Accuracy Of Non-invasive Prenatal Testing Research Articles

Non-invasive prenatal detection of dominant single-gene disorders in fetal structural abnormalities: a clinical feasibility study.

This study evaluated the accuracy of non-invasive prenatal testing (NIPT-SGDs) for dominant monogenic genetic diseases associated with fetal structural abnormalities and to assess the feasibility of clinical application. Pregnant women requiring prenatal diagnosis due to fetal structural abnormalities were enrolled. Maternal peripheral blood was analyzed for cell-free DNA (cfDNA) using coordinative allele-aware target enrichment sequencing (COATE-seq). This assessed fetal allele depth distribution, fraction and variation ratio. The variation's origin was then determined to obtain fetal variation information. Finally, NIPT-SGDs results were confirmed via invasive prenatal diagnosis (IPD). Upon examination of 113 samples using NIPT-SGDs, COATE-seq successfully analyzed 112 for fetal variation, excluding one due to hemolysis. The study detected six positive cases, yielding a 5.36% detection rate. These disorders included tuberous sclerosis complex (TSC1 and TSC2 being its causative genes), Noonan syndrome (PTPN11), polycystic kidney disease (PKD1), and Kabuki syndrome (KMT2D), occurring twice each, except for Noonan and polycystic kidney disease. Two false positives were due to the mother being a genetic mosaicism. Compared to invasive whole-exome sequencing (WES), NIPT-SGDs did not detect nine positive cases of IPD dominant monogenic diseases, accurately identifying 90.18% (101/112) of the actual positive and negative cases. Our findings demonstrate the clinical utility of NIPT-SGDs using COATE-seq in effectively identifying fetuses with dominant single-gene disorders. Furthermore, this method can be applied to all fetuses.

The Role of AI and Machine Learning in Revolutionizing Prenatal Screening and Genetic Analysis

rtificial Intelligence (AI) and Machine Learning (ML) are at the forefront of innovations in medical diagnostics, including prenatal screening and genetic analysis. The development of advanced algorithms, data processing capabilities, and predictive modeling has significantly enhanced the sensitivity, specificity, and accuracy of non-invasive prenatal testing (NIPT). This article explores recent advancements in AI- driven prenatal screening, the methodologies employed, and the future potential of AI in predictive prenatal health diagnostics, with a particular focus on improving genetic disorder detection and fetal health outcomes. The role of ethical considerations in AI-driven diagnostics is also discussed.

Investigational Management for a Positive NIPT Result - Case Report

Non-invasive prenatal testing (NIPT), since its introduction in 2011, has revolutionized prenatal screening, becoming widely used globally and replacing traditional screening methods in developed countries. The accuracy of NIPT in detecting aneuploidies is etremely high and there has been a recent trend towards improving NIPT for detecting microdeletion and microduplication syndromes, monogenic diseases, fetal se determination, and RH genotyping. Significant progress has been made in molecular analysis techniques of fetal DNA, including methods such as massively parallel sequencing, RNA-based testing, digital PCR, and single nucleotide polymorphism analysis. We present the case of a 32-year-old patient who, at 12 weeks of gestation, had a non-invasive prenatal test result showing a maternal 22q11.2 deletion. Following genetic consultation, further investigations were conducted to stratify the fetal risk of inheriting the microdeletion syndrome. As a result, microarray CGH cytoarray from amniotic fluid was performed, and no 22q11.2 deletion was detected. In this case, complete elucidation of the origin of the deletion found in NIPT could not be achieved, as it would require arrayCGH testing for the mother, a test that was not performed due to financial reasons. Given the high rate of genetic syndromes with potential impact on fetal development and familial psychological impact, we wish to emphasize the necessity of financial support from the state to introduce non-invasive prenatal testing into the list of reimbursed analyses covered by health insurance. This would enable superior testing and, implicitly, genetic prevention of all pregnancies, facilitating appropriate risk stratification of pregnancies in our country.

Improving the accuracy of noninvasive prenatal testing through size-selection between fetal and maternal cfDNA.

In general, fetal cfDNA is shorter than maternal cfDNA, and accuracy of noninvasive prenatal testing (NIPT) results can be improved by selecting shorter cfDNA fragments to enrich fetal-derived cfDNA. This study investigated potential improvements in the accuracy of NIPT by performing classification and analysis based on differences in cfDNA size. We performed paired-end sequencing to identify size ranges of fetal and maternal cfDNA from 62,374 pregnant women. We then developed a size-selection method to isolate and analyze both fetal and maternal cfDNA, defining fetal-derived cfDNA as less than 150 bp and maternal-derived cfDNA as greater than 180 bp. By implementing size-selection method, the accuracy of NIPT was improved, resulting in an increase in the overall positive predictive value for all aneuploidies from 89.57% to 97.1%. This was achieved by enriching both fetal and maternal-derived cfDNA, which increased fetal DNA fraction while the number of false positives for all aneuploidies was reduced by more than 70%. We identified the differences in read length between fetal and maternal-derived cfDNA, and selectively enriched both shorter and longer cfDNA fragments for subsequent analysis. Our approach can increase the detection accuracy of NIPT for detecting fetal aneuploidies and reduce the number of false positives caused by maternal chromosomal abnormalities.

Combined fetal fraction to analyze the Z-score accuracy of noninvasive prenatal testing for fetal trisomies 13, 18, and 21

ObjectiveThis study aims to evaluate the correlation combined fetal fraction and Z-score for fetal trisomies 13, 18, and 21 of NIPT by the semiconductor sequencing platform and further analyze the differences of different sequencing depths.MethodsA cohort of 61,581 pregnancies were recruited for NIPT. Invasive prenatal diagnostic confirmation is recommended in all high-risk NIPT cases. Logistic regression and rank correlation analysis were applied to analyze the relationship between different parameters. ROC curve analysis was adopted to analyze the cutoff values of Z-score and fetal fraction.ResultsA total of 278 common trisomy pregnancies were verified in 377 NIPT-positive results. The fitted logistic regression models revealed that Z-scores of NIPT-positive results were significantly associated with PPVs (p < 0.05). The ROC curve analysis showed that the optimal cutoff value of Z-scores for T21, T18, and T13 was 7.597, 4.944, and 9.135 for NIPT and 9.489, 8.004, and 12.4 for NIPT-plus. If combing fetal fraction as another evaluation factor, the PPV of trisomy 21 gradually improved. We analyzed the correlation between the fetal fraction and the PPV, which revealed that the fetal fraction was significantly correlated with PPV. By analyzing the PPV of different groups divided by the associated criteria obtained from ROC curve, the PPV of high Z-score and high fetal fraction is higher in groups of Z-score > the optimal cutoff value.ConclusionThe results of this study show that the fetal fraction is significantly correlated with the PPV. Combining fetal fraction with Z-score is significantly better than in groups of Z-score-associated criteria; clinicians can give more accurate and efficient prenatal genetic counseling.

Accuracy of Non-Invasive Prenatal Testing for Duchenne Muscular Dystrophy in Families at Risk: A Systematic Review.

Methodological advancements, such as relative haplotype and relative mutation dosage analyses, have enabled non-invasive prenatal diagnosis of autosomal recessive and X-linked diseases. Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by progressive proximal muscular dystrophy and a high mortality rate before the age of twenty. We aimed to systematically present obtainable data regarding a non-invasive prenatal diagnosis of DMD and provide a comprehensive resume on the topic. The emphasis was given to the comparison of different available protocols and molecular methods used for fetal inheritance deduction, as well as their correlation with prognostic accuracy. We searched the Scopus and PubMed databases on 11 November 2022 and included articles reporting a non-invasive prenatal diagnosis of DMD in families at risk using relative dosage analysis methods. Of the 342 articles identified, 7 met the criteria. The reported accuracy of NIPT for DMD was 100% in all of the studies except one, which demonstrated an accuracy of 86.67%. The combined accuracy for studies applying indirect RHDO, direct RHDO, and RMD approaches were 94.74%, 100%, and 100%, respectively. Confirmatory results by invasive testing were available in all the cases. Regardless of the technological complexity and low prevalence of the disease that reduces the opportunity for systematic research, the presented work demonstrates substantial accuracy of NIPT for DMD. Attempts for its implementation into everyday clinical practice raise many ethical and social concerns. It is essential to provide detailed guidelines and arrange genetic counseling in order to ensure the proper indications for testing and obtain informed parental consent.

Značaj NIPT U kombinaciji sa serumom bez DNK ćelija, estriolom, AFP i nivoom b-HCG U prepoznavanju trizomije 21 i 18 u drugom trimestru

Background: This study aimed to evaluate the clinical application value of noninvasive prenatal testing from DNA (NIPT) and serum screening for screening in detecting fetal trisomy 21 and 18. Methods: As a retrospective analysis, we collected data from 1383 women (singleton pregnancy) who underwent serum screening and noninvasive prenatal testing from DNA (NIPT) in our department from May 2015 to September 2017 and calculated the diagnostic value of the two methods. Results: In 1383 cases, the sensitivity of serum screening to trisomy 21 and 18 was 76.9%, specificity 74.7%, PPV 2.9%, NPV 99.7%, and AUC 0.758 (95% CI: 0.625-0.891). The sensitivity of noninvasive prenatal testing from DNA (NIPT) to trisomy 21 and 18 was 100%, specificity 99.8%, PPV 81%, NPV 100%, and AUC 0.999 (95% CI: 0.000-1.000). Conclusion: Serum screening can detect high-risk pregnant women in time, but the incidence of false positives and negatives is high; the accuracy of noninvasive prenatal testing from DNA (NIPT) is high, which can effectively reduce the rate of defective babies.

The Clinical Application and Accuracy Evaluation of Noninvasive Prenatal Testing for Common Trisomy and Sex Chromosome Aneuploidy.

Noninvasive prenatal testing (NIPT) has been widely adopted in prenatal examination for fetal chromosomal aneuploidy. The present study aimed to evaluate the clinical features of NIPT for both common trisomy and sex chromosome aneuploidy (SCA). A total of 24,164 pregnant women with NIPT testing from July 2020 to June 2022 were recruited at the Linping Maternity and Child Health Care Hospital. Ninety cases showed high risk of trisomy 21/18/13 with karyotype results available, and the sensitivity, specificity, and positive predictive value (PPV) were 98.41%, 99.88% and 68.89%, respectively. The three most important reasons for screening were advanced maternal age (AMA, 28.06%), intermediate risk of prenatal screening (20.34%) and Multiple of medium (MoM) abnormality of prenatal screening (17.38%). High risk of NIPT results with Z-score ≥15 have a higher PPV when compared to those with 3 ≤ Z-score < 10, and 10 ≤ Z-score < 15. Meanwhile, 97 pregnant women received positive results for fetal sex chromosome aneuploidy (SCA) in NIPT. In addition, the rate for further diagnostics of SCA was 64.95% and the PPV of SCA was 50.79%. Our data show that NIPT has a promising future in prenatal screening for genetic abnormalities of the fetus, and that the accuracy of NIPT is closely related to Z-score.

Combining Z-Score and Maternal Copy Number Variation Analysis Increases the Positive Rate and Accuracy in Non-Invasive Prenatal Testing.

Objective: To evaluate positive rate and accuracy of non-invasive prenatal testing (NIPT) combining Z-score and maternal copy number variation (CNV) analysis. To assess the relationship between Z-score and positive predictive value (PPV). Methods: This prospective study included 61525 pregnancies to determine the correlation between Z-scores and PPV in NIPT, and 3184 pregnancies to perform maternal CNVs analysis. Positive results of NIPT were verified by prenatal diagnosis and/or following-up after birth. Z-score grouping, logistic regression analysis, receiver operating characteristic (ROC) curves, and S-curve trends were applied to correlation analysis of Z-scores and PPV. The maternal CNVs were classified according to the technical standard for the interpretation of ACMG. Through genetic counseling, fetal and maternal phenotypes and family histories were collected. Results: Of the 3184 pregnant women, 22 pregnancies were positive for outlier Z-scores, suggesting fetal aneuploidy. 12 out of 22 pregnancies were true positive (PPV = 54.5%). 17 pregnancies were found maternal pathogenic or likely pathogenic CNVs (> 0.5 Mb) through maternal CNV analysis. Prenatal diagnosis revealed that 7 out of 11 fetuses carried the same CNVs as the mother. Considering the abnormal biochemical indicators during pregnancy and CNV-related clinical phenotypes after birth, two male fetuses without prenatal diagnosis were suspected to carry the maternally-derived CNVs. Further, we identified three CNV-related family histories with variable phenotypes. Statistical analysis of the 61525 pregnancies revealed that Z-scores of chromosomes 21 and 18 were significantly associated with PPV at 3 ≤ Z ≤ 40. Notably, three pregnancies with Z > 40 were both maternal full aneuploidy. At Z < -3, fetuses carried microdeletions instead of monosomies. Sex chromosome trisomy was significantly higher PPV than monosomy. Conclusion: The positive rate of the NIPT screening model combining Z-score and maternal CNV analysis increased from 6.91‰ (22/3184) to 12.25‰ (39/3184) and true positives increased from 12 to 21 pregnancies. We found that this method could improve the positive rate and accuracy of NIPT for aneuploidies and CNVs without increasing testing costs. It provides an early warning for the inheritance of pathogenic CNVs to the next generation.

Evaluation of the Z-score accuracy of noninvasive prenatal testing for fetal trisomies 13, 18 and 21 at a single center.

To assess the correlation between Z-scores of positive noninvasive prenatal testing (NIPT) results and the positive predictive value (PPV) of NIPT. Pregnancies with positive NIPT results at Guangzhou Women and Children's Medical Centre between July 2017 and May 2020 were included in this study. Fetal karyotyping or microarray analysis was provided to patients with abnormal NIPT results for confirmatory testing. Logistic regression analyses was applied to study the relationship between the Z scores and the PPV performance. The optimal cutoff values for indicating fetal common trisomies were obtained based on receiver operating characteristic (ROC) curve analysis, and then the PPV were calculated in pregnancies with positive NIPT results at Z-score greater than or equal to cutoff value and in patients with a Z-score between 3 and cutoff value respectively. A total of 214 pregnancies with positive NIPT results for fetal common trisomies were validated by invasive prenatal diagnosis and follow up in this study. Of these, NIPT indicated trisomy 13 in 25 cases, trisomy 18 in 54 cases and trisomy 21 in 135 patients. Logistic regression analyses showed a significant association (p<0.05) between the Z-scores and true positive results for T21 and T18. For T13, the significant association was not observed (p>0.05). The ROC curve analysis showed that the optimal cutoff Z-score for indicating fetal trisomies 13, 18, and 21 were 6.889, 7.574 and 6.612 respectively, and the corresponding area under curve were 0.706, 0.916, and 0.954. In this cohort with abnormal NIPT results, the cutoff values revealed a sensitivity of 96.8% and a specificity of 90% for indicating trisomies 21, and a sensitivity of 88.9% and a specificity of 92.6% for trisomies 18. However, probably due to the sample size, the sensitivity and specificity for indicating trisomy 13 were lower (85.7% and 61.1%) than that for trisomies 21 and 18. The PPVs in pregnancies with positive NIPT results at Z-score greater than or equal to cutoff value were 99.18% (121/122) for trisomy 21, 92.31% (24/26) for trisomy 18 and 46.15% (6/13) for trisomy 13. In patients with a Z-score between 3 and cutoff Z-score, the PPV of NIPT for trisomies 21, 18, and 13 were 30.77% (4/13), 10.71% (3/28), and 8.33% (1/12) respectively. Moreover, by classifying Z scores as 3≤Z<5, 5≤Z<10, and Z≥10, the majority of Z scores were above 10 with a PPV of 99% for T21 and just 5.2% were between 3 and 5 with a PPV of 14.3%. In contrast for T18, over a third of tests had Z scores between 3 and 5. The PPV in this group is just over 5%. The present results show that the PPV performance of NIPT for fetal trisomies 13, 18, and 21 are closely associated with Z-score. The higher the Z-score, the greater the likelihood that the aneuploidy result is correct. Our experience in evaluating the Z-score accuracy of NIPT in this study could be of use in similar work.

A Novel Graphic-Aided Algorithm (gNIPT) Improves the Accuracy of Noninvasive Prenatal Testing.

Noninvasive Prenatal Testing (NIPT) has advanced the detection of fetal chromosomal aneuploidy by analyzing cell-free DNA in peripheral maternal blood. The statistic Z-test that it utilizes, which measures the deviation of each chromosome dosage from its negative control, is now widely accepted in clinical practice. However, when a chromosome has loss and gain regions which offset each other in the z-score calculation, merely using the Z-test for the result tends to be erroneous. To improve the performance of NIPT in this aspect, a novel graphic-aided algorithm (gNIPT) that requires no extra experiment procedures is reported in this study. In addition to the Z-test, this method provides a detailed analysis of each chromosome by dividing each chromosome into multiple 2 Mb size windows, calculating the z-score and copy number variation of each window, and visualizing the z-scores for each chromosome in a line chart. Data from 13537 singleton pregnancy women were analyzed and compared using both the normal NIPT (nNIPT) analysis and the gNIPT method. The gNIPT method had significantly improved the overall positive predictive value (PPV) of nNIPT (88.14% vs. 68.00%, p = 0.0041) and the PPV for trisomy 21 (T21) detection (93.02% vs. 71.43%, p = 0.0037). There were no significant differences between gNIPT and nNIPT in PPV for trisomy 18 (T18) detection (88.89% vs. 63.64%, p = 0.1974) and in PPV for trisomy 13 (T13) detection (57.14% vs. 50.00%, p = 0.8004). One false-negative T18 case in nNIPT was detected by gNIPT, which demonstrates the potency of gNIPT in discerning chromosomes that have variation in multiple regions with an offsetting effect in z-score calculation. The gNIPT was also able to detect copy number variation (CNV) in chromosomes, and one case with pathogenic CNV was detected during the study. With no additional test requirement, gNIPT presents a reasonable solution in improving the accuracy of normal NIPT.

Clinical experience regarding the accuracy of NIPT in the detection of sex chromosome abnormality.

The present study aimed to determine the accuracy (Z-value) of non-invasive prenatal testing (NIPT) results for sex chromosome aneuploidy (SCA) in routine clinical practice. Among a cohort of 12505 pregnant females, maternal plasma samples collected from our hospital were utilized for SCA analysis by NIPT detection. The positive samples were validated through an invasive procedure and karyotyping analysis. The predictive value from positive samples in sex chromosomes was compared to analyze the accuracy of the Z-value. There were 65 females with sex chromosome abnormalities within 12,505 pregnant females in the NIPT detection, which was validated by karyotype analysis of amniotic fluid puncture through sequencing, as well as bioinformatics analysis, with 18 true-positive samples. The true-positive results with 45,X, 47,XXY, 47,XXX and 47,XYY karyotypes predicted by NIPT were 14.29%, 50.00%, 66.67% and 71.43%, respectively. Among sex chromosome cases, the findings indicated that positive NIPT results with Z ≥ 9 show a higher accuracy. The findings of the present study demonstrate that the positive predictive value of NIPT for sex chromosome abnormalities is distinctive. The positive predictive value was highest for 47,XYY and lowest for 45,X. Additionally, the Z-value results are considered to be correlated with the accuracy of NIPT, although further studies need to be made.

Application values of prenatal screening and non-invasive gene sequencing in fetal birth defects.

Objective:To investigate effects of prenatal screening and non-invasive gene sequencing on the clinical diagnosis of fetal birth defects and the outcome of pregnancy.Methods:Totally 2520 pregnant women who received prenatal screening in our hospital were selected as the research subjects. The high-risk pregnant women were further tested by the non-invasive gene sequencing technology. Pregnant women with positive results were diagnosed by amniocentesis and fetal chromosome karyotype analysis, and the pregnancy outcome was followed up for one year.Results:870 out of the 2520 pregnant women was tested by non-invasive gene sequencing technology; 26 of the 870 women was 13-trisomy-positive and was diagnosed by amniocentesis and fetal chromosome karyotype analysis, 22 of which was diagnosed as 47, XN, +13 and four of which was normal; the diagnosis accuracy of non-invasive prenatal testing (NIPT) was 84.6%. 18 out of the 22 confirmed cases underwent abortion, three cases had termination of embryonic development, and one case had postnatal anomaly. Thirty four out of the 2520 pregnant women was 18-trisomy-positive and was diagnosed by amniocentesis and fetal chromosome karyotype analysis, 31 of which was diagnosed as 47, XN, +18 and three cases were normal; the diagnosis accuracy of NIPT was 91.2%. 29 out of the 31 confirmed cases underwent abortion and two cases had termination of embryonic development. Forty out of the 2520 pregnant women was 21-trisomy-positive and was diagnosed by amniocentesis and fetal chromosome karyotype analysis, 39 of which was diagnosed as 47, XN, +21 and one case was normal; the diagnosis accuracy of NIPT was 97.5%. Thirty four out of the 39 confirmed cases underwent abortion, three cases had termination of embryonic development, and two cases had postnatal anomaly. Twenty eight cases were tested as sex chromosome-positive and were diagnosed by amniocentesis and fetal chromosome karyotype analysis, 25 out of which was diagnosed as abnormal and three cases were normal; the diagnosis accuracy of NIPT was 89.3%. 24 out of the 25 confirmed cases underwent abortion, and one case had termination of embryonic development.Conclusion:Prenatal screening and non-invasive gene sequencing technology have a high accuracy in the diagnosis of fetal birth defects, which can reduce the maternal abortion injury as much as possible and relieve the psychological pressure. The promotion of the mode can be strengthened in clinics.

Antenatal screening for fetal trisomies using microarray-based cell-free DNA testing: A systematic review and meta-analysis.

To evaluate the test accuracy of non-invasive prenatal testing (NIPT) for fetal trisomy 21, 18, and 13 using cell-free (cf) DNA analysis in maternal plasma with microarray quantitation. Systematic review and meta-analysis. Searches in MEDLINE, Pre-MEDLINE, EMBASE, Web of Science, and the Cochrane Library to 09.07.2018. Five studies analyzing 3074 samples, including 187 trisomy 21, 43 trisomy 18, and 19 trisomy 13 cases, were identified. Risk of bias was high in all studies, introduced particularly by exclusions from analysis and by the role of the sponsor. Sensitivity of microarray-based cfDNA testing was 99.5% (95%CI 96.3%-99.9%) for trisomy 21, 97.7% (95%CI 87.9%-99.6%) for trisomy 18, and 100% (95%CI 83.2%-100%) for trisomy 13. Specificity was 100% (95% CI 99.87%-100%) for trisomy 21, 99.97% (95%CI 99.81%-99.99%) for trisomy 18, and 99.97% (95%CI 99.81%-99.99%) for trisomy 13. Pooled test failure rate was 1.1%. A direct comparison of microarray- and sequencing-based cfDNA found equivalent test accuracy. Included studies suggest that NIPT using microarray-based cfDNA testing has high sensitivity and specificity for detecting fetal trisomy 21, 18, and 13. However, the evidence base is small and at high risk of bias.

Progress in non-invasive prenatal testing failure due to less cell-free fetal DNA

Non-invasive prenatal testing (NIPT) has been extensively applied in fetal chromosomal aneuploidy screening with high sensitivity and specificity. The concentration of cell-free fetal DNA (cffDNA) is an important factor related to the accuracy of NIPT. When cffDNA concentration is lower than 4%, accurate results are often unavailable, resulting in failure of NIPT. The factors influencing cffDNA concentration in maternal blood were reviewed and it was found that the concentration of cffDNA was positively correlated with the gestational week at the time of sampling, but negatively correlated with the maternal weight/body mass index. Moreover, gestational complications (such as preeclampsia and polycystic ovary syndrome), maternal age, results of serological screening for Down's syndrome in second trimester, fetal chromosomal karyotype and nuchal translucency thickness were also potential influencing factors. Analysis of the influencing factors of cffDNA concentration helps to improve the NIPT specifications, which is significant for prenatal genetic counselling. Key words: Aneuploidy; Prenatal diagnosis; Cell-free system; DNA; Fetus

A Novel Next-Generation Sequencing Based Assay for Non-Invasive Prenatal Testing of Sickle Cell Disease without Paternal DNA

Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect <5% deviation from 50% allele fraction. To overcome this challenge, we have developed and optimized an NIPT for SCD by assessing the relative mutation dosage of fetal SCD and beta-thalassemia DNA through a novel molecular counting strategy using NGS.Objectives: The primary objective of this study is to evaluate the performance of a novel NIPT for sickle cell disease.Methods: The SCD NIPT assay and associated custom bioinformatics analysis were performed on cfDNA obtained from a training cohort of non-pregnant compound heterozygotes for SCD. The SCD NIPT assay was then performed on a validation cohort of pregnant women with either SCD or sickle cell trait (SCT). The accuracy of the SCD NIPT was evaluated by comparison with newborn screening results.Results: Non-pregnant individuals with genotype HbSE, HbSC, or HbS/beta-thalassemia were included as a training cohort to establish the precision and accuracy of the assay for measuring HbS allele fraction from cfDNA. As expected, the HbS allele fraction in these individuals was 0.500 (standard deviation = 0.011, n = 26), and there was no detectable fetal fraction in these samples. Both training and validation cohort results matched the theoretical limit of detection set by the number of cell-free HBB DNA molecules in plasma. The precision and accuracy of the HBB assay on cfDNA were then used in conjunction with >1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity >98% and specificity >99%, even in the absence of paternal DNA.Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with >20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is <$30 per sample. The ability to ascertain fetal SCD status based only on maternal blood will be valuable in clinical settings where the father is unavailable or sample collection would be inconvenient or time-consuming. Several Phase I/II and Phase III trials for curing SCD or beta-thalassemia using autologous gene-editing of stem cells are currently in progress. SCD NIPT could be particularly useful for deciding to bank umbilical cord blood as a source of stem cells for future gene-editing cures. DisclosuresTsao:BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Atay:BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Analysis of the accuracy of Z-scores of non-invasive prenatal testing for fetal Trisomies 13, 18, and 21 that employs the ion proton semiconductor sequencing platform

BackgroundNon-invasive prenatal testing (NIPT) is frequently being used to screen for trisomies 13, 18 and 21 for prenatal diagnosis. However, NIPT performs poorly when compared with invasive testing and thus should not be used to diagnose trisomies. The result of NIPT for an individual woman in most genome-wide methods is calculated as a Z-score. The aim of this study was to assess the correlation between Z-scores of NIPT results and the accuracy of non-invasive prenatal testing.ResultsWe evaluated 108 pregnant women with positive NIPT results, which were validated through karyotype analysis of amniotic fluid puncture by means of sequencing, bioinformatics analysis, and follow-up. Utilizing the ion proton semiconductor sequencing platform, we report a performance evaluation of NIPT-positive results at Third Affiliated Hospital of Zhengzhou University of Henan Province, China, by classifying Z-scores as 3 ≤ Z<5, 5 ≤ Z < 9 and Z ≥ 9. The findings indicate that positive NIPT results at Z ≥ 9 have a higher accuracy compared with positive NIPT results at 5 ≤ Z < 9 and 3 ≤ Z<5.ConclusionsThe findings show that Z-scores of NIPT results are closely related to the accuracy of non-invasive prenatal testing. However, false-positive NIPT results at 3 ≤ Z<5 may occur due to confined placental mosaicism (CPM).

Clinical experience with sex chromosome aneuploidies detected by noninvasive prenatal testing (NIPT): Accuracy and patient decision-making.

The objectives of the study are to assess the accuracy of noninvasive prenatal testing (NIPT) for sex chromosome aneuploidies (SCAs) and to investigate patient decision-making in clinical practice. This is a retrospective cohort study review of positive NIPT results for SCAs from January 2013 to September 2017. Of the 136 positive NIPT results for SCAs, 73 (53.7%) were positive for 45,X, 62 (45.6%) were a sex chromosome trisomy, and 1 was a sex chromosome tetrasomy. Of the 134 viable pregnancies, 46 (34.3%) elected to pursue prenatal diagnosis. Fewer women underwent invasive prenatal testing when counseled regarding a positive NIPT for monosomy X in the presence of suggestive ultrasound findings (4/23; 17.4%) compared with those who had a positive NIPT result without ultrasound findings (24/46, 52.2%). Abnormal karyotypes consistent with the NIPT result were confirmed in 30/64 (46.9%). Even in the context of ultrasound abnormalities, there was not 100% concordance. The majority (88/134; 65.7%) of patients in our cohort declined prenatal diagnosis even in the presence of associated ultrasound findings. Comprehensive pretest and posttest counseling is recommended and should address the importance of confirmatory testing and benefits of early diagnosis. Practice guidelines are needed to address provider responsibilities about postnatal testing.

Talking Points: Women's Information Needs for Informed Decision-Making About Noninvasive Prenatal Testing for Down Syndrome.

Adequate knowledge is a vital component of informed decision-making; however, we do not know what information women value when making decisions about noninvasive prenatal testing (NIPT). The current study aimed to identify women's information needs for decision-making about NIPT as a first-tier, non-contingent test with out-of-pocket expense and, in turn, inform best practice by specifying the information that should be prioritized when providing pre-test counseling to women in a time-limited scenario or space-limited decision support tool. We asked women (N = 242) in Australia to indicate the importance of knowing 24 information items when making a decision about NIPT and to choose two information items they would most value. Our findings suggest that women value having complete information when making decisions about NIPT. Information about the accuracy of NIPT and the pros and cons of NIPT compared to other screening and invasive tests were perceived to be most important. The findings of this study can be used to maximize the usefulness of time-limited discussions or space-limited decision support tools, but should not be routinely relied upon as a replacement for provision of full and tailored information when feasible.

Non-Invasive Prenatal Testing for Sex Chromosome Aneuploidy in Routine Clinical Practice

Objectives: To assess the accuracy of non-invasive prenatal testing (NIPT) for sex chromosome aneuploidy (SCA) in routine clinical practice and to review counselling and sonographic issues arising in SCA cases. Methods: Three specialist Australian obstetric ultrasound and prenatal diagnosis practices offering NIPT after 10 weeks' gestation participated in this study. NIPT was reported for chromosomes 21, 18, 13, X, and Y. Results: NIPT screening was performed in 5,267 singleton pregnancies. The odds of being affected given a positive screening result (OAPR) was lowest for SCAs, most notably for monosomy X (20%). Fewer women underwent invasive prenatal testing when counselled regarding a high risk for SCA (65.5%) compared with those who had a high risk for another aneuploidy (85%). The positive screening rate of NIPT including SCA was 2.3%, but 1.2% if only the autosomal trisomies were included in the panel. Conclusion: The addition of SCA testing to NIPT doubles the positive screening rate. The OAPR for SCAs (most notably for monosomy X) is reduced compared with the autosomal trisomies. Clinicians need a more extensive discussion with women prior to the inclusion of the X and Y chromosomes in the NIPT panel, given the complexity in counselling regarding further management and the additional anxiety that these abnormal results may cause. A benefit of sex chromosome analysis is an improvement in antenatal diagnosis of some disorders of sexual development.