NIPT: Routine versus Considered Options

Introduction

Congenital diseases, complexes, and syndromes affect 3 to 6% of newborns, with chromosomal abnormalities, teratogenic damage, monogenic disorders, and multigenic diseases being significant contributors to the genetic burden.[1] Specifically, chromosomal disorders account for 6%, teratogenic damage for 7%, monogenic disorders for 8%, and multigenic diseases for 25% of all cases. However, a definitive genetic diagnosis is possible in only about 10% of affected neonates.[2]

Chromosomal abnormalities present a major concern during pregnancy, affecting 1 in every 150 pregnancies.[3] These abnormalities are responsible for half of all early pregnancy losses and are strongly associated with maternal age, regardless of whether the pregnancy involves a singleton or twins.[3] [4] Traditionally, diagnosing these anomalies relied on invasive tests such as amniocentesis and chorionic villus sampling (CVS). While effective, these procedures carry risks of complications, including rare but significant cases of fetal loss.[5]

The field of prenatal diagnostics underwent a revolutionary shift with the discovery of cell-free DNA (cfDNA) in maternal blood in 1997. This breakthrough paved the way for noninvasive prenatal testing (NIPT), which has demonstrated exceptional sensitivity and specificity in detecting fetal aneuploidies. As a result, NIPT has gained rapid acceptance, particularly among high-risk patients. Its remarkable accuracy has significantly reduced the need for invasive diagnostic procedures and the associated risk of fetal loss.

NIPT's transformative impact has earned it the label of a “disruptive innovation” as it continues to reshape prenatal screening and diagnostic practices.[6] However, it is crucial to critically evaluate the claims, promises, and growing excitement surrounding this technology. Such scrutiny is necessary to understand its broader implications, especially given the significant financial investments and strong market demand driving its widespread adoption.[7] [8]

Background

In 1997, Lo and colleagues discovered the presence of fetal DNA fragments, free of cells, circulating in the mother's bloodstream.[9] This cfDNA originates from the placenta and enters maternal blood during pregnancy.[10] Compared to maternal cfDNA, which averages 400 to 500 base pairs in length, fetal DNA is shorter, measuring less than 313 base pairs.[11] [12] The proportion of cfDNA increases with gestational age, reaching 10 to 15% of the total DNA in maternal plasma between 10 and 20 weeks of pregnancy.[12] [13]

cfDNA plays a crucial role in the early detection and diagnosis of fetal chromosomal abnormalities.[14] [15] Its discovery has enabled the development of NIPT, which is now widely used as an initial screening method for detecting conditions such as trisomy 21 in high-risk pregnancies. NIPT results are categorized as either positive, indicating a high likelihood of a chromosomal abnormality, or negative, suggesting that such abnormalities are unlikely. When NIPT yields a positive result, confirmatory invasive procedures, such as amniocentesis, are conducted. This contingent approach significantly reduces the need for invasive diagnostic testing in pregnant women, thereby minimizing the incidence of associated risks of amniocentesis.

Limitations of NIPT

Inconclusive Results

In addition to yielding positive or negative results, NIPT can occasionally produce inconclusive or nonreportable outcomes, most often due to an insufficient amount of fetal cfDNA in the maternal sample. The proportion of fetal DNA in maternal plasma, referred to as the fetal fraction, is not affected by maternal age or the timing of the test. However, it decreases with increasing maternal weight and body mass index (BMI).[16] When the fetal fraction falls below an acceptable threshold (e.g., <4%), NIPT cannot generate reportable results. Reported rates of such outcomes range from 0.32 to 5.4%.[17] [18] An international collaborative study estimated the current rate of nonreportable results at approximately 0.9%. Notably, in regions with a low prevalence of maternal obesity, the test failure rate can be as low as 0.3% after the initial blood collection and further decreases to 0.1% with repeat testing.[19] [20]

Reduced placental size and fetal growth restriction, which are often observed in trisomy 13 and trisomy 18, are other key factors contributing to lower fetal fractions.[21] In contrast, some studies suggest higher concentrations of cfDNA in cases of trisomy 21. The positive predictive value (PPV) of NIPT for trisomy 21 is higher than that for trisomy 13 or trisomy 18, partially due to an increased fetal fraction in samples from affected pregnancies.

Certain maternal health conditions can also significantly impact NIPT results. For example, autoimmune diseases like autoimmune thrombocytopenic purpura (ITP) often lead to recurrent non reportable results. Inflammatory responses in these conditions raise maternal cfDNA levels while lowering fetal cfDNA, thereby reducing the overall fetal fraction in maternal blood.[22] [23] In systemic lupus erythematosus (SLE), abnormal DNA methylation in maternal T cells results in elevated levels of hypomethylated maternal cfDNA and increased apoptotic T cells. Consequently, NIPT utilizing next-generation sequencing (NGS) may reveal unique patterns in SLE patients compared to healthy individuals.[24]

Heparin treatment also influences NIPT outcomes. In individuals treated with heparin, cfDNA fragments have a higher guanine-cytosine content and are shorter than those in healthy participants. Heparin-induced apoptosis of maternal leukocytes increases maternal cfDNA fragments, potentially leading to false positives or nonreportable results.[25]

Additional complexities can occur in cases of vanishing twins (VTs) or multiple pregnancies. Around half of the VTs are associated with chromosomal abnormalities, and the placenta of the resorbed anomalous fetus may continue to release cfDNA into the maternal bloodstream. This can potentially lead to nonreportable results or false-positive findings. In up to 10% of such cases, cfDNA results may be inconsistent with the euploid karyotype of the surviving fetus or neonate.[26] [27] [28]

Source of DNA

It is crucial to emphasize that NIPT primarily analyzes placental DNA, not fetal DNA. While placental DNA typically reflects fetal DNA, discrepancies can occasionally occur. In rare cases, the placental genotype may differ from the fetal genotype, potentially leading to clinical inaccuracies such as false-positive or false-negative results. Therefore, clinical sensitivity and specificity, which evaluate the actual condition of the fetus, hold greater importance than technical sensitivity and specificity ([Fig 1]).

Fig. 1 Fertilized eggs with normal chromosomes at fertilization may experience chromosomal aberrations during the process of recurrent cell division. Fetus A: If all the cells are normal. Fetus B: Chromosomal aberration develops between the two to four divisions and the morula stage, resulting in conventional mosaic including both placenta and the fetus. Fetus C is normal up until the morula stage. However, if cells in the trophectoderm have chromosomal abnormalities (or if abnormality occurs at the morula stage), the placenta will contain the cells with the abnormalities. The placenta is the sole organ that is mosaic; the fetus is normal. (False-positive NIPT). Fetus D: Anomalies arising at the morula stage; chromosomal abnormalities are transported to the placenta. (False-positive NIPT). Fetus E: False-negative NIPT arises when mosaic-type chromosomal aberrations only occur in the fetus and the trophectoderm is normal at the blastocyst stage. In this scenario, the inner cell mass is mosaic.

A notable source of false-positive results in cfDNA testing is confined placental mosaicism (CPM), a condition where chromosomal anomalies are present in part of the placenta but not in the fetus. CPM affects 1 to 2% of pregnancies and is more commonly associated with trisomy 13 and monosomy X than with trisomy 21 or trisomy 18.[27] [29] [30] [31] [32] [33] Another scenario leading to false-positive results is VT syndrome, where a deceased co-twin was aneuploid.[34]

Maternal Factors and Their Impact

Most cfDNA testing methods operate under the assumption that the mother has a normal karyotype. However, maternal mosaicism, where a small proportion of maternal cells carry chromosomal abnormalities, can lead to false-positive results, particularly in older women.[35]

Other maternal conditions can also influence NIPT results. For instance, transplanted tissues or organs from male donors may introduce male cfDNA into the maternal bloodstream, leading to the incorrect identification of a female fetus as male.[36] Similarly, recent blood transfusions from male donors can produce false results.[37] Malignant neoplasia in pregnant women, though rare (affecting 1 in 1,000 women over 40 years), accounts for up to 15% of false positive NIPT results.[27] This is often linked to altered genomic profiles in malignancies like lymphoma, leukemia, or colon cancer.[38] [39] False positive results have also been associated with rare cancers like angiosarcoma and neuroendocrine carcinoma, as well as benign conditions such as fibroids.[40] [41]

False-Negative Results

False-negative results, though rare, are a well-recognized possibility with cfDNA testing. These occur when the fetus has a chromosomal abnormality, but the test fails to detect it. The outcome of NIPT is influenced by the relative proportions of cells with abnormal and normal karyotypes in the placental villi. A high proportion of euploid cells in a mosaic placenta can yield a false-negative result by masking the presence of aneuploid cells. Since negative NIPT results are rarely followed by confirmatory diagnostic tests, such aneuploid fetuses often go undetected. This issue is particularly relevant in cases of trisomies 13 and 18 but is rare in trisomy 21. Data from Japan suggest that false negatives are extremely rare, occurring at a rate of approximately 0.01%.[42]

Limited Scope of NIPT

It is well established that the rarer the disease being tested, the lower the test's performance and the higher the false positive rate.[43] NIPT is widely recognized for its high sensitivity and specificity in detecting the more common trisomy 21.[21] However, its performance is less accurate for comparatively rarer trisomies 13 and 18. Its accuracy is substantially diminished for detecting rare autosomal trisomies (RATs), microdeletions, and sex chromosome aneuploidies (SCAs).

RATs refer to autosomal trisomies other than trisomies 13, 18, and 21, with an incidence ranging from 0.18 to 0.23%.[44]

Owing to their rarity, even with the high sensitivity of NIPT, the likelihood of false positives is higher compared to more common trisomies like trisomy 21. When evaluating the accuracy of a test for rare conditions like RATs, the PPV is the most critical metric. For NIPT, the PPV for detecting RATs is extremely low, typically ranging from 4 to 6%. This indicates that a substantial proportion of positive results for RATs from NIPT may be false positives.[45]

The PPV for detecting microdeletion/microduplication syndromes is also low. For DiGeorge's syndrome, false positive rates of 50% have been recorded.[46] Hence, according to German standards, NIPT should not be used to screen for microdeletion/microduplication disorders.[47]

The accuracy of NIPT in predicting fetal sex chromosome abnormalities (SCAs) is similarly poor, with a very high false positive rate. Some studies have reported a false positive rate of around 91% for monosomy X, which means that a large majority of positive results for monosomy X on NIPT may be false positives. However, the accuracy for detecting other SCAs like XXX or Klinefelter's syndrome can be higher, with a lower false positive rate.[48]

Detecting single gene disorders through NIPT is difficult due to the unique biological characteristics of the placenta. Trophoblast cells, which provide the DNA for NIPT, have higher mutation rates than fetal cells.[49] Moreover, the placenta's clonal processes, which resemble those in cancerous tissues, make it harder to detect single gene mutations. These factors can contribute to high false positive rates in NIPT for single gene disorders. Another challenge arises when the mother and fetus share identical alleles, making it difficult for NIPT to distinguish between their sources.[6] Thus, paternally inherited and de novo autosomal dominant mutations are easier to detect using NIPT[50]

NIPT in Twin Pregnancy

Twin pregnancies, which occur in approximately 1 in 33 births, have become more common due to advanced maternal age and increased use of assisted reproductive technologies (ART).[51] Multifetal pregnancies often pose higher risks of adverse outcomes and complications, thereby increasing the cost of care.[52] [53]

The risk of aneuploidy in twin pregnancies is influenced by zygosity. The age-specific risk of aneuploidy in dizygotic twins is approximately double that of singleton pregnancies. However, the real-world incidence is lower due to higher rates of affected pregnancy loss.[43] [54] Traditional screening methods, such as combined serum and ultrasound, show higher false positive rates and lower detection rates in twins compared to singletons.[55] This is due to the inability of maternal serum markers to differentiate between the monozygotic and dizygotic twins and the confounding effects of ART on markers like PAPP-A and free β-hCG.[56] [57] Monochorionic twins also face higher false positive rates for nuchal translucency screening compared to dichorionic twins.

Additionally, invasive procedures like CVS or amniocentesis in twin pregnancy carry a higher miscarriage risk, although recent data suggest this risk may not be as significant as previously thought.[58] [59] However, this perception deters many women from pursuing these diagnostic options.[58] [59] [60]

In this context, NIPT has emerged as a superior screening tool for aneuploidies in twin pregnancies. Available from late in the first trimester, it reduces the need for invasive procedures and has been endorsed by the American College of Obstetricians and Gynecologists (ACOG)[61] and the International Society for Prenatal Diagnosis (ISPD).[62]

NIPT in Monozygotic Versus Dizygotic Twins

cfDNA offers significant advantages over traditional maternal serum testing in twin pregnancies. The presence of two fetuses results in a higher total cfDNA concentration compared to singleton pregnancies, although it is not quite double.[63] [64] [65] [66] Because of the overall higher concentration and the nearly universal homozygotic genotype of both fetuses, NIPT for genetic disorders in monozygotic pregnancies theoretically should be at par with or even better than that for singletons.[67]

NIPT for dizygotic twins is complex. Each dizygotic twin contributes unique cfDNA and distinct fetal fraction (usually less than that of a singleton). cfDNA from each of the twin needs be analyzed to provide individual risk assessment. This is important as aneuploidy usually affects only one of the fetuses. Here single nucleotide polymorphism (SNP) based NIPT is advantageous. SNPs are the specific variations in the DNA sequences that can be analyzed to determine zygosity, distinguish between maternal and fetal cfDNA, allow assessment of DNA contributed by each dizygotic twin, and measure individual fetal fraction.[67]

The use of SNP-based methods to reliably differentiate between monozygotic and dizygotic twins has implications for prenatal care. For instance, detecting dizygosity suggests a high likelihood of dichorionic pregnancy, while confirmed monozygosity could indicate either monochorionic (75%) or dichorionic (25%) pregnancy. Such distinctions help tailor care, especially when selective fetal growth restriction and structural abnormalities are detected, by limiting the use of CVS or amniocentesis to the abnormal fetus alone.

Limitations of NIPT in Twins

NIPT performance for trisomy 21 in twins aligns closely with singleton pregnancies, despite a higher rate of test failures in twins, often due to low fetal cfDNA fractions. Sensitivity for trisomy 21 in twins has been reported at 99%, while specificity reaches 99.91%.[61] [68] However, there are limited data for trisomies 13 and 18, with results aligning with expectations for singleton pregnancies. Some laboratories restrict testing to monosomy X or avoid sex chromosome aneuploidy screening in twins due to high mosaicism rates and maternal age-related false positives.[35] [69]

Factors such as in vitro fertilization (IVF) conception, higher maternal weight, and gestational age under 12 weeks are associated with reduced fetal cfDNA fractions, further challenging NIPT reliability in twins.[70]

Vanishing Twin Syndrome and Confounding Factors

VT syndrome, where one twin is lost early in pregnancy, complicates cfDNA analysis. Residual cfDNA from the vanished twin can skew NIPT results, leading to false positives. This phenomenon is particularly problematic in sex chromosome aneuploidy detection, where mosaicism further complicates interpretation.[71] [72] National guidelines vary on managing NIPT in VT cases. For example, the UK National Health Service advises against NIPT in VT pregnancies, while ACOG recommends diagnostic testing instead.[4]

Improving Screening Outcomes

To optimize NIPT in twins, genetic counseling and follow up care for intermediate-risk populations (comprising 8–9% of the maternal serum screening population) must be enhanced.[73] This group, twice as large as the high-risk group, requires sufficient testing to avoid missed diagnoses. Additionally, zygosity testing in twins with structural abnormalities could focus invasive testing on the affected fetus alone, improving care efficiency. By addressing these complexities, NIPT can continue to evolve as a valuable tool in managing multifetal pregnancies, complementing invasive diagnostics where necessary and providing reliable, noninvasive screening options.

Discussion

Expectant parents often worry about their unborn child's health, and medical advancements over the decades have provided tools to address these concerns. These include prenatal ultrasound, maternal serum biochemical analysis, and invasive procedures like amniocentesis and CVS. Since its introduction in 2012, NIPT has emerged as a transformative addition to prenatal diagnostics.

Initially developed to screen for common aneuploidies such as trisomies 21, 18, and 13, NIPT demonstrates high sensitivity and specificity in high risk populations. However, its use has expanded rapidly, including screening low risk populations for major aneuploidies. Many laboratories have extended their test menus to cover RATs, microdeletions, duplications, sex chromosome aneuploidies (SCAs), and single gene disorders. This expansion has created a perception that NIPT could replace prenatal imaging and invasive testing. However, it is crucial to maintain a balanced perspective: one that acknowledges its significant advantages while also addressing its methodological limitations and appropriate scope of use.

Although marketed as a test of cfDNA, NIPT primarily analyses placental DNA. This distinction means that various factors can lead to false positive or false-negative results. Thus, it needs to be emphasized that NIPT remains a screening test, not a diagnostic test.[74]

False positive results are particularly troubling, as they may lead to pregnancy terminations without confirmatory invasive testing.[75] [76] [77] For example, approximately 85% of women with a positive result for RATs and 10% with a positive trisomy 21 result could be misled. Many women who experience false positives report that they would not choose NIPT again.[78]

In real-world practice, the test failure (no call) rate—often excluded in studies—significantly affects NIPT performance. Failed tests, even after retesting, often necessitate invasive procedures for medical and psychological reasons.[34] This increases the false-positive rate for trisomy 21, reducing NIPT's PPV. Meta-analyses report that in general populations, NIPT sensitivity drops to 96% for trisomy 21, 87% for trisomy 18, and 77% for trisomy 13.[79]

Consequently, it is contended that NIPT for trisomies 13 and 18 no longer meets international screening standards.[1]

Similarly, the high rate of false positives for microdeletions, microduplications, and SCAs has led to an increase in invasive testing rather than its intended reduction.

Additionally, pregnant women undergoing NIPT often experience higher stress and anxiety levels than those who do not. Genetic counseling and psychological support are essential, as over one third of women report persistent anxiety even after negative results, and around 7% regret undergoing the test.[21] [76]

An unresolved issue in NIPT trials is the lack of follow up data on “abnormal newborns” not accounted for in studies. Conditions such as RATs, SCAs, microdeletions, monogenic disorders, and teratogenic or idiopathic issues remain largely unreported, raising concerns about research comprehensiveness.

The most effective method for detecting chromosomal imbalances is a combination of chromosomal microarray and amniocyte banding cytogenetics.[80] [81] While these methods require invasive procedures, the traditionally cited fetal loss risk of 1% has been updated to less than 1 in 1,000.[5] [82] Therefore, the argument that abortion risk justifies prioritizing NIPT over invasive diagnostics is no longer valid.

Conclusion

NIPT has revolutionized prenatal screening by offering a safer and more accurate method for detecting chromosomal abnormalities. As technology advances, its clinical applications continue to expand, enhancing early detection and informed decision making. However, ethical considerations, accessibility, and result interpretation remain crucial factors in its widespread implementation.

Conflict of Interest

None declared.

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Publication History

Article published online:
30 April 2025