Clinical applications and advancements in noninvasive prenatal diagnosis
Article information
Abstract
Noninvasive prenatal testing (NIPT) is widely performed and enables the detection of fetal chromosomal abnormalities through the analysis of cell-free fetal DNA in maternal blood. Since its introduction in 2011, NIPT has demonstrated high sensitivity and specificity for common trisomies (trisomy 21, trisomy 18, trisomy 13), and its scope has rapidly expanded to include sex chromosome aneuploidies, microdeletion syndromes, and single-gene disorders. However, the widespread adoption of NIPT has brought new challenges, including technical limitations (e.g., low fetal fraction, placental mosaicism), interpretation of variants of uncertain significance, and ethical concerns related to over-screening and patient anxiety. This review summarizes the historical evolution, technical advances, clinical applications, limitations, and future perspectives of NIPT, emphasizing the need for balanced clinical implementation and ongoing innovation.
Introduction
Congenital anomalies, which occur in 2%–4% of all newborns and account for 20.4% of perinatal deaths, represent a significant global health burden with profound psychological, social, and economic implications. Over the past six decades, prenatal genetic testing has revolutionized the identification of chromosomal and genetic abnormalities, facilitated informed reproductive choices and enhanced perinatal outcomes. This review examines the historical evolution, clinical applications, and challenges of prenatal diagnostic technologies, with a particular focus on the transformative role of noninvasive prenatal testing (NIPT) and its integration into modern obstetric practice.
The foundation of prenatal diagnosis was established in the 1960s with amniocentesis, which enabled karyotypic analysis of fetal cells in the amniotic fluid. However, this invasive procedure carried procedural risks and was confined to the second trimester. The introduction of chorionic villus sampling (CVS) in the 1980s expanded diagnostic capabilities to the first trimester, albeit with a 0.5%–1% risk of pregnancy loss. While these methods provided diagnostic accuracy, their invasiveness and low sensitivity (30%) limited their use to high-risk pregnancies. A major advancement emerged in the 1990s with combined first trimester screening (cFTS), which integrated nuchal translucency (NT) measurements with maternal serum biomarkers (pregnancy-associated plasma protein A [PAPP-A], beta-human chorionic gonadotropin [β-hCG]), and achieved 90%–97% detection rates (DRs) for trisomy 21. Despite its efficacy, cFTS maintained a 5% false-positive rate (FPR), necessitating invasive confirmation and perpetuating patient anxiety.
A paradigm shift occurred in 1997 with the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, heralding the era of noninvasive testing. By 2008, massively parallel shotgun sequencing (MPSS) enabled trisomy 21 detection with >99% specificity, positioning NIPT as a cornerstone of prenatal screening. Subsequent innovations have broadened NIPT’s scope to include sex chromosome aneuploidies, microdeletion/microduplication syndromes (MMS), and select single-gene disorders, transforming prenatal care from risk stratification to comprehensive genome-wide fetal assessment.
Advances and clinical impact of noninvasive prenatal diagnosis
Prenatal diagnosis for early detection of congenital anomalies began in the 1960s with amniocentesis, which allowed for fetal sex determination and karyotype analysis. The introduction of CVS followed in the 1980s, which made first trimester testing possible [1-4]. Amniocentesis and chorionic villous sampling, which are invasive to both mother and fetus, have <1% risk of procedure-related pregnancy loss [5,6]. Maternal age-based screening in advanced maternal age has a low sensitivity (approximately 30%) and FPR (15%) [7]. Moreover, maternal age-based screening does not represent a relative risk for sex chromosome aneuploidies or triploidy. In the 1990s, a first trimester integrated test combining NT measurement and biochemical markers (hCG, PAPP-A) was introduced, which has a 90%–97% DR for the chromosomal abnormalities trisomy 21, trisomy 18, and trisomy 13 [8,9]. In 1997, Lo et al. [10] first reported cffDNA in maternal plasma and serum. They also reported that the cffDNA concentration in maternal circulation increases with gestational age and it is suitable for pregnancy tests due to the rapid clearance following the end of the pregnancy. In 2008, Fan et al. [11] and Chiu et al. [12] described the ability to screen for trisomy 21 by sequencing cffDNA in maternal plasma with high specificity by analyzing a blood sample from the pregnant woman [13]. This noninvasive prenatal test was called NIPT (Fig. 1). Generally, NIPT protocols are based on comparisons between specific chromosomes in the analyzed sample and an internal control, which may be another chromosome or a disomy control, which may be a pool of disomy pregnancy samples. With the remarkable development of genetic testing, the carrier states of parents can be determined (Table 1) [14,15]. NIPT is based on analyzing the entire circulating DNA fragment without distinguishing between the fetus and the mother. NIPT should be considered a screening test rather than a diagnostic test. While NIPT is considered safer than invasive testing, it does not replace the role of diagnostic testing. A positive result may require additional invasive testing (e.g., amniocentesis). Thus, parents can be informed about the genetic risk for congenital abnormalities and make informed decisions during the early period of pregnancy.
Non invasive prenatal screening process schematic diagram. cffDNA, cell-free fetal DNA; CNVs, copy number variants; SNPs, single nucleotide polymorphisms.
Diagnostic modalities for congenital genetic abnormalities
However, the ethical implications of selective abortion, as well as the challenges posed by the proliferation of complex genetic information that may hinder truly informed decision-making, must be carefully considered in the context of NIPT and related technologies [16].
Fetal fraction and failure rate
cffDNA can be isolated at clinically acceptable levels by the 10th week of gestation. NIPT can be performed during any of three points of gestation: before an ultrasound, after an ultrasound, or after a first trimester screening test. This provides great flexibility for physicians and parents. The proportion of cffDNA, termed the fetal fraction (FF), varies widely from less than 4% (clinically insufficient for reliable diagnosis) to approximately 40% [17].
A higher FF is associated with increased reliability of test results, as it facilitates more accurate discrimination between fetal and maternal cell-free DNA (cfDNA). A study reported that the DR was only 62.1% when the FF was 4%, but reached 100% when the FF exceeded 9% [18]. CffDNA can be detected in the maternal plasma as early as 5–7 weeks of gestation [19]. It increases by 0.1% per week between 10 and 21 weeks of gestation, and then by 1% per week beyond 21 weeks of gestation [20]. To ensure an adequate FF, it is recommended that NIPT screening be performed after 10 weeks of gestation [20].
Several studies have demonstrated that the FF in cases of trisomy 21 is comparable to, or occasionally higher than, that observed in euploid fetuses. In contrast, the FF in trisomy 18, trisomy 13, and triploidy is generally lower than that in euploid fetuses; higher levels of PAPP-A in these anomalies are associated with reduced placental size, which in turn contributes to the decreased FF in trisomy 18 and trisomy 13 [21-23]. The American College of Obstetricians and Gynecologists (ACOG) recommends diagnostic testing, rather than repeat NIPT testing, in cases of low FF. ACOG further advises that patients should be informed of the possibility of fetal chromosomal abnormalities and that comprehensive ultrasound evaluation, genetic counseling, and diagnostic testing should be offered. The reported success rate of repeat sampling is approximately 70% for singleton pregnancies and about 55% for twin pregnancies [22,24]. In multiple pregnancies, the failure rate is higher, and the FF in twin pregnancies is lower than that observed in singleton pregnancies. The FF is lower in twins compared to singletons and in dichorionic compared to monochorionic twins [25]. In twin pregnancies, the FF contributed by each fetus can vary significantly, with up to twofold differences observed between co-twins [26]. Notably, in dichorionic twins, if the aneuploid fetus contributes a FF below the diagnostic threshold of 4%, the total FF may exceed 8% due to the proportionally higher contribution from the euploid co-twin. This imbalance can mask chromosomal abnormalities in the affected fetus, thereby increasing the risk of false-negative results [17,25].
Other factors that affect FF include maternal body mass index (BMI), and maternal age, although the impact of BMI and maternal age remains controversial. FF is also directly related to fetal crown-rump length, PAPP-A, and free β-hCG multiples of the median (MoM), which are relatively high in East Asians (Chinese, Japanese) due to race. In contrast, smokers tend to have higher FF due to decreased maternal cfDNA. As maternal age and BMI increase, FF decreases relatively due to increased maternal cfDNA [25,27]. Studies have also shown that FF below the 10th percentile is associated with an increased risk of preeclampsia or preterm birth, and FF below the 5th percentile is associated with low birth weight [28]. Failures can also be caused by errors during blood collection and sample transport, as well as handling errors within the laboratory.
Placental mosaicism can cause additional complications. Placental mosaicism can occur in both the fetus and the placenta. This can result in false-positive results, requiring additional testing for an accurate diagnosis. Placental mosaicism can also lead to false-positive results, lead to confirmatory testing to establish an accurate diagnosis [29-32].
Technologies available for noninvasive prenatal diagnosis
Recent technologies are summarized in Table 2 and are discussed below. MPSS sequences millions of cfDNA fragments from the mother and fetus, assigns each fragment to its original chromosome, and statistically analyzes whether the number of fragments for that chromosome is increased in trisomy fetuses compared to normal fetuses [33]. Chromosome-selective sequencing (CSS) employs targeted amplification of predefined chromosomal regions, including chromosomes 13, 18, 21, X, and Y, which are subsequently subjected to high-resolution sequencing to enhance detection of fetal aneuploidies. This method is cost-effective, but has a failure rate that is about 2% higher than MPSS [34].
Performance comparison of NIPT, NIPT-plus, and GW-NIPT in detecting chromosomal abnormalities
Genome-wide cfDNA analysis showed high sensitivity (approximately 100%) and specificity (approximately 99.87%) for effectively detecting trisomy 21, 18, and 13 compared to conventional NIPT [35]. It can also effectively detect additional clinically important chromosomal abnormalities (rare autosomal trisomies, segmental imbalances, etc.) and copy number variants (CNVs) [35]. This approach enables the effective detection of other clinically relevant chromosomal abnormalities that may not be identified by conventional NIPT, such as rare autosomal trisomies, segmental imbalances, and CNVs. More than 15% of all congenital abnormalities are associated with structural abnormalities such as MMS [36]. MMS are frequently undetectable by routine prenatal ultrasound. Approximately 25% of all positive results have been reported to be identified exclusively through genome-wide analysis [35]. However, it should be noted that some variants of uncertain significance and false positives have also been observed. The introduction of whole-genome sequencing and high-resolution analytical algorithms has enabled the simultaneous detection of rare autosomal trisomies and MMS [37]. Genome-wide screening based on cffDNA has demonstrated sensitivity and specificity for the detection of chromosomal aneuploidy in patients with early pregnancy loss and recurrent pregnancy loss that are comparable to those of product of conception-based cytogenetic analysis [38].
The enhanced resolution of next-generation sequencing (NGS) has enabled NIPT to detect sex chromosome abnormalities, recurrent CNVs (e.g., 22q11.2 deletions), and targeted monogenic conditions [39]. Nevertheless, structural variants <3 Mb, which account for about 15% of pathogenic CNVs, are not currently included in the scope of the application [40]. NIPT combined with NGS is utilized to detect chromosomal aneuploidies either through whole-genome (shotgun) sequencing of fetal DNA or by targeted sequencing of selected chromosomes associated with aneuploidy, such as chromosomes 13, 18, X, and Y [33]. Another approach involves the selective amplification and sequencing of specific genomic loci on the chromosome of interest [41]. This technique is more cost-effective due to the reduced number of regions requiring sequencing; however, its primary limitation is that only a limited number of preselected target regions can be analyzed.
Multiplex polymerase chain reaction (PCR) techniques based on the amplification of single nucleotide polymorphisms (SNPs) at multiple loci on the chromosome of interest can be used to distinguish between maternal and fetal DNA [42].
Digital PCR was initially validated for trisomy 21 detection and is faster and more cost-effective than NGS. However, digital PCR requires sufficient cffDNA and has limitations in large-scale analysis or in detecting structural abnormalities such as low-grade mosaicism or balanced translocations [43].
Microarray quantification has recently been proposed as an alternative to CSS, which may be cost- and time-saving, and reduce the risk of PCR contamination and assay variability [41].
The genetic diseases studied using cyclic single-molecule amplification and resequencing technology were phenylketonuria and Wilson disease [44]. Genotyping is considered a reliable method for some recessive diseases such as duchenne muscular dystrophy [45]. Recent studies have demonstrated that the integration of NGS-based NIPT with quantitative template DNA analysis achieves high diagnostic sensitivity and specificity for detecting the most prevalent genetic disorders worldwide (sickle cell anemia, cystic fibrosis, spinal muscular atrophy, alpha thalassemia, and beta thalassemia) [46].
Reliability of testing
Placental-confined mosaicism is a primary contributor to false-positive NIPT results and a key source of discordance between noninvasive screening and invasive diagnostic findings. Placental mosaicism has been reported to underlie >50% of sex chromosome aneuploidies [47]. Diagnostic confirmation of placental mosaicism requires amniocentesis rather than CVS. Maternal mosaicism may contribute to false-positive fetal abnormalities via maternal X chromosome fragments in cfDNA, requiring maternal karyotyping to clarify discordant results [48].
Low FF, maternal chromosomal abnormalities, fetal mosaicism, pathogenic CNVs, and vanishing twins are recognized as the main causes of false-positive and false-negative results in NIPT. Additionally, false-positive results may also arise from maternal malignancies of unknown origin [38].
Among sex chromosome aneuploidies, the majority of validated data pertains to 45,X (Turner syndrome), demonstrating a DR of 95.8% and a FPR of 0.14% in singleton pregnancies [49]. The overall prevalence of sex chromosome abnormalities-including Turner syndrome, Klinefelter syndrome (47,XXY or 48,XXYY), triple X syndrome (47,XXX), and 47,XYY-is approximately 1 in 500, making these conditions more common than the major autosomal trisomies [48]. Although cases of 47,XYY, 47,XXY, and 47,XXX have been identified, the overall number of reported cases remains small, making it difficult to draw definitive conclusions regarding the performance of NIPT for these sex chromosome aneuploidies [48].
Increased NT identified on a first trimester ultrasound or the presence of cystic edema or hydrocele on a second trimester ultrasound indicates the need for invasive diagnostic testing (rather than cffDNA analysis) for a more accurate evaluation [50].
Triploidy is classified into paternal (diandric) and maternal (digynic) origins based on the parental source from an extra chromosome set. Maternal origin triploidy presents with a markedly small placenta, severe early-onset fetal growth restriction, normal NT, and markedly reduced free β-hCG and PAPP-A levels (<0.1 MoM). In contrast, paternal-origin triploidy features placental enlargement with hydatidiform mole-like changes, elevated free β-hCG (more than 10 times the normal), and increased NT. SNP analysis reliably identifies paternal-origin cases, while maternal origin triploidy is suggested by an extremely low FF [48,49,51,52].
NIPT in clinical practice
Universal screening: NIPT is performed on all patients at 10 weeks of pregnancy, followed by a combined screening test and first trimester ultrasound at 12 weeks of pregnancy [53].
Contingent screening: This method considers the results of combined screening with the first trimester ultrasound, and performs NIPT based on the results [53]. This method has the advantage of reducing the cost burden compared to general screening, while maintaining the main advantages of NIPT (improved DR and reduced FPR) [53].
During contingent testing, a detailed ultrasound examination should be performed to confirm the date of pregnancy, exclude major abnormalities, and screen for specific abnormalities (e.g., megabladder, cranial abnormalities, gastrointestinal malformations, etc.) or pregnancy complications (e.g., preterm birth, preeclampsia, etc).
Structural abnormalities suspected on first trimester ultrasound require confirmation through invasive diagnostic testing. In the absence of major abnormalities on ultrasound, a second NIPT sample collection may be considered. If the second attempt is unsuccessful, invasive testing can be performed again, or testing may be discontinued with subsequent management determined according to the results of the combined screening test [22]. In the absence of significant abnormalities on first trimester combined screening or suspicious ultrasound findings following two NIPT failures, the risk of trisomy 18 and trisomy 13 is classified as low, and further diagnostic testing may be omitted. However, in cases with an elevated risk for trisomy 21, invasive diagnostic procedures such as amniocentesis are indicated. The success rate of repeat sampling is approximately 70% in singleton pregnancies and 55% in twin pregnancies [22,24].
Challenges of noninvasive prenatal diagnosis for single-gene disorders
NIPT remains predominantly utilized for screening chromosomal aneuploidies. Advances in genomic analysis have enabled its application in detecting select single-gene disorders through targeted sequencing of cffDNA. Recently, advanced exome sequencing technologies have helped to identify the presence of single-gene disorders in fetuses with abnormal ultrasound findings despite a normal karyotype [54,55]. Innovative technologies such as NGS enable noninvasive prenatal sequencing of multiple single-gene disorders using circulating cffDNA in maternal plasma, and therefore facilitate early diagnosis. Although cfDNA analysis uses fetal DNA fragments, the haplotyping strategy used in preimplantation genetic diagnosis is being studied. Relative haplotype dosage, which indirectly estimates the fetal haplotype from the maternal plasma by multi-analyzing parental haplotypes, especially heterozygous SNPs associated with mutations. This technology can identify paternal fetal alleles and mutations that are absent from the mother, such as cystic fibrosis [56].
One of the key advantages of NGS is that it can simultaneously analyze multiple mutations in a single panel. In the UK, noninvasive prenatal diagnosis is already being performed for several genetic disorders (obvious dominant, recessive by paternal allele deletion, X-linked inheritance), providing results without the need for additional confirmation by invasive testing [57].
The positive predictive value (PPV) of cffDNA-based analysis for microdeletion syndromes remains low. For example, the PPV for major microdeletion syndromes such as DiGeorge syndrome, Prader-Willi/Angelman syndrome, Cri-du-chat syndrome, and 1p36 deletion syndrome is approximately 13%, which is partly attributable to the low prevalence of these conditions [37].
22q11.2 deletion syndrome is the most prevalent microdeletion syndrome and is associated with a broad spectrum of clinical phenotypes, including congenital heart defects and developmental delay. In pregnancies with confirmed cardiac anomalies, targeted cfDNA analysis demonstrates high specificity (100%) for detecting fetal 22q11.2 deletions (DiGeorge syndrome), although sensitivity remains moderate at 69.6% [39].
Widespread implementation of NIPT has introduced new challenges. Additional genomic variants, such as CNVs, are detected in approximately 0.36% of cases. Up to 50% of these genomic variants represent false positives that are attributable to confined placental mosaicism (CPM) or maternal origin. Notably, the PPV for rare disorders may fall below 50%, resulting in unnecessary invasive procedures and increased maternal anxiety. Moreover, some individuals recommended NIPT for nonmedical, preventive purposes, leading to excessive and potentially unnecessary screening tests [58].
A recent study found that among the cases of CPM detected by NIPT, 13.6% had birth weight below the 2.3 percentile and 8.5% had preeclampsia, which is a 5.5-fold and 18.5-fold increased risk, respectively, compared to the general pregnancy group. These findings suggest an association with placental insufficiency and highlight the necessity of comprehensive clinical evaluations that account for potential genetic discordance between placental and fetal tissues when interpreting NIPT results [28].
Future advancements in NIPT will likely focus on enhancing precision through single-cell genomic analysis and artificial intelligence analytical platforms, as well as leveraging methylation profiling to identify fetal-specific epigenetic signatures. Concurrently, standardization of testing protocols and cost-reduction strategies are critical to improving global accessibility. A balanced approach is essential: prioritizing the detection of clinically actionable variants while mitigating unnecessary patient anxiety via comprehensive pre- and posttest counseling frameworks.
NIPT has been extensively validated for detecting chromosomal aneuploidies (e.g., trisomy 21, 18, and 13), sex chromosome aneuploidies, CNVs, and recurrent microdeletion syndromes. However, its primary diagnostic challenge lies in the reliable detection of single-gene disorders using cffDNA, which requires overcoming technical limitations in sensitivity and specificity due to a low FF and maternal DNA background interference.
Emerging molecular techniques such as chromosomal microarray analysis and NGS demonstrate potential for prenatal detection of chromosomal and genetic anomalies that are not identifiable through cffDNA screening; however, their application remains contingent upon invasive sampling procedures.
Conclusions
NIPT is currently widely used for nondrainage screening. With the advancement of genetic technology, the scope of application for early detection of congenital diseases through NIPT is expanding, and more clinical applications are expected in the future.
The number of women availing themselves of NIPT screening will progressively rise, as will the number of conditions being tested. That undoubtedly represents a great opportunity for prospective mothers to make informed reproductive decisions, but it also poses issues and concerns about the routinization of testing and negative impacts on informed consent and decision-making at its core [59].
Notes
Conflicts of interest
Jin Hyuk Choi, Jihun Kang, and Chang Zoo Kim are editorial board members of the journal but were not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.
Funding
None.
Author contributions
Conceptualization: HY. Data curation: HY. Formal analysis: SL. Investigation: TYK. Methodology: JY, CZK. Project administration: JK. Resources: SHK. Software: JHY. Supervision: JHC. Validation: SL. Visualization: SL. Writing – original draft: SL, HY. Writing – review & editing: JHC, JHY, SHK, JY, JK, CZK, TYK. All authors read and approved the final manuscript.