Anticoagulation use is associated with lower fetal fraction and more indeterminate results.

BACKGROUND: Maternal anticoagulation use may increase indeterminate result rates on cell-free DNA-based screening, but existing studies are confounded by inclusion of individuals with autoimmune disease, which alone is associated with indeterminate results. Changes in chromosome level Z-scores are proposed by others as a reason for indeterminate results, but the etiology of this is uncertain. OBJECTIVE: This study aimed to evaluate differences in fetal fraction, indeterminate result rate, and total cell-free DNA concentration in individuals on anticoagulation without autoimmune disease compared with controls undergoing noninvasive prenatal screening. Secondly, using a nested case-control design, we evaluated differences in fragment size, GC-content, and Z-scores to evaluate laboratory-level test characteristics. STUDY DESIGN: This was a retrospective single-institution study of pregnant individuals undergoing cell-free DNA-based noninvasive prenatal screening using low-pass whole-genome sequencing between 2017 and 2021. Individuals with autoimmune disease, suspected aneuploidy, and cases where fetal fraction was not reported were excluded. Anticoagulation included heparin-derived products (unfractionated heparin, low-molecular-weight heparin), clopidogrel, and fondaparinux, with a separate group for those on aspirin alone. An indeterminate result was defined as fetal fraction <4%. We evaluated the association between maternal anticoagulation or aspirin use, and fetal fraction, indeterminate results, and total cell-free DNA concentration using univariate and multivariate analyses, controlling for body mass index, gestational age at sample collection, and fetal sex. For the anticoagulation cohort, we compared laboratory-level test characteristics among cases (on anticoagulation) and a subset of controls. Lastly, we evaluated for differences in chromosome level Z-scores among those on anticoagulation with and without indeterminate results. RESULTS: A total of 1707 pregnant individuals met the inclusion criteria. Of those, 29 were on anticoagulation and 81 were on aspirin alone. For those on anticoagulation, the fetal fraction was significantly lower (9.3% vs 11.7%; P<.01), the indeterminate result rate was significantly higher (17.2% vs 2.7%; P<.001), and the total cell-free DNA concentration was significantly higher (218 pg/µL vs 83.7 pg/µL; P<.001). Among those on aspirin alone, the fetal fraction was lower (10.6% vs 11.8%; P=.04); however, there were no differences in the rate of indeterminate results (3.7% vs 2.7%; P=.57) or total cell-free DNA concentration (90.1 pg/µL vs 83.8 pg/µL; P=.31). After controlling for maternal body mass index, gestational age at sample collection, and fetal sex, anticoagulation was associated with an >8-fold increase in the likelihood of an indeterminate result (adjusted odds ratio, 8.7; 95% confidence interval, 3.1-24.9; P<.001), but not aspirin (adjusted odds ratio, 1.2; 95% confidence interval, 0.3-4.1; P=.8). Anticoagulation was not associated with appreciable differences in cell-free DNA fragment size or GC-content. Although differences in chromosome 13 Z-scores were observed, none were observed for chromosomes 18 or 21, and this difference did not contribute to the indeterminate result call. CONCLUSION: In the absence of autoimmune disease, anticoagulation use, but not aspirin, is associated with lower fetal fraction, higher total cell-free DNA concentration, and higher rates of indeterminate results. Anticoagulation use was not accompanied by differences in cell-free DNA fragment size or GC-content. Statistical differences in chromosome level Z-scores did not clinically affect aneuploidy detection. This suggests a likely dilutional effect by anticoagulation on cell-free DNA-based noninvasive prenatal screening assays contributing to low fetal fraction and indeterminate results, and not laboratory or sequencing-level changes.

Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

Noninvasive prenatal screening (NIPS) for fetal chromosome abnormalities in a general-risk population: An evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: This workgroup aimed to develop an evidence-based clinical practice guideline for the use of noninvasive prenatal screening (NIPS) for pregnant individuals at general risk for fetal trisomy 21, trisomy 18, or trisomy 13 and to evaluate the utility of NIPS for other chromosomal disorders. METHODS: The NIPS Evidence-Based Guideline Work Group (n = 7) relied on the results from the recent American College of Medical Genetics and Genomics (ACMG) systematic review to form the evidentiary basis of this guideline. Workgroup members used the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework to draft recommendations. The guideline underwent extensive internal and external peer review with a public comment period before approval by the ACMG Board of Directors. RESULTS: Evidence consistently demonstrated improved accuracy of NIPS compared with traditional screening methods for trisomies 21, 18, and 13 in singleton and twin gestations. Identification of rare autosomal trisomies and other microdeletion syndromes with NIPS is an emerging area of interest. CONCLUSION: ACMG strongly recommends NIPS over traditional screening methods for all pregnant patients with singleton and twin gestations for fetal trisomies 21, 18, and 13 and strongly recommends NIPS be offered to patients to screen for fetal sex chromosome aneuploidy.

Obstetrical, perinatal, and genetic outcomes associated with nonreportable prenatal cell-free DNA screening results.

BACKGROUND: The clinical implications of nonreportable cell-free DNA screening results are uncertain, but such results may indicate poor placental implantation in some cases and be associated with adverse obstetrical and perinatal outcomes. OBJECTIVE: This study aimed to assess the outcomes of pregnancies with nonreportable cell-free DNA screening in a cohort of patients with complete genetic and obstetrical outcomes. STUDY DESIGN: This was a prespecified secondary analysis of a multicenter prospective observational study of prenatal cell-free DNA screening for fetal aneuploidy and 22q11.2 deletion syndrome. Participants who underwent cell-free DNA screening from April 2015 through January 2019 were offered participation. Obstetrical outcomes and neonatal genetic testing results were collected from 21 primary-care and referral centers in the United States, Europe, and Australia. The primary outcome was risk for adverse obstetrical and perinatal outcomes (aneuploidy, preterm birth at <28, <34, and <37 weeks' gestation, preeclampsia, small for gestational age or birthweight <10th percentile for gestational week, and a composite outcome that included preterm birth at <37 weeks, preeclampsia, small for gestational age, and stillbirth at >20 weeks) after nonreportable cell-free DNA screening because of low fetal fraction or other causes. Multivariable analyses were performed, adjusting for variables known to be associated with obstetrical and perinatal outcomes, nonreportable results, or fetal fraction. RESULTS: In total, 25,199 pregnant individuals were screened, and 20,194 were enrolled. Genetic confirmation was missing in 1165 (5.8%), 1085 (5.4%) were lost to follow-up, and 93 (0.5%) withdrew; the final study cohort included 17,851 (88.4%) participants who had cell-free DNA, fetal or newborn genetic confirmatory testing, and obstetrical and perinatal outcomes collected. Results were nonreportable in 602 (3.4%) participants. A sample was redrawn and testing attempted again in 427; in 112 (26.2%) participants, results were again nonreportable. Nonreportable results were associated with higher body mass index, chronic hypertension, later gestational age, lower fetal fraction, and Black race. Trisomy 13, 18, or 21 was confirmed in 1.6% with nonreportable tests vs 0.7% with reported results (P=.013). Rates of preterm birth at <28, 34, and 37 weeks, preeclampsia, and the composite outcome were higher among participants with nonreportable results, and further increased among those with a second nonreportable test, whereas the rate of small for gestational age infants was not increased. After adjustment for confounders, the adjusted odds ratios were 2.2 (95% confidence interval, 1.1-4.4) and 2.6 (95% confidence interval, 0.6-10.8) for aneuploidy, and 1.5 (95% confidence interval, 1.2-1.8) and 2.1 (95% confidence interval, 1.4-3.2) for the composite outcome after a first and second nonreportable test, respectively. Of the patients with nonreportable tests, 94.9% had a live birth, as opposed to 98.8% of those with reported test results (adjusted odds ratio for livebirth, 0.20 [95% confidence interval, 0.13-0.30]). CONCLUSION: Patients with nonreportable cell-free DNA results are at increased risk for a number of adverse outcomes, including aneuploidy, preeclampsia, and preterm birth. They should be offered diagnostic genetic testing, and clinicians should be aware of the increased risk of pregnancy complications.

Cell-free DNA screening for trisomy 21 in twin pregnancy: a large multicenter cohort study.

BACKGROUND: Analysis of cell-free DNA from maternal blood provides effective screening for trisomy 21 in singleton pregnancies. Data on cell-free DNA screening in twin gestations are promising although limited. In previous twin studies, cell-free DNA screening was primarily performed in the second trimester and many studies did not report chorionicity. OBJECTIVE: This study aimed to evaluate the screening performance of cell-free DNA for trisomy 21 in twin pregnancies in a large, diverse cohort. A secondary aim was to evaluate screening performance for trisomy 18 and trisomy 13. STUDY DESIGN: This was a retrospective cohort study of twin pregnancies from 17 centers for which cell-free DNA screening was performed from December 2011 to February 2020 by one laboratory using massively parallel sequencing technology. Medical record review was conducted for all newborns and data on the birth outcome, the presence of any congenital abnormalities, phenotypic appearance at birth, and any chromosomal testing that was undertaken in the antenatal or postnatal period were extracted. Cases with a possible fetal chromosomal abnormality with no genetic test results were reviewed by a committee of maternal-fetal medicine geneticists. Cases with a vanishing twin and inadequate follow-up information were excluded. A minimum of 35 confirmed cases of trisomy 21 was required to capture a sensitivity of at least 90% with a prevalence of at least 1.9% with 80% power. Test characteristics were calculated for each outcome. RESULTS: A total of 1764 samples were sent for twin cell-free DNA screening. Of those, 78 cases with a vanishing twin and 239 cases with inadequate follow-up were excluded, leaving a total of 1447 cases for inclusion in the analysis. The median maternal age was 35 years and the median gestational age at cell-free DNA testing was 12.3 weeks. In total, 81% of the twins were dichorionic. The median fetal fraction was 12.4%. Trisomy 21 was detected in 41 of 42 pregnancies, yielding a detection rate of 97.6% (95% confidence interval, 83.8-99.7). There was 1 false negative and no false positive cases. Trisomy 21 was detected in 38 out of 39 dichorionic twin pregnancies, yielding a detection rate of 97.4% (95% confidence interval, 82.6-99.7). Trisomy 18 was detected in 10 of the 10 affected pregnancies. There was 1 false positive case. Trisomy 13 was detected in 4 of the 5 cases, yielding a detection rate of 80% (95% confidence interval, 11.1-99.2). There was one false negative and no false positive cases. The nonreportable rate was low at 3.9 %. CONCLUSION: Cell-free DNA testing is effective in screening for trisomy 21 in twin gestations from the first trimester of pregnancy. Detection of trisomy 21 was high in dichorionic and monochorionic twins, and the nonreportable result rates were low. This study included high numbers of cases of trisomy 18 and 13 when compared with the current literature. Although screening for these conditions in twins seems to be promising, the numbers were too small to make definitive conclusions regarding the screening efficacy for these conditions. It is possible that cell-free DNA testing performance may differ among laboratories and vary with screening methodologies.

Defining the scope of extended NIPS in Western China: evidence from a large cohort of fetuses with normal ultrasound scans.

BACKGROUND: Standard noninvasive prenatal screening(NIPS) is an accurate and reliable method to screen for common chromosome aneuploidies, such as trisomy 21, 18 and 13. Extended NIPS has been used in clinic for not only aneuploidies but also copy number variants(CNVs). Here we aim to define the range of chromosomal abnormalities that should be able to identify by NIPS in order to be an efficient extended screening test for chromosomal abnormalities. METHODS: A prospective study was conducted, involving pregnant women without fetal sonographic structural abnormalities who underwent amniocentesis. Prenatal samples were analyzed using copy number variation sequencing(CNV-seq) to identify fetal chromosomal abnormalities. RESULTS: Of 28,469 pregnancies included 1,022 (3.59%) were identified with clinically significant fetal chromosome abnormalities, including 587 aneuploidies (2.06%) and 435 (1.53%) pathogenic (P) / likely pathogenic (LP) CNVs. P/LP CNVs were found in all chromosomes, but the distribution was not uniform. Among them, P/LP CNVs in chromosomes 16, 22, and X exhibited the highest frequencies. In addition, P/LP CNVs were most common on distal ends of the chromosomes and in low copy repeat regions. Recurrent microdeletion/microduplication syndromes (MMS) accounted for 40.69% of total P/LP CNVs. The size of most P/LP CNVs (77.47%) was < 3 Mb. CONCLUSIONS: In addition to aneuploidies, the scope of extended NIPS should include the currently known P/LP CNVs, especially the regions with recurrent MMS loci, distal ends of the chromosomes, and low copy repeat regions. To be effective detection should include CNVs of < 3 Mb. Meanwhile, sufficient preclinical validation is still needed to ensure the clinical effect of extended NIPS.

Ethical concerns surrounding sex prediction using noninvasive prenatal screening from pediatric endocrinologists' perspective.

Noninvasive prenatal screening (NIPS) with predicted fetal sex chromosomes included in the results has become increasingly available for pregnant individuals. Predicted fetal sex chromosome results from NIPS are interpreted so as to equate sex chromosomes with sex and gender. As pediatric endocrinologists, we worry about how this use of NIPS harmfully reinforces sex and gender binaries and sets potentially inaccurate assumptions about what the identified chromosomes mean. We use a hypothetical case based on our clinical experience in which the NIPS report of fetal sex does not conform to expectations at birth to highlight ethical concerns surrounding this practice. The use of NIPS for fetal sex chromosome prediction has the potential to perpetuate stigma and bring psychological harm to parents and their future children, particularly those who are intersex, transgender, and gender diverse. The medical community should adopt an approach to the use of NIPS for fetal sex chromosome prediction that recognizes the spectrums of sex and gender to avoid reproducing stigma towards sex- and gender-diverse individuals and associated harms.

Noninvasive prenatal screening (NIPS) results for participants of the eXtraordinarY babies study: Screening, counseling, diagnosis, and discordance.

Sex chromosome aneuploidies (SCAs), including 47,XXY, 47,XXX, 47,XYY, and supernumerary variants, occur collectively in approximately one of 500 live births. Clinical phenotypes are highly variable resulting in previous ascertainment rates estimated to be only 10%-25% during a lifetime. Historically, prenatal SCA diagnoses were incidental findings, accounting for ≤10% of cases, with the majority of diagnoses occurring postnatally during evaluations for neurodevelopmental, medical, or infertility concerns. The initiation of noninvasive prenatal screening (NIPS) in 2012 and adoption into standardized obstetric care provides a unique opportunity to significantly increase prenatal ascertainment of SCAs. However, the impact NIPS has had on ascertainment of SCAs is understudied, particularly for those who may defer diagnostic testing until after birth. This study evaluates the timing of diagnostic testing following positive NIPS in 152 infants with SCAs and potential factors influencing this decision. Eighty-seven (57%) elected to defer diagnostic testing after a positive NIPS until birth, and 8% (7/87) of those confirmed after birth were found to have discordant results on postnatal diagnostic testing, most of which would have influenced genetic counseling.

Noninvasive prenatal screening with conventional sequencing depth to screen fetal copy number variants: A retrospective study of 19 144 pregnant women.

AIM: To investigate the detectability of noninvasive prenatal screening (NIPS) with conventional sequencing depth to detect fetal copy number variants. METHODS: We performed a retrospective study in a total of 19 144 pregnant women. Their cell-free plasma DNA were assessed for trisomy 21, trisomy 18, trisomy 13, sex chromosome aneuploidies, and genome-wide copy number variants by NIPS at conventional sequencing depth. RESULTS: Three hundred seventy-four cases (2.0%, 374/19 144) with abnormal results were detected, which including 84 cases (0.4%, 84/19 144) with high risk of trisomy 21, 18, and 13, 90 cases (0.5%, 90/19 144) with high risk of sex chromosome abnormalities (SCA), and 44 cases (0.2%, 44/19 144) with high risk of other chromosome aneuploidies. One hundred fifty-six cases (0.8%, 156/19 144) with high risk of copy number variations (CNVs) were also detected. In following prenatal diagnosis, composite positive predictive value (PPV) of trisomy 21, 18, and 13 was 69.6% (48/69). The PPV of SCAs was 37.3% (19/51). And the PPVs for CNVs was detected as 51.0% (<5 Mb), 71.4% (5 Mb ≤ CNV ≤10 Mb), 56.5% (>10 Mb). Finally, a follow-up about the pregnancy outcomes were conducted for all available cases. CONCLUSIONS: NIPS yielded high PPVs for trisomy 21, 18, and 13 aneuploidies and moderate PPVs for SCAs and CNVs. The screening effectiveness was closely related to the size of CNV fragments. Larger CNVs, especially larger than 5 Mb, could be detected more accurately by NIPS in our analytic technique. Meanwhile, diagnostic confirmation by microarray analysis was highly recommended.

Comparison of noninvasive prenatal screening with combined first-trimester screening as a frontline screening approach for common trisomies in a public hospital in Australia.

BACKGROUND: Combined first-trimester screening (cFTS) for fetal anomalies involves maternal serum screening for biochemical markers and measurement of the nuchal translucency (NT) by ultrasound. Noninvasive prenatal screening (NIPS) analyses cell-free DNA present in a maternal blood sample for presence of fetal chromosomal aneuploidies. AIMS: To compare NIPS with cFTS as frontline screening in a public hospital in Australia. MATERIALS AND METHODS: Women were offered NIPS in addition to the usual cFTS routinely offered to all women at a public hospital in NSW, Australia. The cFTS sample was collected at ten weeks' gestation and the NIPS sample at 12 weeks' gestation at the ultrasound appointment. RESULTS: In a low-risk population of 997 women, frontline NIPS had a screen-positive rate of 0.5% (5/997) vs 4.2% (42/997) with cFTS. cFTS correctly identified one trisomy 21 case and one trisomy 18 case; however, there were two trisomy 18 false negatives. Of five positive NIPS calls, four were correctly identified as trisomy 21 (one) and trisomy 18 (three); there were no NIPS false negatives. Overall, the false-positive rate with NIPS was 0.1% vs 4.0% by cFTS. CONCLUSIONS: The lower screen-positive rate with NIPS for common trisomies was a result of the significantly lower false-positive rate with NIPS. Consequently, NIPS as first-line screening, even if funded by the hospital, may provide cost savings. We believe NIPS should be used from ten weeks' gestation in conjunction with morphology ultrasound for routine first-trimester prenatal management.

Does isolated nuchal translucency from 2.5 to 2.9 mm increase the risk of fetal chromosome disease?

Increased fetal nuchal translucency (NT) is a common ultrasonic manifestation during pregnancy. Many studies have confirmed that NT ≥ 3 mm is a high risk factor for adverse pregnancy outcome. However, when NT is between 2.5 and 2.9 mm, will it increase the risk of fetal chromosome abnormalities and other diseases? What is the most appropriate method for prenatal chromosome evaluation? At present, it has not been widely reported in the literature, and the conclusion is also controversial. This prospective cohort study included fetal samples from women who underwent amniocentesis from 2017 to 2020. The samples of the experimental group were fetuses with NT ≥ 2.5 mm at 11 to 13 + 6 weeks of gestation, with or without ultrasonographic anomaly. The control group contained fetal NT < 2.5 mm without ultrasonographic anomalies. All amniotic fluid samples were tested by copy number variants sequencing. In 262 fetal samples with isolated NT from 2.5 to 2.9 mm, the detection rate of aneuploidy was 3.4% (9/262), and the risk of aneuploidy was significantly higher than that of the control group (1.4%, 32/2331) (relative risk 2.5, 95% CI 1.2-5.2). The detection rates of other pathogenic/likely pathogenic copy number variants in the two groups were 0.8% (2/262) and 1.3% (31/2331), respectively, which was not statistically significant (relative risk 0.6, 95% CI 0.1-2.4). Our results showed that isolated NT from 2.5 to 2.9 mm increased the risk of fetal chromosome aneuploidy. Therefore, noninvasive prenatal screening is recommended as the first choice for prenatal chromosome evaluation in this population.

Systematic evidence-based review: The application of noninvasive prenatal screening using cell-free DNA in general-risk pregnancies.

PURPOSE: Noninvasive prenatal screening (NIPS) using cell-free DNA has been assimilated into prenatal care. Prior studies examined clinical validity and technical performance in high-risk populations. This systematic evidence review evaluates NIPS performance in a general-risk population. METHODS: Medline (PubMed) and Embase were used to identify studies examining detection of Down syndrome (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies, rare autosomal trisomies, copy number variants, and maternal conditions, as well as studies assessing the psychological impact of NIPS and the rate of subsequent diagnostic testing. Random-effects meta-analyses were used to calculate pooled estimates of NIPS performance (P < .05). Heterogeneity was investigated through subgroup analyses. Risk of bias was assessed. RESULTS: A total of 87 studies met inclusion criteria. Diagnostic odds ratios were significant (P < .0001) for T21, T18, and T13 for singleton and twin pregnancies. NIPS was accurate (≥99.78%) in detecting sex chromosome aneuploidies. Performance for rare autosomal trisomies and copy number variants was variable. Use of NIPS reduced diagnostic tests by 31% to 79%. Conclusions regarding psychosocial outcomes could not be drawn owing to lack of data. Identification of maternal conditions was rare. CONCLUSION: NIPS is a highly accurate screening method for T21, T18, and T13 in both singleton and twin pregnancies.

Residual risk for clinically significant copy number variants in low-risk pregnancies, following exclusion of noninvasive prenatal screening-detectable findings.

BACKGROUND: Chromosomal microarray analysis detects a clinically significant amount of copy number variants in approximately 1% of low-risk pregnancies. As the constantly growing use of noninvasive prenatal screening has facilitated the detection of chromosomal aberrations, defining the rate of abnormal chromosomal microarray analysis findings following normal noninvasive prenatal screening is of importance for making informed decisions regarding prenatal testing and screening options. OBJECTIVE: To calculate the residual risk for clinically significant copy number variants following theoretically normal noninvasive prenatal screening. STUDY DESIGN: The chromosomal microarray results of all pregnancies undergoing amniocentesis between the years 2013 and 2021 in a large hospital-based laboratory were collected. Pregnancies with sonographic anomalies, abnormal maternal serum screening, or multiple fetuses were excluded. Clinically significant (pathogenic and likely pathogenic) copy number variants were divided into the following: 3-noninvasive prenatal screening-detectable (trisomies 13, 18, and 21), 5- noninvasive prenatal screening-detectable (including sex chromosome aberrations), 5-noninvasive prenatal screening and common microdeletion-detectable (including 1p36.3-1p36.2, 4p16.3-4p16.2, 5p15.3-5p15.1, 15q11.2-15q13.1, and 22q11.2 deletions), and genome-wide noninvasive prenatal screening-detectable (including variants >7 Mb). The theoretical residual risk for clinically significant copy number variants was calculated following the exclusion of noninvasive prenatal screening-detectable findings. RESULTS: Of the 7235 pregnancies, clinically significant copy number variants were demonstrated in 87 cases (1.2%). The residual risk following theoretically normal noninvasive prenatal screening was 1.07% (1/94) for 3-noninvasive prenatal screening, 0.78% (1/129) for 5- noninvasive prenatal screening, 0.74% (1/136) for 5- noninvasive prenatal screening including common microdeletions, and 0.68% (1/147) for genome-wide noninvasive prenatal screening. In the subgroup of 4048 pregnancies with advanced maternal age, the residual risk for clinically significant copy number variants following theoretically normal noninvasive prenatal screening ranged from 1.36% (1/73) for 3- noninvasive prenatal screening to 0.82% (1/122) for genome-wide noninvasive prenatal screening. In 3187 pregnancies of women <35 years, this residual risk ranged from 0.69% (1/145) for 3- noninvasive prenatal screening to 0.5% (1/199) for genome-wide noninvasive prenatal screening. CONCLUSION: The residual risk of clinically significant copy number variants in pregnancies without structural sonographic anomalies is appreciable and depends on the noninvasive prenatal screening extent and maternal age. This knowledge is important for the patients, obstetricians, and genetic counselors to facilitate informed decisions regarding prenatal testing and screening options.

Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology.

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35-100.00%); 100.00% sensitivity for T18 (71.51-100.00%); and 100.00% sensitivity for T13 analyses (66.37-100.00%). The specificities were >99% for each trisomy (99.7% (99.01-99.97%) for T21; 99.5% (98.62-99.85%) for T18; 99.7% (99.03-99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.

High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.

PURPOSE: The percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body mass index (and >5% overall) receive a test failure due to low FF (<4%). METHODS: A scalable fetal fraction amplification (FFA) technology was analytically validated on 1264 samples undergoing whole-genome sequencing (WGS)-based NIPS. All samples were tested with and without FFA. RESULTS: Zero samples had FF < 4% when screened with FFA, whereas 1 in 25 of these same patients had FF < 4% without FFA. The average increase in FF was 3.9-fold for samples with low FF (2.3-fold overall) and 99.8% had higher FF with FFA. For all abnormalities screened on NIPS, z-scores increased 2.2-fold on average in positive samples and remained unchanged in negative samples, powering an increase in NIPS sensitivity and specificity. CONCLUSION: FFA transforms low-FF samples into high-FF samples. By combining FFA with WGS-based NIPS, a single round of NIPS can provide nearly all women with confident results about the broad range of potential fetal chromosomal abnormalities across the genome.

The yield of chromosomal microarray in pregnancies with congenital cardiac defects and normal noninvasive prenatal screening.

BACKGROUND: Evidence comparing the yield of chromosomal microarray analysis to noninvasive prenatal screening in pregnancies with congenital heart anomalies is currently limited. OBJECTIVE: This study aimed to examine the residual risk of clinically significant chromosomal microarray analysis results in fetuses with congenital heart defects by its various subtypes following a normal noninvasive prenatal screening. STUDY DESIGN: Using a population-based, countrywide computerized database, we retrieved the reports of all pregnancies undergoing chromosomal microarray analysis because of congenital heart defects through the years 2013-2019. We examined the risk of clinically significant (pathogenic and likely pathogenic) chromosomal microarray analysis results and compared it with the results of a local cohort of low-risk pregnancies. Of 5541 fetuses, 78 (1.4%) showed abnormal results. The residual risk of abnormal chromosomal microarray analysis results was calculated using several options-trisomies 21, 18, and 13; sex chromosome aneuploidies; 22q11.2 deletion, and deletions and duplications of at least 10 MB in size (genome-wide noninvasive prenatal screening)-following the exclusion of theoretically detectable noninvasive prenatal screening anomalies. RESULTS: Of the 1728 fetuses with congenital heart defects, 93 (5.4%) showed clinically significant chromosomal microarray analysis results (relative risk, 2.7; 95% confidence interval, 2.3-3.1). The result of pregnancies with fetuses with congenital heart defects was compared with the results of the control population. Unique variants were found in 15 pregnancies (16.1%). The detection rate of noninvasive prenatal screening in isolated congenital heart defects varied from 1.0% (aimed at 3 common trisomies) to 2.2% (aimed at 5 common aneuploidies and 22q11.2 deletion) using noninvasive prenatal screening. In nonisolated congenital heart defects, the noninvasive prenatal screening detection rates ranged from 7.8% (aimed at common autosomal trisomies) to 9.2% using genome-wide noninvasive prenatal screening. The residual risk of clinically significant chromosomal microarray analysis results following normal noninvasive prenatal screening ranged from 2.0% to 2.8% in isolated congenital heart defects and 4.5% to 5.9% in nonisolated cases and was significantly higher than those of the control cohort in all noninvasive prenatal screening options. In addition, the residual risk following noninvasive prenatal screening aimed at chromosomes 13, 18, 21, X, and Y was significantly higher than those of the control cohort for most specific congenital heart defect subtypes, except for ventricular septal defects and aberrant right subclavian artery. CONCLUSION: The residual risk of clinically significant chromosomal microarray analysis results in pregnancies with fetuses with congenital heart defects following normal noninvasive prenatal screening was higher than those in pregnancies with normal ultrasound in most isolated and nonisolated congenital heart defect subtypes. This information should be taken into account by obstetricians and genetic counselors when considering the option of diagnostic testing.

Non-Invasive Prenatal Screening: Navigating the Relevant Legal Norms.

The implementation of non-invasive prenatal screening (NIPS) in Canada will be affected by legal norms. The law can shape physician behaviour, help to crystallize standards of care, influence utilization patterns, and reflect and reinforce patient expectations. In Canada, failure to inform a patient about NIPS, or misinterpretation of NIPS results, could result in a successful "wrongful birth" claim if the patient subsequently gives birth to a child with a condition that had been detectable. Given that research shows that physicians' decisions are influenced by concerns about liability, malpractice law seems likely to encourage increased recommendation and use of NIPS. Physicians' fiduciary and negligence-based disclosure standards require they consider both objective factors as well as a specific patient's subjective and reasonable beliefs, fears, desires, and expectations. Thus, physicians likely must address dominant public discourses and controversies relevant to NIPS. Given the existence of spin, hype, and misinformation about NIPS, there is an increasing need for a robust consent process and, when appropriate, genetic counselling. In sum, the law will define and bound the acceptable behaviour of physicians recommending or administering NIPS, and nudge the technology's implementation forward. Physicians and policymakers should be aware of the potential impact of these legal norms on both utilization and public expectations.

Alternative option labeling impacts decision-making in noninvasive prenatal screening.

Prenatal genetic screening should be an informed, autonomous patient choice. Extrinsic factors which influence patient decision-making threaten the ethical basis of prenatal genetic screening. Prior research in the area of medical decision-making has identified that labeling may have unanticipated effects on patient perceptions and decision-making processes. This Internet-administered study explored the impact of option labeling on the noninvasive prenatal screening (NIPS) selections of US adults. A total of 1,062 participants were recruited through Amazon Mechanical Turk (MTurk) and randomly assigned to one of three possible label sets reflecting provider-derived and industry-derived option labels used in prenatal screening. Multinomial logistic regression analysis showed option labeling had a statistically significant impact on the NIPS selections of study participants (p = .0288). Outcomes of the Satisfaction with Decision Scale (SWD) indicated option labels did not play a role in participant satisfaction with screening selection. The results of this study indicate a need for further evaluation of the impact NIPS option labeling has on patient screening decisions in real-world clinical interactions. Clinical providers and testing laboratories offering NIPS should give careful consideration to the option labels used with prenatal screening so as to minimize influence on patient screening selection and decision-making processes.

Technology-Driven Noninvasive Prenatal Screening Results Disclosure and Management.

Background: Noninvasive prenatal screening (NIPS) utilization has grown dramatically and is increasingly offered to the general population by nongenetic specialists. Web-based technologies and telegenetic services offer potential solutions for efficient results delivery and genetic counseling. Introduction: All major guidelines recommend patients with both negative and positive results be counseled. The main objective of this study was to quantify patient utilization, motivation for posttest counseling, and satisfaction of a technology platform designed for large-scale dissemination of NIPS results. Methods: The technology platform provided general education videos to patients, results delivery through a secure portal, and access to telegenetic counseling through phone. Automatic results delivery to patients was sent only to patients with screen-negative results. For patients with screen-positive results, either the ordering provider or a board-certified genetic counselor contacted the patient directly through phone to communicate the test results and provide counseling. Results: Over a 39-month period, 67,122 NIPS results were issued through the platform, and 4,673 patients elected genetic counseling consultations; 95.2% (n = 4,450) of consultations were for patients receiving negative results. More than 70% (n = 3,370) of consultations were on-demand rather than scheduled. A positive screen, advanced maternal age, family history, previous history of a pregnancy with a chromosomal abnormality, and other high-risk pregnancy were associated with the greatest odds of electing genetic counseling. By combining web education, automated notifications, and telegenetic counseling, we implemented a service that facilitates results disclosure for ordering providers. Discussion: This automated results delivery platform illustrates the use of technology in managing large-scale disclosure of NIPS results. Further studies should address effectiveness and satisfaction among patients and providers in greater detail. Conclusions: These data demonstrate the capability to deliver NIPS results, education, and counseling-congruent with professional society management guidelines-to a large population.

Noninvasive Prenatal DNA Testing: The Vanguard of Genomic Medicine.

Noninvasive prenatal DNA testing is the vanguard of genomic medicine. In only four years, this screening test has revolutionized prenatal care globally and opened up new prospects for personalized medicine for the fetus. There are widespread implications for increasing the scope of human genetic variation that can be detected before birth, and for discovering more about maternofetal and placental biology. These include an urgent need to develop pretest education for all pregnant women and consistent post-test management recommendations for those with discordant test results. The reduction in invasive testing has had downstream effects on specialist training and caused many countries to re-examine their national approaches to prenatal screening. Finally, the accumulating datasets of genomic information on pregnant women and their fetuses raise ethical issues regarding consent for future data mining and intellectual property.