Search Results (22)

Background: Non-invasive prenatal testing (NIPT) based on analysis of Cell-Free Fetal DNA has transformed screening for common aneuploidies and is increasingly extended to genome-wide detection of copy number variants (CNVs). However, CNV detection remains constrained by analytical limitations and biological signal complexity. Methods: This review evaluates the analytical validity, biological constraints, and clinical interpretation challenges of CNV detection by NIPT, framing it as a probabilistic genomic inference rather than a direct measure of fetal copy number. Results: Performance depends on sequencing depth, bin resolution, fetal fraction, guanine–cytosine correction, and reference modeling, leading to variable detection thresholds. The predominantly placental origin of cfDNA introduces discordance through Confined Placental Mosaicism, post-zygotic events, and clonal variation. Maternal CNVs, mosaicism, vanishing twin, and occult malignancy further complicate interpretation and may cause false positives. Clinical validity is heterogeneous, with positive predictive value dependent on CNV size, genomic context, and prevalence. Reporting practices remain inconsistent. Conclusions: CNV detection by NIPT is fundamentally limited by interpretation of a composite maternal–placental signal. Progress requires improved tissue-of-origin discrimination, multi-omic integration, and standardized reporting to ensure responsible clinical implementation. Full article

Objectives: The overall objective of this study is to examine prenatal patients ascertained without an abnormal ultrasound (US) or an abnormal cell-free DNA (cfDNA) finding to provide a unique understanding of pathogenic copy number variants, identity by descent (IBD) and variants of uncertain significance (VUSs) in a normal population. Methods: This study retrospectively provides an analysis of over 28,362 prenatal specimens ascertained without an abnormal US or abnormal cfDNA finding utilizing an SNP microarray. These specimens include at least 10 different ascertainment groups, including advanced maternal age (AMA), anxiety, abnormal maternal serum screen (MSS) with/without AMA, and a previous or familial child/pregnancy with a chromosome abnormality or a genetic disorder. Results: This study provides a basic understanding of pathogenic copy number variants (CNVs), homozygosity and VUSs in an essentially normal population. This low-risk population has a frequency of pathogenic CNVs of ~1.26%; however, ~52% were associated with neurodevelopmental microdeletions/microduplications and ~13% were associated with incidental findings. Overall, ~1.32% of these patients showed an increase in homozygosity, the majority due to consanguinity. Lastly, VUSs were seen in 1.41% of this group, of which ~90% were familial. Conclusions: Overall, these findings provide a better estimate of the baseline frequencies and types of pathogenic CNVs and homozygosity in a low-risk population. It provides insight into the distribution of stretches of homozygosity associated with identity by descent in this population and gives a better understanding of the extent of variants of uncertain significance in phenotypically unaffected individuals. Full article

Background: Non-invasive prenatal testing (NIPT) based on cell-free fetal DNA (cfDNA) in maternal blood has revolutionized prenatal screening for trisomies 21, 18, and 13. This approach, based on next-generation sequencing (NGS), usually allows the detection of other chromosomal abnormalities; however, their clinical value in routine practice requires further evidence. Objectives: This study aimed to assess the experience and clinical utility of genome-wide NIPT in pregnant women at intermediate risk in the autonomous communities of Aragón and Valencia, Spain. Methods: For this purpose, a retrospective cohort study was conducted between 2020 and 2024 across two public hospitals. Pregnant women at intermediate risk for trisomies 21, 18, or 13, were included, as well as those meeting specific clinical criteria. Participants were offered either basic or expanded NIPT, and positive results were confirmed by invasive prenatal testing or placental analysis. Results: Among 9,059 expanded NIPT tests, 132 (1.45%) indicated a high-risk result for less common chromosomal anomalies, comprising 60 rare autosomal aneuploidies (RAAs), 39 copy number variants (CNVs), 23 sex chromosome aneuploidies (SCAs), and 10 multiple abnormalities. The positive predictive value (PPV) was 5.5% for RAAs in the fetus, 12.8% for CNVs (31% for deletions), and 58% for SCAs. Conclusions: Several confirmed anomalies were clinically significant and would not have been detected through conventional screening. Opportunistic use of expanded NIPT enables the detection of additional clinically relevant abnormalities, potentially improving obstetric management without substantially increasing invasive testing. Full article

Advancements in genomic technologies have transformed prenatal genetic testing, offering more accurate, comprehensive, and noninvasive approaches to reproductive care. This review provides an in-depth overview of current methodologies and emerging innovations, including expanded carrier screening (ECS), cell-free DNA (cfDNA) testing, chromosomal microarray analysis (CMA), and sequencing-based diagnostics. We highlight how next-generation sequencing (NGS) technologies have revolutionized carrier screening and fetal genome analysis, enabling detection of a broad spectrum of genetic conditions. The clinical implementation of cfDNA has expanded from common aneuploidies to include copy number variants (CNVs), and single-gene disorders. Diagnostic testing has similarly evolved, with genome sequencing outperforming traditional CMA and exome sequencing through its ability to detect both sequence and structural variants in a single assay. Emerging tools such as optical genome mapping, RNA sequencing, and long-read sequencing further enhance diagnostic yield and variant interpretation. This review summarizes major technological advancements, assesses their clinical utility and limitations, and outlines future directions in prenatal genomics. Full article

Introduction: For pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the fetus and newborn (HDFN), prenatal intervention in subsequent pregnancies may be necessary to prevent complications for the fetus. A non-invasive prenatal diagnostic procedure (NIPD) is recommended for fetal blood group genotyping. RT-PCR is used for fetal RHD determination as a reliable screening method with high sensitivity and specificity. For other antigens with variants involving single-base substitutions, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) are recommended to reduce the risk of false-negative results. Only NGS offers the possibility of determining the cell-free fetal DNA (cffDNA) fraction in maternal plasma by sequencing additional gene fragments in parallel, but no standard exists for assay validation. Material and Methods: A custom-made primer panel was designed to target the common platelet and red cell antigens involved in fetal red cell and platelet incompatibilities, as well as additional anonymous single-nucleotide polymorphism (SNP) targets for use as an internal control. Amplicon-based NGS was carried out using semiconductor sequencing. For HPA-1a (HPA*1A, ITGB3) and K (KEL*01.01, KEL) assay validation, the limit of detection (LOD) and limit of quantification (LOQ) were estimated, as were false-positive antithetic alleles, linearity, and inter-assay variation, using cell-free DNA (cfDNA) extracted from the blood samples of healthy blood donors. An additional analysis was performed using 23 diagnostic samples from 21 pregnant women. Results: Regression analysis of dilution series using HPA-1a- and K-positive cell-free plasma samples in antigen-negative donor plasma showed that recovery is definitely feasible up to an HPA*1A and KEL*01.01 allele frequency of 1%. Base calls of false-positive antithetic alleles were detected with a maximum of 0.25% using 21 healthy blood donors. The LOD was estimated to be 0.2057% (mean + 3 SD) for HPA*1A with a LOQ of 0.6298% (mean + 10 SD). For KEL*01.01, the LOD was 0.1706% (mean + 3 SD) and the LOQ was 0.5314% (mean + 10 SD). The analysis of 15 of 21 cases with diagnostic samples from pregnant women with neonatal blood available for confirmatory testing resulted in 100% concordant results. The fetal fraction of these samples was calculated with a median of 11.03% (95% CI: 8.89, 13.20). Conclusions: NGS for non-invasive fetal blood group genotyping is an accurate and reliable method. In-house validation of the used assays can be performed using healthy donors to determine the LOD, LOQ and sensitivity. The threshold for paternally inherited fetal HPA*1A and KEL*01.01 alleles could be set at 1% (i.e., 2% fetal fraction) to obtain reliable test results. Internal controls for assessing the fetal fraction are essential to avoid false-negative test results. Full article

Background/Objectives: Non-invasive prenatal testing (NIPT) has become a widely used method for screening common fetal aneuploidies due to its high sensitivity and specificity compared to traditional screening methods. With various NIPT technologies available, such as whole-genome sequencing (WGS), single nucleotide polymorphisms (SNPs), microarray, and rolling circle amplification (RCA), understanding the accuracy and reliability of each method is critical for clinical decision-making. However, comprehensive evaluations comparing the performance of these NIPT methods, especially in terms of predictive values for trisomy detection, remain limited. A systematic review of the difference in accuracy of the different NIPT methods used for common aneuploidy screening. Methods: A systematic review of former clinical studies using different NIPT methods, such as whole-genome sequencing (WGS), a targeted method of single nucleotide polymorphisms (SNPs), microarray and rolling circle amplification (RCA). We collected data from the PubMed, Embase, Web of Science, Scopus, clinicaltrials.gov, and Cochrane library from the last 20 years, between 2003 January and 2023 October, without any language, search filter or publication type restrictions. Results: Two authors selected twenty articles including twenty-one studies to perform the systematic review. In these studies, altogether 92,164 pregnant women were tested by genomics-based non-invasive prenatal testing (NIPT). We extracted data on true positive, false positive, false negative, and true negative values from each study, and calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) from them. We collected data regarding trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13) detection from all studies. Conclusions: As a conclusion, for the detection of common fetal trisomies, different methods of NIPT perform similarly in terms of clinical sensitivity, specificity and NPV. However, the tests utilizing SNP and RCA had lower PPV values than other NIPT methods. Our research indicates all NIPT methods showed greater sensitivity for the detection of T21, above 97%, than traditional screening tests. For T18 detection, the targeted method with the microarray had a lower sensitivity compared to other tests. The SNP and the microarray-based test had high PPV, whilst the other tests, utilizing WGS, and the test with RCA had quite low PPV. Regarding T13 detection, all of the tests performed similarly in terms of clinical sensitivity, specificity, PPV, and NPV (with one exception—one of the tests using WGS had lower PPV). According to these results, there was no significant difference between the methods of NIPT, such as WGS, SNPs, microarray, and RCA, used to detect common trisomies, but the variation in PPV underlines the importance of invasive tests to derive positive NIPT results. We suggest that NIPT combined with US screening for structural abnormalities could further improve the utility of the non-invasive tests in pregnancy. This is the first independent systematic review into the efficacy of the different NIPT methods. Full article

We introduce an innovative, non-invasive prenatal screening approach for detecting fetal monogenic alterations and copy number variations (CNVs) from maternal blood. Method: Circulating free DNA (cfDNA) was extracted from maternal peripheral blood and processed using the VeriSeq NIPT Solution (Illumina, San Diego, CA, USA), with shallow whole-genome sequencing (sWGS) performed on a NextSeq550Dx (Illumina). A customized gene panel and bioinformatics tool, named the “VERA Revolution”, were developed to detect variants and CNVs in cfDNA samples. Results were compared with genomic DNA (gDNA) extracted from fetal samples, including amniotic fluid and chorionic villus sampling and buccal swabs. Results: The study included pregnant women with gestational ages from 10 + 3 to 15 + 2 weeks (mean: 12.1 weeks). The fetal fraction (FF), a crucial measure of cfDNA test reliability, ranged from 5% to 20%, ensuring adequate DNA amount for analysis. Among 36 families tested, 14 showed a wild-type genotype. Identified variants included two deletions (22q11.2, and 4p16.3), two duplications (16p13 and 5p15), and eighteen single-nucleotide variants (one in CFTR, three in GJB2, three in PAH, one in RIT1, one in DHCR7, one in TCOF1, one in ABCA4, one in MYBPC3, one in MCCC2, two in GBA1 and three in PTPN11). Significant concordance was found between our panel results and prenatal/postnatal genetic profiles. Conclusions: The “VERA Revolution” test highlights advancements in prenatal genomic screening, offering potential improvements in prenatal care. Full article

Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the β-globin gene cluster. To identify highly heterozygous SNVs in the population, 96 randomly selected samples were processed using Illumina DNA-prep NGS chemistry. A custom, high-density NGS genotyping panel, named HAPLONID, was designed with 169 SNVs, including 15 common pathogenic ones. The AmpliSeq for Illumina assay was then applied to cfDNA to evaluate the panel’s efficiency in performing NIPT for β-thalassaemia. Analysis revealed 219 highly polymorphic SNVs, and the sequencing of 17 families confirmed successful paternal allele determination. The NIPH assay demonstrated 100% success in diagnostic interpretation. This study achieved the advancement of an integrated NGS-NIPT assay for β-thalassaemia, bringing it one step closer to being a diagnostic assay and thereby enabling a reduction in the number of risky invasive prenatal sampling procedures in Cyprus and elsewhere. Full article

Background: Arising in the late 1990s, when a promising role in prenatal diagnostics was first delineated for circulating fetal DNA, non-invasive prenatal tests (NIPTs) have been increasingly used with more frequency and popularity. These exams have been used as a prenatal screening tests for genetic diseases. Initially, they were developed for the investigation of the main fetal chromosomal aneuploidies, but lately they have also been used to rule out genomic microrearrangements and monogenic conditions. However, along with great opportunities and potential, the tests can show inconclusive or unexpected results. Several studies have shown that the current pre-test counseling is often insufficient, and more oriented at providing pieces of information about the identifiable diseases rather than providing extensive information on all possible scenarios which may affect both the fetus and the pregnant mother, especially in the case of an invasive test for the pregnant mother. Methods and Results: We have gathered from the literature on NIPT the main pitfalls, imperfections, and particular cases associated with this innovative diagnostic procedure. Conclusions: In view of further improvements in the methods that can limit the inconclusive or unexpected results, this paper aims to reinforce the importance of more accurate pre-test counseling with comprehensive information about the above-mentioned questions, as well as ultrasound use and also the creation of an international consensus statement concerning these topics. Full article

Background/Objectives: Cell-free DNA (cfDNA) is a non-invasive prenatal test used to screen for common trisomies (target cfDNA) that can be expanded to assess all autosomal chromosomes (genome-wide cfDNA). As cfDNA testing gains popularity, it is crucial to examine the factors influencing the decision-making process of pregnant individuals when choosing between these two approaches. Methods: In this prospective cohort study, 190 individuals undergoing cfDNA testing for aneuploidy screening, according to the current screening protocol, were allowed to make their own choice between target and genome-wide cfDNA testing. They were asked to complete a first survey at 11–13 weeks, designed to explore their characteristics, preferences, and satisfaction with the prenatal genetic counseling session, as well as a Decisional Conflict Scale. A postnatal survey was administered three months after delivery, including the Decisional Regret Scale and two open questions. Results: 84% of participants opted for genome-wide cfDNA. However, 17% found the decision challenging, and 14% felt that the results might increase anxiety. No significant differences in participant characteristics were found when comparing decisions between genome-wide and target cfDNA. However, significant differences were observed regarding ethnicity (p = <0.001), educational level (p = 0.029), previous cfDNA experience (p = 0.004), and having sufficient information when comparing termination options (p = 0.002). After delivery, only 4% would have changed their decision. Conclusions: Individuals, regardless of their characteristics, prefer genome-wide cfDNA; however, the complexity of the results necessitates enhanced genetic education for prenatal care clinicians. Full article

Sex chromosome aneuploidies (SCAs) collectively occur in 1 in 500 livebirths, and diagnoses in the neonatal period are increasing with advancements in prenatal and early genetic testing. Inevitably, SCA will be identified on either routine prenatal or newborn screening in the near future. Tetrasomy SCAs are rare, manifesting more significant phenotypes compared to trisomies. Prenatal cell-free DNA (cfDNA) screening has been demonstrated to have relatively poor positive predictive values (PPV) in SCAs, directing genetic counseling discussions towards false-positive likelihood rather than thoroughly addressing all possible outcomes and phenotypes, respectively. The eXtraordinarY Babies study is a natural history study of children prenatally identified with SCAs, and it developed a longitudinal data resource and common data elements with the Newborn Screening Translational Research Network (NBSTRN). A review of cfDNA and diagnostic reports from participants identified a higher than anticipated rate of discordance. The aims of this project are to (1) compare our findings to outcomes from a regional clinical cytogenetic laboratory and (2) describe discordant outcomes from both samples. Twenty-one (10%), and seven (8.3%) cases were found to be discordant between cfDNA (result or indication reported to lab) and diagnosis for the Babies Study and regional laboratory, respectively. Discordant results represented six distinct discordance categories when comparing cfDNA to diagnostic results, with the largest groups being Trisomy cfDNA vs. Tetrasomy diagnosis (66.7% of discordance in eXtraordinarY Babies study) and Mosaicism (57.1% in regional laboratory). Traditional genetic counseling for SCA-related cfDNA results is inadequate given a high degree of discordance that jeopardizes the accuracy of the information discussed and informed decision making following prenatal genetic counseling. Full article

(1) Background: Non-invasive prenatal testing (NIPT) is a screening test for fetal aneuploidy using cell-free fetal DNA. The fetal fragments (FF) of cell-free DNA (cfDNA) are derived from apoptotic trophoblast of the placenta. The level of fetal cfDNA is known to be influenced by gestational age, multiple pregnancies, maternal weight, and height. (2) Methods: This study is a single-center retrospective observational study which examines the relationship between the fetal fraction (FF) of cell-free DNA in non-invasive prenatal testing (NIPT) and adverse pregnancy outcomes in singleton pregnancies. A total of 1393 samples were collected between 10 weeks and 6 days, and 25 weeks and 3 days of gestation. (3) Results: Hypertensive disease of pregnancy (HDP) occurred more frequently in the low FF group than the normal FF group (5.17% vs. 1.91%, p = 0.001). Although the rates of small for gestational age (SGA) and placental abruption did not significantly differ between groups, the composite outcome was significantly higher in the low FF group (7.76% vs. 3.64%, p = 0.002). Furthermore, women who later experienced complications such as HDP or gestational diabetes mellitus (GDM) had significantly lower plasma FF levels compared to those without complications (p < 0.001). After adjustments, the low FF group exhibited a significantly higher likelihood of placental compromise (adjusted odds ratio: 1.946). (4) Conclusions: Low FF in NIPT during the first and early second trimesters is associated with adverse pregnancy outcomes, particularly HDP, suggesting its potential as a predictive marker for such outcomes. Full article

Genome-wide prenatal cell-free DNA (cfDNA) screening can be used to screen for a wide range of fetal chromosomal anomalies in pregnant patients. In this study, we describe our clinical experience with a genome-wide cfDNA assay in screening for common trisomies, sex chromosomal aneuploidies (SCAs), rare autosomal aneuploidies (RAAs), and copy-number variations (CNVs) in about 6000 patients over a three-year period at our hospital’s Prenatal Diagnostic Unit in Spain. Overall, 204 (3.3%) patients had a high-risk call, which included 76 trisomy 21, 21 trisomy 18, 7 trisomy 13, 29 SCAs, 31 RAAs, 31 CNVs, and 9 cases with multiple anomalies. The diagnostic outcomes were obtained for the high-risk cases when available, allowing for the calculation of positive predictive values (PPVs). Calculated PPVs were 95.9% for trisomy 21, 77.8% for trisomy 18, 66.7% for trisomy 13, 10.7% for RAAs, and 10.7% for CNVs. Pregnancy and birth outcomes were also collected for the majority of RAA and CNV cases. Adverse perinatal outcomes for some of these cases included preeclampsia, fetal growth restriction, preterm birth, reduced birth weight, and major congenital structural abnormalities. In conclusion, our study showed strong performance for genome-wide cfDNA screening in a large cohort of pregnancy patients in Spain. Full article

Prenatal cell-free DNA screening (cfDNA) can identify fetal chromosome abnormalities beyond common trisomies. Emanuel syndrome (ES), caused by an unbalanced translocation between chromosomes 11 and 22, has lacked a reliable prenatal screening option for families with a carrier parent. A cohort of cases (n = 46) sent for cfDNA screening with indications and/or results related to ES was queried; diagnostic testing and pregnancy outcomes were requested and analyzed. No discordant results were reported or suspected; there were ten true positives with diagnostic confirmation, six likely concordant positives based on known translocations and consistent cfDNA data, and twenty-six true negatives, by diagnostic testing or birth outcomes. For cases with parental testing, all affected ES cases had maternal translocation carriers. Expanded cfDNA may provide reassurance for t(11;22) carriers with screen negative results, and screen positive results appear to reflect a likely affected fetus, especially with a known maternal translocation. Current society guidelines support the use of expanded cfDNA screening in specific circumstances, such as for translocation carriers, with appropriate counseling. Diagnostic testing is recommended for prenatal diagnosis of ES and other chromosome abnormalities in pregnancy. To our knowledge, this cohort is the largest published group of cases with prenatal screening for carriers of t(11;22). Full article

Cell-free DNA (cfDNA) screening for normal fetal aneuploidy has been widely adopted worldwide due to its convenience, non-invasiveness, and high positive predictive rate. We retrospectively evaluated 9327 Korean women with single pregnancies who underwent a non-invasive prenatal test (NIPT) to investigate how various factors such as maternal weight, age, and the method of conception affect the fetal fraction (FF). The average FF was 9.15 ± 3.31%, which decreased significantly as the maternal body mass index (BMI) increased (p < 0.001). The highly obese group showed a ‘no-call’ rate of 8.01%, which is higher than that of the normal weight group (0.33%). The FF was 8.74 ± 3.20% when mothers were in their 40s, and lower than that when in their 30s (9.23 ± 3.34, p < 0.001) and in the natural pregnancy group (9.31% ± 3.33). The FF of male fetuses was observed to be approximately 2.76% higher on average than that of female fetuses. As the gestational age increased, there was no significant increase in the fraction of fetuses up to 21 weeks compared to that at 10–12 weeks, and a significant increase was observed in the case of 21 weeks or more. The FFs in the NIPT high-risk result group compared to that in the low-risk group were not significantly different (p = 0.62). In conclusion, BMI was the factor most associated with the fetal fraction. Although the NIPT is a highly prevalent method in prenatal analysis, factors affecting the fetal fraction should be thoroughly analyzed to obtain more accurate results. Full article