Public Opinions and Attitudes toward Noninvasive Prenatal Testing on Reddit: Content and Sentiment Analysis.

INTRODUCTION: Noninvasive prenatal testing (NIPT) can be used to detect fetal chromosomal abnormalities early in pregnancy. As eligibility criteria broaden and screening targets expand, gauging public acceptability of NIPT becomes increasingly important. Leveraging social media as a rich source of public discourse, the purpose of this study was to understand public opinions and attitudes toward NIPT on the social media platform Reddit. METHODS: We applied content and natural language processing techniques (i.e., sentiment analysis) to textual data collected from 4 Reddit communities focusing on the NIPT content posted from September 2012 to September 2022 (367 posts and 7,822 comments in total). RESULTS: Content analysis findings indicated that social media users consider NIPT to be worthwhile. Reasons NIPT was perceived to be not worthwhile related to unwanted anxiety, and the fact that NIPT results would not change anything about their approach to pregnancy were also expressed. The sentiment analysis identified more positive than negative emotions; the mean sentiment scores ranged from 0.48 to 1.22, depending on the specific Lexicon used. Specific emotions (i.e., trust, fear) were also identified. CONCLUSION: Our novel approach to understanding public perception and attitudes toward NIPT yielded results that are consistent with conventional patient-oriented research methods. These findings may not only contribute to ongoing improvements in prenatal patient care, research, and policy but also indicate that sentiment analysis applied to social media data can serve as a suitable means to assess public acceptability of NIPT, particularly as public dialogue on this topic increases over time.

Exploring factors impacting haplotype-based noninvasive prenatal diagnosis for single-gene recessive disorders.

Haplotype-based noninvasive prenatal diagnosis (NIPD) is applicable for various recessive single-gene disorders in proband families. However, a comprehensive exploration of critical factors influencing the assay performance, such as fetal fraction, informative single nucleotide polymorphism (SNP) count, and recombination events, has yet to be performed. It is critical to identify key factors affecting NIPD performance, including its accuracy and success rate, and their impact on clinical diagnostics to guide clinical practice. We conducted a prospective study, recruiting 219 proband families with singleton pregnancies at risk for eight recessive single-gene disorders (Duchenne muscular dystrophy, spinal muscular atrophy, phenylketonuria, methylmalonic acidemia, hemophilia A, hemophilia B, non-syndromic hearing loss, and congenital adrenal hyperplasia) at 7-14 weeks of gestation. Haplotype-based NIPD was performed by evaluating the relative haplotype dosage (RHDO) in maternal circulation, and the results were validated via invasive prenatal diagnosis or newborn follow-ups. Among the 219 families, the median gestational age at first blood draw was 8+5 weeks. Initial testing succeeded for 190 families and failed for 29 due to low fetal fraction (16), insufficient informative SNPs (9), and homologous recombination near pathogenic variation (4). Among low fetal fraction families, successful testing was achieved for 11 cases after a redraw, while 5 remained inconclusive. Test failures linked to insufficient informative SNPs correlated with linkage disequilibrium near the genes, with F8 and MMUT exhibiting the highest associated failure rates (14.3% and 25%, respectively). Homologous recombination was relatively frequent around the DMD and SMN1 genes (8.8% and 4.8%, respectively) but led to detection failure in only 44.4% (4/9) of such cases. All NIPD results from the 201 successful families were consistent with invasive diagnostic findings or newborn follow-up. Fetal fraction, informative SNPs count, and homologous recombination are pivotal to NIPD performance. Redrawing blood effectively improves the success rate for low fetal fraction samples. However, informative SNPs count and homologous recombination rates vary significantly across genes, necessitating careful consideration in clinical practice. We have designed an in silico method based on linkage disequilibrium data to predict the number of informative SNPs. This can identify genomic regions where there might be an insufficient number of SNPs, thereby guiding panel design. With these factors properly accounted for, NIPD is highly accurate and reliable.

Haplotype-based noninvasive prenatal diagnosis of methylmalonic acidemia and the discovery of a recurrent pathogenic haplotype associated with c.609G>A.

BACKGROUND: Early diagnosis and intervention are crucial for the prognosis of methylmalonic acidemia (MMA). However, research focused on early prenatal diagnosis of MMA is limited. METHODS: A 161.89kb capture panel was designed for selectively enriching highly heterozygous SNPs. Fetal genotypes were inferred using relative haplotype dosage (RHDO) and Bayes factor, followed by invasive prenatal diagnosis (IPD) for validation. A core pathogenic haplotype associated with c.609G>A was identified based on the frequency differences between pathogenic and normal haplotypes. RESULTS: We recruited 41 pregnancies at risk of MMA with a median gestational age of 8+2  weeks. The assay success rate of NIPD-MMA for maternal variants was 92.7% (38/41), and after incorporating the paternal result, the overall assay success rate reached 100% (41/41). All NIPD results were concordant with IPD. Notably, a core haplotype (hap_2), comprising 28 SNPs, demonstrates significant enrichment within pathogenic haplotypes bearing the c.609G>A variation. On average, c.609G>A carriers had 22.38 heterozygous loci within these 28 SNPs. CONCLUSION: NIPD-MMA presents a viable choice for early, accurate, and safe prenatal diagnosis. Furthermore, the discovery of the recurrent core pathogenic haplotype provides a novel approach for haplotype phasing and has the potential for realizing proband-independent NIPD in the future.

Liquid Biopsy in Adverse Neurodevelopment of Children: Problems and Prospects.

Neurodevelopmental disorders in children have an important impact on the quality of life in the whole life cycle. Severe neurodevelopmental disorders will become a serious social and family burden and an important social and economic problem. The early and middle childhood is the critical period of children's neurodevelopment. Early diagnosis of neurological disorders plays an important role in guiding children's neurological development. Existing monitoring tools lack prenatal and even early assessment of children's neurodevelopment, so reliable biomarkers are conducive to personalized care at an earlier stage. In this review, we will discuss different methods of neurodevelopmental monitoring at different times and the role and evaluation of liquid biopsy in neurodevelopmental monitoring.

Evaluation of noninvasive prenatal screening for copy number variations among screening laboratories.

OBJECTIVE: To evaluate the current situation of expanded noninvasive prenatal screening (NIPS) for copy number variations (CNVs) in laboratories in China, the National Center of Clinical Laboratories conducted an externalqualityassessment (EQA) program. METHODS: The EQA panel consisted of 12 artificial samples associated with different syndromes, which were mixed with maternal plasma collected from pregnant women and enzyme-digested cell-free DNA (cfDNA) from cell lines with different fetal fractions (FFs) ranging from 5% to 15%. The panel was validated by next-generation sequencing and distributed to laboratories, along with questionnaires and case scenarios. RESULTS: Sixty-nine laboratories participated in the EQA program, and 91.30% (63/69) of laboratories correctly identified all samples. A total of 7.25% (5/69) of the laboratories reported false-negative results, and 2.90% (2/69) of the laboratories reported unexpected CNVs. The correct rates of the 22q11.2 deletion syndrome, Cri-du-chat syndrome, 1p36 deletion syndrome and Angelman/Prader-Willi syndrome samples were 97.46%, 98.55%, 100%, and 100%, respectively. With the increase in the FF, deletion size, and read depth, the detection rate increased. For results reports, only five laboratories reported FF values, one laboratory reported the CNV classification type, and none reported sensitivity, specificity, positive predictive values, and negative predictive values. CONCLUSION: The detection capabilities of NIPS for CNVs still need to be improved and standardized, and FF, deletion size, and read depth are factors that affect the detection rate.

Early noninvasive prenatal paternity testing by targeted fetal DNA analysis.

Today the challenge in paternity testing is to provide an accurate noninvasive assay that can be performed early during pregnancy. This requires the use of novel analytical methods capable of detecting the low fraction of circulating fetal DNA in maternal blood. We previously showed that forensic compound markers such as deletion/insertion polymorphisms-short tandem repeats (DIP-STR) can efficiently resolve complex mixed biological evidence including the target analysis of paternally inherited fetal alleles. In this study, we describe for the first time the validation of this type of markers in the first trimester of pregnancies, in addition to defining the statistical framework to evaluate paternity. To do so, we studied 47 DIP-STRs in 87 cases, with blood samples collected throughout gestation starting from the seven weeks of amenorrhea. Fetal DNA detection in the first trimester shows a false negative rate as low as 6%. The combined paternity index (CPI) results indicate that seven markers with fully informative genotypes are sufficient to determine the paternity. This study demonstrates that DIP-STR markers can be used from early pregnancy and that a small set of markers (about 40) is sufficient to address the question of paternity. The novel method offers substantial improvements over similar approaches in terms of reduced number of markers, lower costs and increased accuracy.

The predictive value of noninvasive prenatal screening for copy number variations: a cohort study and a systematic meta-analysis.

OBJECTIVE: To assess the diagnostic accuracy of noninvasive prenatal screening (NIPS) in screening for copy number variations (CNVs). METHODS: We conducted a systematic review and meta-analysis by combining our study results with those reported in other articles. We retrospectively collected the data of pregnant women with NIPS testing in the Hangzhou Women's Hospital from December 2019 to February 2022. Simultaneously, a systematic search of PubMed, EMBASE, and Web of Science was carried out to identify all relevant peer-reviewed publications. Statistical analysis was performed based on the random-effects model to determine a pooled estimate of the positive predictive value (PPV). RESULTS: A total of 29 studies involving 2,667 women were included for analysis. The pooled PPV of NIPS in the detection of CNVs was 32.86% (95% confidence interval [24.61-41.64]). Statistical heterogeneity was high, while no significant publication bias was found in this meta-analysis. There were insufficient data to accurately determine sensitivity and specificity, as most studies only performed confirmatory tests on high-risk women. CONCLUSIONS: The PPV of NIPS in screening for CNVs was approximately 33%. Cautions should be kept in mind for the pretest guidance and subsequent after-test counseling when offering such genome-wide NIPS tests.

Non-invasive prenatal diagnosis of single gene disorders with enhanced relative haplotype dosage analysis for diagnostic implementation.

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

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.

Discovery of Cell-Free Fetal DNA in Maternal Blood and Development of Noninvasive Prenatal Testing: 2022 Lasker-DeBakey Clinical Medical Research Award.

In this Viewpoint, 2022 Lasker-DeBakey Clinical Medical Research Award winner Y. M. Dennis Lo discusses his discovery and application of cell-free fetal DNA for noninvasive prenatal testing.

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.

Clinical utility of expanded NIPT for chromosomal abnormalities and etiology analysis of cytogenetic discrepancies cases.

PURPOSE: This study is to assess the performance of expanded noninvasive prenatal testing (NIPT) in detecting chromosome aneuploidies and chromosome copy number variants (CNVs), and elucidate the discordant cases between NIPT and fetal karyotype. METHODS: A total of 2139 single pregnancies have been recruited and sequenced with expanded NIPT. Karyotype analysis and CNV sequencing (CNV-seq) of amniotic fluid were performed in 22 of 23 high-risk, three low-risk NIPT pregnant women with abnormal ultrasound findings in the follow-up, and three non-reportable NIPT pregnant women. The genetic investigation of discordant results between NIPT and amniocytes in three cases was proceeded. Placental samples, fetal samples from the limb, hip, umbilical cord, and maternal peripheral blood leukocytes were collected for CNV-Seq. RESULTS: Expanded NIPT revealed a total of 23 positive pregnancies and yielded the overall positive predictive value (PPV) 65.2%. For T21, T18, and XXY, all the PPV was 100% respectively. For CNVs > 10 Mb and 5-10 Mb, the PPV was 42.8% and 16.7%, respectively. The genetic investigation of placental and fetal samples indicated different levels of placental and fetal mosaicism contributing to two of three verified discordant results. CONCLUSIONS: The results showed that screening for CNVs with expanded NIPT is promising although the accuracy rate remains insufficient. The different occurring time of mitotic non-disjunction of different chromosome in early development of embryo results in varying levels of chromosomal mosaicism in different placental and fetal tissues. The result highlights the significance of comprehensive cytogenetic validation of placental and fetal specimens with an inconsistent NIPT results.

A microfluidic platform for high-purity cell free DNA extraction from plasma for non-invasive prenatal testing.

OBJECTIVES: Increase the yield and purity of cell-free DNA (cfDNA) extracted from plasma for non-invasive prenatal testing (NIPT) as inefficiencies in this extraction and purification can dramatically affect the sensitivity and specificity of the test. METHODS: This work integrates cfDNA extraction from plasma with a microfluidic chip platform by combining magnetic bead-based extraction and electroosmotic flow on the microfluidic chip. Various wash buffers and voltage conditions were simulated using COMSOL Multiphysics Modeling and tested experimentally. RESULTS: When performing the first wash step of this assay on the microfluidic chip with 300 V applied across the channel there was a six-fold increase in the A260 /A230 ratio showing a significant improvement (p value 0.0005) in the purity of the extracted sample all while maintaining a yield of 68.19%. These values are critical as a high yield results in more sample to analyze and an increase in A260 /A230 ratio corresponds to a decrease in salt contaminants such as guanidinium thiocyanate which can interfere with downstream processes during DNA library preparation and potentially hinder the NIPT screening results. CONCLUSIONS: This technique has the potential to improve NIPT outcomes and other clinically relevant workflows that use cfDNA as an analyte such as cancer detection.

Knowledge, attitudes, and practices of healthcare professionals working in prenatal diagnosis toward expanded non-invasive prenatal testing in China.

OBJECTIVES: To investigate the knowledge, attitudes, and practices of healthcare professionals (HCPs) working in prenatal diagnosis toward expanded non-invasive prenatal testing (NIPT) in China. METHODS: We conducted a national online survey among HCPs working in prenatal diagnosis, including specialists in prenatal diagnosis and foetal medicine, obstetricians and gynaecologists, nurses in obstetrics and gynaecology, obstetric ultrasound doctors, and technicians in prenatal diagnosis laboratories. A total of 1882 questionnaires were collected, among which 1822 questionnaires met the research criteria and were included in the analysis. RESULTS: More than 99% of all participants opted for NIPT for trisomies 21, 18, and 13. The rates of support for expanded NIPT for sex chromosome aneuploidies, rare autosomal trisomies, microdeletions and microduplications, and single-gene disorders were 93.9%, 88.6%, 89.4%, and 86.8%, respectively. Specialists in prenatal diagnosis and foetal medicine had greater knowledge but were less likely to support expanded NIPT compared to other participants. Knowledge increased with educational level, whereas support for expanded NIPT decreased with educational level. CONCLUSIONS: More than 80% of HCPs working in prenatal diagnosis in China expressed support for expanding NIPT to conditions other than common trisomies. The degree of knowledge was negatively associated with the rate of support.

Two cases of microvillus inclusion disease caused by MYO5B deficiency with prenatal abnormalities.

BACKGROUNDS: Microvillus inclusion disease (MVID) characterizes as intractable life-threatening watery diarrhea malnutrition after birth. MATERIALS & METHODS: Here we describe two patients with prenatal ultrasound findings of bowel dilation or increased amniotic fluid volume presented intractable diarrhea after birth. Exome sequencing and Intestinal biopsy were performed for the patients and their parents to reveal the underlying causes. The mutations were verified by Sanger sequencing and quantitative polymerase chain reaction. RESULTS: Exome sequencing revealed that both of the patients carrying MYO5B compound heterozygote mutations that were inherited from their parents. CONCLUSION: Here we describe two cases with MVID caused by MYO5B deficiency, which was the most common caused with prenatal ultrasound findings of bowel dilation and increased amniotic fluid volume. Due to the lack of effective curative therapies, early diagnosis even in prenatal of MVID can provide parents with better genetic counseling on the fetal prognosis.

What's out there for parents? A systematic review of online information about prenatal microarray and exome sequencing.

OBJECTIVE: To identify what online patient information (presented in English) is available to parents about prenatal microarray (CMA) and exome sequencing (ES), and evaluate its content, quality, and readability. METHOD: Systematic searches (Google and Bing) were conducted, and websites were categorised according to their purpose. Websites categorised as patient information were included if they were: in English, directed at patients, or were a text, video, or online version of an information leaflet. Author-developed content checklists, the DISCERN Genetics tool, and readability tests (the Flesch Reading Ease Score, the Gunning Fog Index, and the Simple Measure of Gobbledygook Index) were then used to assess those sources of patient information. RESULTS: Of the 665 websites screened, 18 met the criteria. A further 8 sources were found through a targeted search of professional organisations, resulting in 26 sources available for further evaluation. In general, this was found to be low in quality, omitted details recommended by national or international guidance, and was written at a level too advanced for average readers. CONCLUSION: Improvements should be made to the content, quality, and readability of online information so that it both reinforces and complements the discussions between parents and clinicians about testing options during pregnancy.

A systematic review of maternal TORCH serology as a screen for suspected fetal infection.

BACKGROUND: The acronym 'TORCH' refers to well-recognised causes of perinatal infections: toxoplasmosis, rubella, cytomegalovirus (CMV) and herpes simplex virus (HSV). A TORCH serology panel is often used to test for maternal primary infection following detection of ultrasound abnormalities in pregnancy. AIM: This review aims to estimate the diagnostic yield of maternal TORCH serology in pregnancy following fetal ultrasound abnormalities. MATERIALS AND METHODS: Primary studies published since 2000 that assessed maternal TORCH serology for suspected fetal infection and included information on indications for testing, definition of positive TORCH serology results, and perinatal outcomes were included. RESULTS: Eight studies with a total of 2538 pregnancies were included. The main indications for testing were polyhydramnios, fetal growth restriction and hyperechogenic bowel. There were 26 confirmed cases of congenital CMV, of which 15 had multiple ultrasound abnormalities. There were no cases of congenital toxoplasmosis, rubella or HSV confirmed in any of the eight studies. CONCLUSIONS: The clinical utility of TORCH serology for non-specific ultrasound abnormalities such as isolated fetal growth restriction or isolated polyhydramnios is low. It is time to retire the TORCH acronym and the reflex ordering of 'TORCH' panels, as their continued use obscures, rather than illuminates, appropriate investigation for fetal ultrasound abnormalities.

Clinical experience with non-invasive prenatal screening for single-gene disorders.

OBJECTIVE: To assess the performance of a non-invasive prenatal screening test (NIPT) for a panel of dominant single-gene disorders (SGD) with a combined population incidence of 1 in 600. METHODS: Cell-free fetal DNA isolated from maternal plasma samples accessioned from 14 April 2017 to 27 November 2019 was analyzed by next-generation sequencing, targeting 30 genes, to look for pathogenic or likely pathogenic variants implicated in 25 dominant conditions. The conditions included Noonan spectrum disorders, skeletal disorders, craniosynostosis syndromes, Cornelia de Lange syndrome, Alagille syndrome, tuberous sclerosis, epileptic encephalopathy, SYNGAP1-related intellectual disability, CHARGE syndrome, Sotos syndrome and Rett syndrome. NIPT-SGD was made available as a clinical service to women with a singleton pregnancy at ≥ 9 weeks' gestation, with testing on maternal and paternal genomic DNA to assist in interpretation. A minimum of 4.5% fetal fraction was required for test interpretation. Variants identified in the mother were deemed inconclusive with respect to fetal carrier status. Confirmatory prenatal or postnatal diagnostic testing was recommended for all screen-positive patients and follow-up information was requested. The screen-positive rates with respect to the clinical indication for testing were evaluated. RESULTS: A NIPT-SGD result was available for 2208 women, of which 125 (5.7%) were positive. Elevated test-positive rates were observed for referrals with a family history of a disorder on the panel (20/132 (15.2%)) or a primary indication of fetal long-bone abnormality (60/178 (33.7%)), fetal craniofacial abnormality (6/21 (28.6%)), fetal lymphatic abnormality (20/150 (13.3%)) or major fetal cardiac defect (4/31 (12.9%)). For paternal age ≥ 40 years as a sole risk factor, the test-positive rate was 2/912 (0.2%). Of the 125 positive cases, follow-up information was available for 67 (53.6%), with none classified as false-positive. No false-negative cases were identified. CONCLUSIONS: NIPT can assist in the early detection of a set of SGD, particularly when either abnormal ultrasound findings or a family history is present. Additional clinical studies are needed to evaluate the optimal design of the gene panel, define target populations and assess patient acceptability. NIPT-SGD offers a safe and early prenatal screening option. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

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.