) and probability risk score incorporating adjustment for maternal age (cut-off ≥ 1/150 considered high risk for trisomy). The false-positive rate (FPR) was 0% for trisomies 18 and 13 with both analysis outputs. For trisomy 21, a FPR of 0.3% was observed for the likelihood ratio, but became 0% with adjustment for maternal age.Conclusion: This study indicates that the IONA test is suitable for trisomy screening in a high-risk screening population. The result-interpretation feature of the IONA software should facilitate wider implementation, particularly in local laboratories, and should be a useful addition to the current screening methods for trisomies 21, 18 and 13. [ABSTRACT FROM AUTHOR]

Objective: To assess the potential performance of screening for fetal trisomies 21, 18 and 13 by cell-free DNA (cfDNA) analysis of maternal blood using the IONA® test.Methods: This was a nested case-control study of cfDNA analysis of maternal plasma using the IONA test. Samples were obtained at 11-13 weeks' gestation, before chorionic villus sampling, from 201 euploid pregnancies, 35 with trisomy 21, four with trisomy 18 and two with trisomy 13. Laboratory personnel were blinded to the fetal karyotype.Results: Probability scores for trisomies 21, 18 and 13 were given for 241/242 samples analyzed. No probability score was provided for one (0.5%) euploid pregnancy because of low fetal fraction. In all 35 cases of trisomy 21 the probability score for trisomy 21 was > 95% and the scores for trisomies 18 and 13 were ≤ 0.0001%. In all four cases of trisomy 18, the probability score for trisomy 18 was > 77% and the scores for trisomies 21 and 13 were ≤ 0.0001%. In the two cases of trisomy 13, the probability score for trisomy 13 was > 59% and the scores for trisomies 21 and 18 were ≤ 0.0001%. In the 200 euploid pregnancies with a test result, the probability score was < 0.08% for trisomy 21, < 0.001% for trisomy 18 and < 0.002% for trisomy 13. Therefore, the IONA test detected 100% of all three trisomies, with a false-positive rate of 0%.Conclusion: The IONA test successfully differentiated all cases of trisomies 21, 18 and 13 from euploid pregnancies. [ABSTRACT FROM AUTHOR]

Objective To investigate the prevalence of soft markers identified at second trimester ultrasound in a low-risk population and the association of these markers with trisomies and invasive testing. Design Prospective observational study. Setting Swedish University Hospital. Population All women with fetuses examined by ultrasound at 15+0-22+0 weeks gestation between July 2008 and March 2011. Methods Cases with soft markers were compared with non-cases with regard to trisomies and invasive testing. Main outcome measures Prevalence of soft markers, likelihood ratio for trisomies and risk ratio for invasive tests after detection of soft markers. Results Second trimester ultrasound was performed on 10 710 fetuses. Markers were detected in 5.9% of fetuses. 5.1% were isolated, 0.7% were multiple and 0.1% were combined with an anomaly. Presence of markers showed a positive likelihood ratio for Down syndrome, but the association (likelihood ratio = 7.1) was only statistically significant for the combined category of any marker (isolated, multiple or combined with anomaly). The risk ratio for invasive testing after the second trimester ultrasound was 24.0 in pregnancies with isolated soft markers compared with those without markers. Conclusion In a low-risk population, soft markers were found in 5.9% of fetuses at second trimester ultrasound. The likelihood ratio for Down syndrome was significant only for any marker (isolated, multiple or combined with anomaly). The presence of soft markers increased the incidence of invasive procedures substantially. Soft markers should be noted when information on second trimester ultrasound is formulated, and all units performing fetal ultrasound examinations should have established routines concerning information management when soft markers are identified. [ABSTRACT FROM AUTHOR]

ABSTRACT Objectives We performed a meta-analysis to examine the performance of second-trimester (14-24 weeks' gestation) isolated fetal pyelectasis as a marker for trisomy 21 and to calculate its associated weighted pooled likelihood ratios. Methods PubMed, Ovid MEDLINE and Cochrane databases were searched using the terms 'pyelectasis' and 'pelviectasis'. Studies were included if fetuses with isolated pyelectasis were reported separately from fetuses with other soft markers of aneuploidy and/or structural anomalies and if knowledge of the fetal karyotype was unknown at the time of ultrasound examination. Results Individual study statistics were pooled as weighted positive and negative likelihood ratios with 95% CIs, using a random-effects model. Ten observational studies were included (2148 cases of isolated pyelectasis). Isolated fetal pyelectasis was defined in seven out of 10 studies as a renal pelvis anteroposterior diameter of ≥ 4 mm. Isolated fetal pyelectasis was associated with pooled positive and negative likelihood ratios of 2.78 (95% CI, 1.75-4.43) and 0.99 (95% CI, 0.98-1.00), respectively. Conclusions The detection of isolated fetal pyelectasis on mid-trimester ultrasound is associated with an increased likelihood of trisomy 21. If the finding of isolated fetal pyelectasis is used to adjust the trisomy 21 risk from maternal serum screening tests, a positive likelihood ratio of 2.78 should be used in the calculation. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Objectives To determine whether there is an association between sonographically assessed hyper- or hypocoiling of the umbilical cord and the presence of trisomy 21, to provide reference values for the antenatal umbilical coiling index ( aUCI) at a gestational age of 16-21 weeks and to determine whether these measurements are reliable and reproducible. Methods This was a prospective study of 737 pregnancies in which the aUCI was measured between 16 and 21 weeks of gestation by ultrasound at the time of amniocentesis. The aUCI was calculated as the reciprocal value of the mean length of one complete coil in centimeters. We created reference curves and studied the relationship with trisomy 21 and other chromosomal defects. In 30 pregnancies we studied the intra- and interobserver variation in measurements using Bland-Altman plots with associated 95% limits of agreement and intraclass correlation coefficients. Results aUCI was found to be non-linearly related to gestational age at 16-21 weeks and reference curves were created for the mean aUCI and the 2.3rd, 10th, 90th and 97.7th percentiles. There was no significant difference in aUCI values between the reference group (n = 714) and cases with trisomy 21 (n = 16) or other aneuploidies (n = 7) (one-way ANOVA, P = 0.716). There was good intra- and interobserver agreement in aUCI measurements. Conclusions The aUCI can be measured reliably and varies according to gestational age at 16-21 weeks. The aUCI was not significantly associated with trisomy 21 or other chromosomal defects. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Non-invasive prenatal testing ( NIPT) for aneuploidy using cell-free DNA in maternal plasma is revolutionizing prenatal screening and diagnosis. We review NIPT in the context of established screening and invasive technologies, the range of cytogenetic abnormalities detectable, cost, counseling and ethical issues. Current NIPT approaches involve whole-genome sequencing, targeted sequencing and assessment of single nucleotide polymorphism ( SNP) differences between mother and fetus. Clinical trials have demonstrated the efficacy of NIPT for Down and Edwards syndromes, and possibly Patau syndrome, in high-risk women. Universal NIPT is not cost-effective, but using NIPT contingently in women found at moderate or high risk by conventional screening is cost-effective. Positive NIPT results must be confirmed using invasive techniques. Established screening, fetal ultrasound and invasive procedures with microarray testing allow the detection of a broad range of additional abnormalities not yet detectable by NIPT. NIPT approaches that take advantage of SNP information potentially allow the identification of parent of origin for imbalances, triploidy, uniparental disomy and consanguinity, and separate evaluation of dizygotic twins. Fetal fraction enrichment, improved sequencing and selected analysis of the most informative sequences should result in tests for additional chromosomal abnormalities. Providing adequate prenatal counseling poses a substantial challenge given the broad range of prenatal testing options now available. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Calabrese G, Baldi M, Fantasia D, Teresa Sessa M, Kalantar M, Holzhauer C, Alunni-Fabbroni M, Palka G, Sitar G. Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single-chromosome dual-probe FISH analysis. Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing. [ABSTRACT FROM AUTHOR]

Objectives To study the ratio of prenasal thickness (PT) to nasal bone length (NBL) in normal and trisomy-21 fetuses in the second and third trimesters of pregnancy. Methods The PT and NBL were measured retrospectively in 106 normal fetuses (in three-dimensional (3D) volumes) and in 30 fetuses with trisomy 21 (10 on two-dimensional (2D) images and 20 in 3D volumes). Results In normal fetuses the mean PT and NBL increased between 15 and 33 weeks' gestation from 2.3 to 6.1 mm ( r = 0.85, P < 0.001) and from 3.3 to 9.6 mm ( r = 0.87, P < 0.001), respectively. The PT : NBL ratio was stable throughout gestation, with a mean of 0.61 (95% CI, 0.59-0.63; r = − 0.04, P = 0.7). The 5 th and 95 th percentiles were 0.48 and 0.80, respectively. In trisomy-21 fetuses the mean PT and NBL increased between 14 and 34 weeks from 3.0 to 9.2 mm ( r = 0.86, P < 0.001) and from 1.9 to 7.8 mm ( r = 0.85, P < 0.001), respectively. The PT : NBL ratio was significantly higher than in normal fetuses ( P < 0.001) but also stable throughout gestation, with a mean of 1.50 (95% CI, 1.20-1.80; r = − 0.35, P = 0.07). Twenty-three (77%) of the 30 fetuses with trisomy 21 had a PT above the 95 th percentile and 20 (67%) had an NBL below the 5 th percentile. All the trisomy-21 fetuses had a PT : NBL ratio above the 95 th percentile. When the 95 th percentile of the PT : NBL ratio was used as a cut-off value the detection and false positive rates for trisomy 21 were 100 (95% CI, 89-100)% and 5 (95% CI, 2-11)%, respectively. The positive likelihood ratio was 21.2. Conclusions The PT : NBL ratio is stable in the second and third trimesters of pregnancy in both normal and trisomy-21 fetuses, but all trisomy-21 fetuses in this series had a PT : NBL ratio above the 95 th percentile. The ratio is therefore a strong marker for trisomy 21. Copyright © 2011 ISUOG. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Tetrasomy 21 is a very rare aneuploidy which could clinically resemble a Down syndrome. It was most often described in its partial form than complete. We report the prenatal, pathological and genetic characteristics of a fetus with mosaic complete tetrasomy 21. This is the second well‐documented description of a complete tetrasomy 21 in the literature. Methods: Prenatal and fetal pathological examinations, cytogenetic and molecular analyses were performed to characterize fetal features with tetrasomy 21. Results: Prenatal ultrasound examination revealed an isolated complete atrioventricular septal defect with normal karyotype on amniotic fluid. After termination of pregnancy, clinical examination of the fetus evoked trisomy 21 or Down syndrome. Chromosomal microarray analysis and FISH on lung tissue showed a mosaicism with four copies of chromosome 21 (tetrasomy 21). Conclusion: Our observation and the review of the literature reported the possibility of very weak mosaicism and disease‐causing confined tissue‐specific mosaicism in fetus or alive patients with chromosome 21 aneuploidy, mainly Down syndrome. In case of clinical diagnosis suggestive of Down syndrome, attention must be paid to the risk of false‐negative test due to chromosomal mosaicism (very weak percentage, different tissue distribution). To overcome this risk, it is necessary to privilege the diagnostic techniques without culture step and to increase the number of cells and tissues analyzed, if possible. This study highlights the limits of microarray as the unique diagnostic approach in case of weak mosaic and French cytogenetics guidelines recommend to check anomalies seen in microarray by another technique on the same tissue. [ABSTRACT FROM AUTHOR]