Detection Of Fetal Aneuploidy Research Articles

Cell-Free DNA Analysis of Fetal Aneuploidies in Early Pregnancy Loss.

Background: Products of conception samples are often collected and analyzed to try to determine the cause of an early pregnancy loss. However, sample collection may not always be possible, and maternal cell contamination and culture failure can affect the analysis. Cell-free DNA-based analysis of a blood sample could be used as an alternative method in early pregnancy loss cases to detect if aneuploidies were present in the fetus. Methods: In this prospective study, blood samples from early pregnancy loss patients were analyzed for the presence of fetal aneuploidies using a modified version of a noninvasive prenatal testing assay for cell-free DNA analysis. Results from cell-free DNA analysis were compared against the gold standard, microarray analysis of products of conception samples. This study was registered with ClinicalTrials.gov, identifier: NCT04935138. Results: Of the 76 patient samples included in the final study cohort, 11 were excluded from performance calculations. The 65 patient samples included in the final analysis included 49 with an abnormal microarray result and 16 with a normal microarray result. Based on results from these 65 samples, the study found that genome-wide cell-free DNA analysis had a sensitivity of 73.5% with a specificity of 100% for the detection of fetal aneuploidies in early pregnancy loss cases. Conclusions: This prospective study provides further support for the utility of cell-free DNA analysis in detecting fetal aneuploidies in early pregnancy loss cases. This approach could allow for a noninvasive method of investigating the etiology of miscarriages to be made available clinically.

MaterniCode: New Bioinformatic Pipeline to Detect Fetal Aneuploidies and Rearrangements Using Next-Generation Sequencing.

Objective: The present study is aimed at introducing and evaluating MaterniCode, a state-of-the-art bioinformatic pipeline for noninvasive prenatal testing (NIPT) that leverages the Ion Torrent semiconductor sequencing platform. The initiative strives to revolutionize prenatal diagnostics by offering a rapid and cost-effective method without sacrificing accuracy. Methods: Two distinct bioinformatic strategies were employed for fetal sex determination, one of which achieved 100% accuracy. We analyzed 1225 maternal blood samples for fetal aneuploidies, benchmarking against the industry standard Illumina VeriSeq™ NIPT Solution v2. The capability of MaterniCode to detect and characterize complex chromosomal anomalies was also assessed. Results: MaterniCode achieved near-perfect accuracy in fetal sex determination through chromosome Y (chrY )-specific gene analysis, whereas the alternative method, employing the ratio of high-quality mapped reads on chrY relative to all reads, delivered 100% accuracy. For fetal aneuploidy detection, both the integrated WisecondorX and NIPTeR algorithms demonstrated a 100% sensitivity and specificity rate, consistent with Illumina VeriSeq™ NIPT Solution v2. The pipeline also successfully identified and precisely mapped significant chromosomal abnormalities, exemplified by a 2.4 Mb deletion on chromosome 13 and a 3 Mb duplication on chromosome 2. Conclusion: MaterniCode has proven to be an innovative and highly efficient tool in the domain of NIPT, demonstrating excellent sensitivity and specificity. Its robust capability to effectively detect a wide range of complex chromosomal aberrations, including rare and subtle variations, positions it as a promising and valuable addition to prenatal diagnostic technologies. This enhancement to diagnostic precision significantly aids clinicians in making informed decisions during pregnancy management.

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.

A Systematic Review and Meta-analysis of Cell-Free DNA Testing for Detection of Fetal Sex Chromosome Aneuploidy

ABSTRACT Noninvasive prenatal screening has become a valuable tool in detecting a variety of disorders and abnormalities, including sex chromosome aneuploidies (SCAs). The test uses cell-free DNA (cfDNA) analysis to identify risk for chromosomal abnormalities, which can then be more fully investigated using other tests. Much of previous research focused on cfDNA has assessed autosomal aneuploidies, but few studies have specifically focused on SCAs. The aim of this systematic review and meta-analysis was to assess the accuracy of cfDNA testing in identifying SCAs in singleton pregnancies. Studies were identified by keywords associated with cfDNA, SCA, and noninvasive prenatal testing, and studies were included based on relevance. Inclusion criteria were limited to singleton pregnancies that underwent cfDNA testing with confirmation by diagnostic genetic testing between January 1, 2010 and December 1, 2021. A total of 21 articles were selected for the final meta-analysis. All included studies reported results for monosomy X (45,X), representing nearly 13,000 tests with 254 confirmed positive results, 133 false-positives, and 18 false-negatives. Estimated sensitivity based on this sample was 98.8%, with specificity of 99.4%. Positive and negative predictive values were 14.5% and 100%, respectively. Of the 21 included studies, 16 also reported results for Klinefelter syndrome (47,XXY), representing just over 11,000 tests with 62 confirmed positive results, 10 false-positives, and 11 false-negatives. Estimated sensitivity was 100% and specificity was 100%, with positive predictive value of 97.7% and negative predictive value of 100%. Thirteen of 21 studies included reported results for triple X syndrome (47,XXX), representing roughly 10,000 tests with 38 confirmed positive results, 18 false-positives, and 1 false-negative. This showed an estimated sensitivity of 100% and specificity of 99.9%, with positive predictive value of 61.6% and negative predictive value of 100%. Finally, 9 of the studies included in this meta-analysis reported results for Jacobs syndrome (47,XYY), representing approximately 8500 tests with 22 confirmed positive results, 2 false-positives, and no false-negatives. This led to an estimated sensitivity and specificity of 100%, with positive and negative predictive values also 100%. These results provide evidence that cfDNA testing is effective in identification of SCAs in pregnancies at high risk. This particular population limits the generalizability of the findings but adds to the literature surrounding the effectiveness and accuracy of this method. The analysis used strict criteria to define the studies that were included, yet still included a large sample of tests (12,000), which adds to the statistical and practical significance of the analysis. Generalizing the findings of this study is difficult based on both the fact that the sample did not include low-risk individuals and that positive predictive values varied greatly with prevalence, impacting clinical significance. Overall, cfDNA testing for SCAs has both high sensitivity and specificity, along with a high negative predictive value of 99.99% or above for each SCA examined. Further research is needed to confirm these findings and extend knowledge to a more diverse population.

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

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A systematic review and meta-analysis of cell-free DNA testing for detection of fetal sex chromosome aneuploidy.

The aim was to determine the accuracy of cell-free DNA testing (cfDNA) for detecting sex chromosome aneuploidies (SCA) in singleton pregnancies. A systematic review and meta-analysis was performed to assess cfDNA accuracy for prenatal detection of 45,X, 47,XXY, 47,XXX and 47,XYY. Inclusion was restricted to studies published between January 2010 and December 2021 reporting both cfDNA and confirmatory diagnostic test results. For 45,X, the sensitivity was 98.8% (95%CI 94.6%-100%), specificity 99.4% (95%CI 98.7%-99.9%) and positive predictive value (PPV) 14.5% (95%CI 7.0%-43.8%). For 47,XXY, the sensitivity was 100% (95%CI 99.6%-100%), specificity 100% (95%CI 99.9%-100%) and PPV 97.7% (95%CI 78.6%-100%). For 47,XXX, the sensitivity was 100% (95%CI 96.9%-100%), specificity 99.9% (95%CI 99.7%-100%) and PPV 61.6% (95%CI 37.6%-95.4%). For 47,XYY, the sensitivity was 100% (95%CI 91.3%-100%), specificity 100% (95% CI 100%-100%) and PPV 100% (95%CI 76.5%-100%). All four SCAs had estimated negative predictive values (NPV) exceeding 99.99%, though false negatives were reported. This analysis suggests that cfDNA is a reliable screening test for SCA, though both false negatives and false positives were reported. These estimates of test performance are derived from pregnancies at high pretest risk for aneuploidy, limiting the generalisability to average risk pregnancies.

Non-invasive prenatal testing for everybody or contingent screening?

To compare the advantages and disadvantages of two main implementation strategies for non-invasive prenatal testing (NIPT) for the detection of trisomies 21, 13 and 18 in public healthcare settings. These concern NIPT as a first-tier screening test for all pregnant women, or NIPT contingent on an increased risk first-trimester combined test (FCT) result. Comparison based on (un)published data and literature. Despite the lower prevalence of trisomies in the low-risk population, data show that the positive predictive value (PPV) and detection-miscarriage ratio of first-tier NIPT are comparable to NIPT used as contingency test. Advantages of NIPT as a first-tier test compared to FCT with contingent NIPT are that there is no time-window for testing, sensitivity is higher, fewer women need follow-up testing (PPV of NIPT is higher than PPV of FCT), less anxiety for pregnant women and partners. When given the choice, pregnant women prefer first-tier NIPT. Although contingency testing still seems to be the cheaper option, the differences in costs are becoming increasingly smaller and might soon disappear. We argue that NIPT should preferably be offered as a first-tier test for the detection of fetal aneuploidies for all pregnant women, provided that women are supported to make informed decisions. This article is protected by copyright. All rights reserved.

A fetal fraction enrichment method reduces false negatives and increases test success rate of fetal chromosome aneuploidy detection in early pregnancy loss

ObjectiveWe and others have previously demonstrated that the size-selection enrichment method could remarkably improve fetal fraction (FF) in the early gestational age (GA, 12–13 weeks), suggesting that 9 or 10 weeks should not be used as a threshold for GA in size-selection noninvasive prenatal screening (NIPS). Here, we assessed whether this method was reliable for detecting fetal chromosomal aneuploidy at the earliest GA (6–8 weeks).MethodsSize-selection NIPS for fetal chromosomal aneuploidy was applied to 208 pregnancy plasma samples (102 male and 106 female fetuses), while the 169 pregnancy samples with male fetuses also underwent standard NIPS. Multivariable linear regression models were used to evaluate the association between fold-change of FF and experimental factors.ResultsThe sensitivity of the cell-free DNA (cfDNA) test in detecting aneuploidy was 100% when screened with FF enrichment, whereas the sensitivity of the same patients was only 62.5% (5/8) without FF enrichment. In the 102 pregnancy samples with male fetuses, FF increased from 6.1% to 15.7%, and the median increase in FF was 2.8-fold with enrichment. Moreover, there was a trend toward an increasing success rate of the cfDNA test from 6 to 13 weeks of gestation, especially when the test success rate reached 100% after 7 weeks with FF enrichment. Multivariate linear regression analysis demonstrated that a lower initial FF, shorter cfDNA size, increased body mass index (BMI), and later GA were all independent predictors of a higher fold-change of FF. Compared with ≤ 120 bp cfDNA fragments, the mean fold-change of FF differences was 0.820 for 121–125 bp, 0.229 for 126–130 bp, − 0.154 for 131–135 bp, − 0.525 for 136–140 bp and − 0.934 for > 140 bp (Ptrend < 0.0001), suggesting that fold-change of FF significantly decreased with cfDNA fragments > 125 bp. These results were statistically significant after adjusting for confounding factors in the models for fold-change of FF.ConclusionsThe FF enrichment method is a reasonable strategy to detect fetal chromosomal aneuploidy in early pregnancy loss with reduced false negatives and increased test success rate after 7 weeks of GA and should be recommended for patients with early pregnancy loss.

A dPCR-NIPT assay for detections of trisomies 21, 18 and 13 in a single-tube reaction-could it replace serum biochemical tests as a primary maternal plasma screening tool?

BackgroundThe next generation sequencing (NGS) based non-invasive prenatal test (NIPT) has outplayed the traditional serum biochemical tests (SBT) in screen of fetal aneuploidies with a high sensitivity and specificity. However, it has not been widely used as a primary screen tool due to its high cost and the cheaper SBT is still the choice for primary screen even with well-known shortages in sensitivity and specificity. Here, we report a multiplex droplet digital PCR NIPT (dPCR-NIPT) assay that can detect trisomies 21, 18 and 13 (T21, T18 and T13) in a single tube reaction with a better sensitivity and specificity than the SBT and a much cheaper price than the NGS-NIPT.MethodsIn this study, the dPCR-NIPT assay’s non-clinical characteristics were evaluated to verify the cell free fetal DNA (cffDNA) fraction enrichment efficiencies, the target cell free DNA (cfDNA) concentration enrichment, the analytical sensitivity, and the sample quality control on the minimum concentration of cfDNA required for the assay. We validated the clinical performance for this assay by blindly testing 283 clinical maternal plasma samples, including 36 trisomic positive samples, from high risk pregnancies to access its sensitivity and specificity. The cost effectiveness of using the dPCR-NIPT assay as the primary screen tool was also analyzed and compared to that of the existing contingent strategy (CS) using the SBT as the primary screen tool and the strategy of NGS-NIPT as the first-tier screen tool in a simulating situation.ResultsFor the non-clinical characteristics, the sample processing reagents could enrich the cffDNA fraction by around 2 folds, and the analytical sensitivity showed that the assay was able to detect trisomies at a cffDNA fraction as low as 5% and the extracted cfDNA concentration as low as 0.2 ng/μL. By testing the 283 clinical samples, the dPCR-NIPT assay demonstrated a detection sensitivity of 100% and a specificity of 95.12%. Compared to the existing CS and the NGS-NIPT as the first-tier screen strategy, dPCR-NIPT assay used as a primary screen tool followed by the NGS-NIPT rescreen is the most economical approach to screen pregnant women for fetal aneuploidies without sacrificing the positive detection rate.ConclusionThis is the first report on a dPCR-NIPT assay, consisting of all the necessary reagents from sample processing to multiplex dPCR amplification, can detect T21, T18 and T13 in a single tube reaction. The study results reveal that this assay has a sensitivity and specificity superior to the SBT and a cost much lower than the NGS-NIPT. Thus, from both the test performance and the economic benefit points of views, using the dPCR-NIPT assay to replace the SBT as a primary screen tool followed by the NGS-NIPT rescreen would be a better approach than the existing CS for detection of fetal aneuploidies in maternal plasma.

Year-end academic review of 2021: Advances in the field of birth defect prevention and control in China as of 2021

Year-end academic review of 2021: Advances in the field of birth defect prevention and control in China as of 2021

Cell-free DNA screening for prenatal detection of 22q11.2 deletion syndrome

Historically, prenatal screening has focused primarily on the detection of fetal aneuploidies. Cell-free DNA now enables noninvasive screening for subchromosomal copy number variants, including 22q11.2 deletion syndrome (or DiGeorge syndrome), which is the most common microdeletion and a leading cause of congenital heart defects and neurodevelopmental delay. Although smaller studies have demonstrated the feasibility of screening for 22q11.2 deletion syndrome, large cohort studies with confirmatory postnatal testing to assess test performance have not been reported. This study aimed to assess the performance of single-nucleotide polymorphism-based, prenatal cell-free DNA screening for detection of 22q11.2 deletion syndrome. Patients who underwent single-nucleotide polymorphism-based prenatal cell-free DNA screening for 22q11.2 deletion syndrome were prospectively enrolled at 21 centers in 6 countries. Prenatal or newborn DNA samples were requested in all cases for genetic confirmation using chromosomal microarrays. The primary outcome was sensitivity, specificity, positive predictive value, and negative predictive value of cell-free DNA screening for the detection of all deletions, including the classical deletion and nested deletions that are ≥500 kb, in the 22q11.2 low-copy repeat A-D region. Secondary outcomes included the prevalence of 22q11.2 deletion syndrome and performance of an updated cell-free DNA algorithm that was evaluated with blinding to the pregnancy outcome. Of the 20,887 women enrolled, a genetic outcome was available for 18,289 (87.6%). A total of 12 22q11.2 deletion syndrome cases were confirmed in the cohort, including 5 (41.7%) nested deletions, yielding a prevalence of 1 in 1524. In the total cohort, cell-free DNA screening identified 17,976 (98.3%) cases as low risk for 22q11.2 deletion syndrome and 38 (0.2%) cases as high risk; 275 (1.5%) cases were nonreportable. Overall, 9 of 12 cases of 22q11.2 were detected, yielding a sensitivity of 75.0% (95% confidence interval, 42.8-94.5); specificity of 99.84% (95% confidence interval, 99.77-99.89); positive predictive value of 23.7% (95% confidence interval, 11.44-40.24), and negative predictive value of 99.98% (95% confidence interval, 99.95-100). None of the cases with a nonreportable result was diagnosed with 22q11.2 deletion syndrome. The updated algorithm detected 10 of 12 cases (83.3%; 95% confidence interval, 51.6-97.9) with a lower false positive rate (0.05% vs 0.16%; P<.001) and a positive predictive value of 52.6% (10/19; 95% confidence interval, 28.9-75.6). Noninvasive cell-free DNA prenatal screening for 22q11.2 deletion syndrome can detect most affected cases, including smaller nested deletions, with a low false positive rate.

Simple and rapid detection of common fetal aneuploidies using peptide nucleic acid probe-based real-time polymerase chain reaction

To examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies. Using amniotic fluid samples, PNA probe based real-time PCR (Patio DEP Detection Kit; SeaSun Biomaterials, Korea) assay was performed. PNA probe was designed to hybridize to similar sequences located on different segments of target chromosomes (21, 18, and 13) and a reference chromosome. Amplification of target sequences and melting curve analysis was performed. When analyzing the melting curve, the ratio of the peak height of the target and reference chromosome was calculated and determined as aneuploidy if the ratio of peak height was abnormal. All the results from the PNA probe-based real-time PCR and melting curve analyses were compared to those from conventional karyotyping. Forty-two cases with common aneuploidies (24 of trisomy 21, 12 of trisomy 18, and 6 of trisomy 13) and 131 cases with normal karyotype were analyzed. When comparing the karyotyping results, the sensitivity and specificity of the PNA probe-based real-time PCR assay were both 100%. The level of agreement was almost perfect (k = 1.00). PNA real-time PCR assay is a rapid and easy method for detecting common aneuploidies.

Fetal anatomy scan with integrated nuchal translucency and combination of PAPP-A and fβhCG for prediction of aneuploidy.

Nuchal translucency (NT) is an important finding of early fetal anatomy scan because of the association with genetic and structural anomalies. Enlarged nuchal translucency can be easily detected even without measurement on fetal anatomy scan as a neck pathology. Because of demanding criteria for measurning NT in established prenatal aneuploidy screening we came with an idea of improvement and simplification with availabe methods. The aim of this study is to compare established screening methods with new model of screening composed of fetal anatomy scan with integrated nuchal translucency and combination of PAPP-A and fβhCG. A prospective one center study analyzed atotal of 351 pregnancies between January 2017 and December 2020. Sonographic measurement of NT and fetal anatomy scan (FAS) were performend with biochemical testing from blood sample in the first trimester. Combined screening and fetal anatomy scan was performed. Patients with a pathological screening or with structural defects underwent an invasive procedure. In patient with positive screenining who missed the first trimester invasive procedure, amniocentesis was performed. Fetuses were divided into two groups according to positive or negative karyotype and to calculate sensitivity and specificity of screening methods. From statistical methods regression analysis, significance p of individual predictor, sensitivity and specificity with graphic drawing of ROC charts were used. Data were analyzed using statistical tools of Microsoft Excel 365 and BESH stat. Four models for aneuploidy screening were tested. 1) Model of "Age at the time of diagnosis" was slightly significant predictor with insignificant odds ratio (P=0.04, OR=1). 2) Model of" First trimester biochemical screening" (age, free beta human chorionic gonadotropine - fβhCG and pregnancy associated plasmatic protein A -PAPP-A) were significant (P=0.0001; LR=21) with sensitivity of 87.5 % and specificity of 65.7 %. 3) Model of "First trimester combined test" (age of patients at the time of diagnosis, fβhCG, PAPP-A, NT) was significant (P=7.9 x10-14, LR=67, sensitivity 87 %, specificity 80 %). 4) Model of "Fetal anatomy scan with biochemistry" (structural abnormality finding with combination including age, fβhCG and PAPP-A) was significant (P=4.9x10-18, LR=87, sensitivity 95 %, specificity 80 %). Fetal anatomy scan combined with age, fβhCG and PAPP-A has the highest sensitivity and specificity for both, the detection of fetal aneuploidies and structural abnormalities. Our study shows that fetal anatomy scan is the best possible option for first trimester diagnostics (Tab. 4, Fig. 5, Ref. 16).

WisecondorFF: Improved Fetal Aneuploidy Detection from Shallow WGS through Fragment Length Analysis.

In prenatal diagnostics, NIPT screening utilizing read coverage-based profiles obtained from shallow WGS data is routinely used to detect fetal CNVs. From this same data, fragment size distributions of fetal and maternal DNA fragments can be derived, which are known to be different, and often used to infer fetal fractions. We argue that the fragment size has the potential to aid in the detection of CNVs. By integrating, in parallel, fragment size and read coverage in a within-sample normalization approach, it is possible to construct a reference set encompassing both data types. This reference then allows the detection of CNVs within queried samples, utilizing both data sources. We present a new methodology, WisecondorFF, which improves sensitivity, while maintaining specificity, relative to existing approaches. WisecondorFF increases robustness of detected CNVs, and can reliably detect even at lower fetal fractions (<2%).

Systematic evaluation of NIPT aneuploidy detection software tools with clinically validated NIPT samples.

Non-invasive prenatal testing (NIPT) is a powerful screening method for fetal aneuploidy detection, relying on laboratory and computational analysis of cell-free DNA. Although several published computational NIPT analysis tools are available, no prior comprehensive, head-to-head accuracy comparison of the various tools has been published. Here, we compared the outcome accuracies obtained for clinically validated samples with five commonly used computational NIPT aneuploidy analysis tools (WisecondorX, NIPTeR, NIPTmer, RAPIDR, and GIPseq) across various sequencing depths (coverage) and fetal DNA fractions. The sample set included cases of fetal trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome). We determined that all of the compared tools were considerably affected by lower sequencing depths, such that increasing proportions of undetected trisomy cases (false negatives) were observed as the sequencing depth decreased. We summarised our benchmarking results and highlighted the advantages and disadvantages of each computational NIPT software. To conclude, trisomy detection for lower coverage NIPT samples (e.g. 2.5M reads per sample) is technically possible but can, with some NIPT tools, produce troubling rates of inaccurate trisomy detection, especially in low-FF samples.

The Influence of Maternal Cell Contamination on Fetal Aneuploidy Detection Using Chip-Based Digital PCR Testing.

Prenatal samples obtained by amniocentesis or chorionic villus sampling are at risk of maternal cell contamination (MCC). In traditional prenatal analysis, MCC is recommended to be assayed by special tests, such as the short tandem repeat analysis and, if detected at a high level, may result in failed analysis report. The objective of this study was to test the ability of chip-based digital PCR to detect fetal aneuploidies in the presence of MCC. To determine the level of accuracy of MCC detection, an aneuploid male sample was subjected to serial dilution with an euploid female sample. DNA was extracted from prenatal samples and analyzed with QuantStudio 3D Digital PCR. Digital PCR analysis allowed the detection of trisomy 21, trisomy 18, and X monosomy accurately in samples with 90%, 85%, and 92% of MCC, respectively. Moreover, our results indicated that digital PCR was able to accurately confirm the presence of Y chromosome at up to 95% contamination. The amniotic fluid and chorionic villus sampling (CVS) received in our clinical laboratory was subjected to further analysis of MCC based on the aneuploidy assessment algorithm, resulting in the identification of 10 contaminated samples and four cases of true fetal mosaicism. We conclude that chip-based digital PCR analysis enables the detection of fetal aneuploidy with high levels of accuracy, even in cases of significant MCC. Importantly, the algorithm eliminates the need for maternal DNA and additional MCC tests, which reduces costs and simplifies the diagnostic procedure. The method is easy to set up and suitable for routine clinical practice.

Performance and Diagnostic Value of Genome-Wide Noninvasive Prenatal Testing in Multiple Gestations

To evaluate the accuracy and diagnostic value of genome-wide noninvasive prenatal testing (NIPT) for the detection of fetal aneuploidies in multiple gestations, with a focus on dichorionic-diamniotic twin pregnancies. We performed a retrospective cohort study including data from pregnant women with a twin or higher-order gestation who underwent genome-wide NIPT at one of the eight Belgian genetic centers between November 1, 2013, and March 1, 2020. Chorionicity and amnionicity were determined by ultrasonography. Follow-up invasive testing was carried out in the event of positive NIPT results. Sensitivity and specificity were calculated for the detection of trisomy 21, 18, and 13 in the dichorionic-diamniotic twin cohort. Unique NIPT analyses were performed for 4,150 pregnant women with a multiple gestation and an additional 767 with vanishing gestations. The failure rate in multiple gestations excluding vanishing gestations ranged from 0% to 11.7% among the different genetic centers. Overall, the failure rate was 4.8%, which could be reduced to 1.2% after single resampling. There were no common fetal trisomies detected among the 86 monochorionic-monoamniotic and 25 triplet cases. Two monochorionic-diamniotic twins had an NIPT result indicative of a trisomy 21, which was confirmed in both fetuses. Among 2,716 dichorionic-diamniotic twin gestations, a sensitivity of 100% (95% CI 74.12-100%) and a specificity of 100% (95% CI 99.86-100%) was reached for trisomy 21 (n=12). For trisomy 18 (n=3), the respective values were 75% (95% CI 30.06-95.44%) sensitivity and 100% (95% CI 99.86-100%) specificity, and for trisomy 13 (n=2), 100% (95% CI 20.65-100%) sensitivity and 99.96% (95% CI 99.79-99.99%) specificity. In the vanishing gestation group, 28 NIPT results were positive for trisomy 21, 18, or 13, with only five confirmed trisomies. Genome-wide NIPT performed accurately for detection of aneuploidy in dichorionic-diamniotic twin gestations.

Results of non-invasive prenatal testing for 2473 women with twin pregnancy

To assess the value of non-invasive prenatal testing (NIPT) for the detection of fetal chromosomal aneuploidies in women with twin pregnancy. A total of 2473 women with twin pregnancy underwent the NIPT test to assess the risk for fetal chromosomal aneuploidies from January 2016 to September 2019. Those with a high risk by NIPT were confirmed by amniocentesis or chorionic villus sampling. All cases were followed up to evaluate the positive prediction value of NIPT for twin pregnancies. Among the 2473 women, the NIPT test has identified 31 cases (1.25%) with a high risk for fetal chromosomal aneuploidies, which included 5 cases of trisomy 21, 1 case of chromosome 21 deletion, 4 cases of trisomy 18, 7 cases of sex chromosome abnormality and 14 cases of microdeletion and microduplication. By invasive prenatal diagnosis or chromosomal karyotyping analysis of neonates, 5 cases of trisomy 21, 3 cases of trisomy 18, 1 case of sex chromosome abnormality, and 2 cases of microdeletion and microduplication were confirmed, which yielded a positive predictive value of 100%, 75%, 25% and 25%, respectively. NIPT can be used for the screening of fetal chromosomal aneuploidies in women with twin pregnancy with high accuracy. The method is non-invasive, safe and effective for the screening of fetal chromosomal aneuploidies, in particular trisomy 21.

EP503 - Clinical validation and reporting of noninvasive prenatal testing for fetal aneuploidy detection

eP503 - Clinical validation and reporting of noninvasive prenatal testing for fetal aneuploidy detection

Simultaneous detection of fetal aneuploidy, de novo FGFR3 mutations and paternally derived β-thalassemia by a novel method of noninvasive prenatal testing.

ObjectiveThe aim is to develop a novel noninvasive prenatal testing (NIPT) method that simultaneously performs fetal aneuploidy screening and the detection of de novo and paternally derived mutations.MethodsA total of 68 pregnancies, including 26 normal pregnancies, 7 cases with fetal aneuploidies, 7 cases with fetal achondroplasia or thanatophoric dysplasia, 18 cases with fetal skeletal abnormalities, and 10 cases with β‐thalassemia high risk were recruited. Plasma cell‐free DNA was amplified by Targeted And Genome‐wide simultaneous sequencing (TAGs‐seq) to generate around 99% of total reads covering the whole‐genome region and around 1% covering the target genes. The reads on the whole‐genome region were analyzed for fetal aneuploidy using a binary hypothesis T‐score and the reads on target genes were analyzed for point mutations by calculating the minor allelic frequency of loci on FGFR3 and HBB. TAGs‐seq results were compared with conventional NIPT and diagnostic results.ResultsIn each sample, TAGs‐seq generated 44.7–54 million sequencing reads covering the whole‐genome region of 0.1–3× and the target genes of >1000×depth. All cases of fetal aneuploidy and de novo mutations of achondroplasia/thanatophoric dysplasia were identified with high sensitivities and specificities except for one false‐negative paternal mutation of β‐thalassemia.ConclusionsTAGs‐seq is a novel NIPT method that combines the fetal aneuploidy screening and the detection of de novo FGFR3 mutations and paternal HBB mutations.