Non-invasive prenatal testing (NIPT): limitations on the way to become diagnosis

Introduction

Fetal aneuploidy and other chromosomal aberrations affect 9 out of 1000 live births, due to various mechanisms and advanced maternal age [1, 2]. In women identified by biochemical and/or sonographic screening to be at increased risk for such aneuploidies, prenatal diagnosis then involves invasive testing by chorionic villus sampling or amniocentesis. The gold standard for prenatal diagnosis of chromosome abnormalities is the conventional cytogenetic analysis using culture of fetal nucleated cells retrieved mostly by amniocentesis or chorionic villus biopsy. However, invasive testing carries a 1% risk of causing miscarriage.

The 1997 discovery of free fetal DNA in maternal plasma launched scientists’ efforts to establish a reliable method for non-invasive prenatal diagnosis [3]. Several recent studies have demonstrated that the most effective screening method for trisomy 21, with a detection rate of more than 99% and false-positive rate of about 0.1%, is derived from examination of cell-free DNA (cf DNA) in maternal plasma [4–13].

Current prenatal diagnosis

Prenatal diagnosis is a medical procedure that employs a variety of techniques to determine the health and condition of an unborn fetus. The knowledge gained prevents an untoward outcome either for the fetus or the mother considering the fact that congenital anomalies account for 20%–25% of perinatal deaths. Specifically, prenatal diagnosis is helpful for:

• Managing the remaining weeks of the pregnancy

• Determining the outcome of the pregnancy

• Planning for possible complications with the birth process

• Planning for problems that may occur in the newborn infant

• Providing parents the chance to “prepare” psychologically, socially, financially, and medically for a baby with a health problem or disability, or for the likelihood of a stillbirth

• Deciding whether to continue the pregnancy

• Finding conditions that may affect future pregnancies

There is a spectrum of techniques that are used for prenatal diagnosis, each one performed at a different level of invasiveness and sampling time. The available methods are presented in Table 1. A relatively reliable non-invasive screening approach is the combination of first trimester Pregnancy-associated Plasma Protein A (PAPP-A) and free β-subunit of Human Chorionic Gonadotropin (free-β-HCG) biochemical measurements with nuchal translucency that can detect, e.g., 88% of trisomies 21 with a 5% false-positive rate [14]. Prenatal diagnosis of chromosome abnormalities is performed by conventional cytogenetic analysis using in vitro culture of fetal nucleated cells retrieved by amniocentesis, chorionic biopsy or fetal blood sampling. The need for rapid prenatal diagnosis led to the use of the FISH assay; however, the cost of this procedure has limited its application in specific cases [3]. The quantitative fluorescent PCR (QF-PCR) test allows prenatal diagnosis of major numerical abnormalities of chromosomes 21, 18, 13, X and Y in a few hours after sampling [15]. Chromosomal microarray analysis is a technique that identifies chromosomal abnormalities, including submicroscopic ones that are too small to be detected by conventional karyotyping [16]. This analysis requires direct testing of fetal tissue and thus can be offered only with chorionic villus sampling or amniocentesis. This analysis is most beneficial when ultrasonographic examination identifies fetal structural anomalies. However, the potential of complex results and detection of clinically uncertain findings can result in substantial patient anxiety. This underscores the critical need for patient pretest and post test genetic counseling about the benefits, limitations and results of testing.

Cell-free DNA

Non-invasive prenatal testing (NIPT) uses fetal genetic material obtained from a maternal blood sample to detect certain genetic conditions during pregnancy. Current literature often refers also to NIPT as non-invasive prenatal diagnosis (NIPD). This terminology may be misleading given that, at the time of this writing, the technology is recommended only as a highly specific screening measure, which requires follow-up diagnostic testing.

The first evidence for circulating nucleic acids in the peripheral blood was shown by Mandel and Metals in 1948 [17]. The identification of fetal cells from maternal blood by Bianchi et al. in 1997 [18] and ways to isolate them [19] have paved the way for NIPT until 1997, when the group of Dennis Lo reported for the first time the presence of circulating cell-free fetal DNA (ccffDNA) in the plasma of pregnant women [4].

It is essential to elucidate on the origin of the amounts of cell free DNA in maternal plasma (Figure 1). Circulating cell-free fetal DNA originates from the trophoblasts making up the placenta [20] and can comprise approximately 3–13% of the total cell free DNA in most of the samples [21]. Studies have shown that ccffDNA can first be observed as early as 4 gestation weeks and the amount of ccffDNA increases as the pregnancy progresses [22] but diminishes quickly after the birth of the baby, so that it is no longer detectable in the maternal blood approximately 2 h after birth. The fetal DNA is significantly smaller than the maternal DNA in the bloodstream, with fragments with a median size of 146 bp and makes its way into the maternal bloodstream via shedding of the placental microparticles into the maternal bloodstream [23]. Fetal ccffDNA is heavily diluted with maternal plasma and this “fetal fraction” is positively correlated with gestational age while preliminary evidence suggests that it is negatively correlated with maternal weight [7, 24, 25]. In general, the fetal fraction percentage has to be provided and to be estimated with real-time quantitative PCR [22, 26]: over 8% is considered satisfactory for most NIPT methods, while a 4–8% is considered marginal [11]. There are now research efforts ongoing for the extraction of the fetal DNA from the maternal plasma based on its size in order to distinguish it from the maternal DNA and/or accurately estimate it [23].

Figure 1:

Origin of cell free DNA.

The limited amounts of cffDNA as well as the facts that it co-exists with maternal DNA and shares 50% identical sequences posed major challenges toward the development of NIPT. In 2008, two research groups applied the novel massive parallel sequencing (MPS) technology for the first time in maternal plasma in order to detect an overrepresentation of material from chromosome 21 in pregnancies affected with trisomy 21 (Down syndrome) [24, 27]. Another study then, demonstrated the ability of MPS of maternal plasma to detect fetal trisomy 21 with a near 99% sensitivity and specificity in high-risk pregnancies, defined by maternal age, family history or positive serum and/or sonographic screening tests [7]. The group then published an analysis from the same study demonstrating the detection of trisomy 18 (Edwards syndrome) at 100% sensitivity with a false-positive rate of 0.28%, and trisomy 13 (Patau syndrome) at 91.7% sensitivity with a false-positive rate of 0.97% [28]. The overall detection rate for trisomy 13, 18, and 21 was reported as 98.9% sensitivity with a false-positive rate of 1.4%. Bianchi et al. also examined the use of MPS in maternal serum of high-risk pregnancies, using a slightly different algorithm for analysis [12]. In this study, NIPT detected trisomy 21 with 100% sensitivity, trisomy 18 with 97.2% sensitivity, and trisomy 13 with 78.6% sensitivity – all with a specificity of 100%. They also reported monosomy X detection with 93.8% sensitivity and 99.8% specificity. Sparks et al. proposed sequencing analysis, for selected loci from specific chromosomes of interest in cell-free DNA from maternal as a more efficient option for NIPT [29]. A multicenter cohort study from Norton et al., evaluated the performance of chromosome-selective sequencing on chromosomes 21 and 18 in a population of women undergoing CVS or amniocentesis for any indication [11]. Using a predefined cut-off value of 1 in 100 (1%) for classifying a sample as high risk vs. low risk, the sensitivity and specificity for trisomy 21 were 100% and 99.97%. The sensitivity and specificity for trisomy 18 were 97.4% and 99.93%. For trisomy 13, Ashoor et al. reported 80% sensitivity and 99.95% specificity [10]. These studies validate NIPT as a reliable screen for trisomies 21, 13, and 18 and monosomy X in high-risk pregnancies. A blinded validation study from the group of K.H. Nicolaides in 2013, has demonstrated that ccffDNA testing in maternal blood using targeted sequencing of SNPs at chromosomes 13, 18, 21, X, and Y and use of the specific algorithm holds promise as an accurate method for detecting fetal autosomal aneuploidies, sex chromosome aneuploidies and triploidy in the first trimester of pregnancy [30].

The medical significance of the development of NIPT is of great importance as it could potentially be offered to all pregnancies (assuming that it will become cheaper in the future), it presents no risk of pregnancy loss and it provides a more effective prenatal diagnosis compared to currently-used invasive methods.

Available NIPT techniques

As mentioned earlier, the overall amount of fetal ccffDNA is small (<1 μg in 20 mL whole blood) and there are still no reliable routine methods to separate fetal from maternal ccffDNA, precluding fetal ccffDNA isolation prior to analysis [9, 31–33]. Thus, detection of fetal chromosomal copy number based on ccffDNA analysis requires polymerase chain reaction (PCR) amplification and analysis of the full (maternal+fetal) ccffDNA complement [21]. In most of the available methods, reactions can be prepared by massive parallel anchoring of numerous amplified molecules in a next generation sequencing (NGS) platform [24, 27]. Thus, the number of DNA molecules that can be sequenced and consequently the amount of information produced is dramatically increased when compared to “first-generation” Sanger sequencing methods [34, 35]. There are two different approaches in this concept: quantitative (either shotgun or targeted) and qualitative targeted single nucleotide polymorphism (SNP)-based methods [36]. A different approach originates from the epigenetics field: the methylation DNA immunoprecipitation (MeDIP) combined with real time qPCR is based on the identification of differentially methylated regions (DMRs) and their use in discriminating normal from abnormal cases [37]. All NIPT methods have several advantages and limitations that are analyzed in depth in the following section:

Epigenetic-based NIPT approaches

MeDIP (methylated DNA immunoprecipitation) employs a 5′- methyl cytidine specific antibody to capture methylated sites and was originally used to study levels of DNA methylation [48]. Then, this methodology was employed by Papageorgiou et al. in 2009 with the aim of investigating and identifying differentially methylated regions (DMRs) between placenta and female peripheral blood towards the development of a NIPT test [49]. They used MeDIP in combination with chromosome-specific high-resolution oligo arrays for the investigation of the methylation pattern of chromosomes 13, 18, 21, X and Y. More than 10,000 DMRs were identified for chromosomes 13, 18, 21, X and Y.

To provide chromosome dosage information, the ccffDNA has to be hypermethylated compared to the maternal DNA. This is crucial in order to achieve fetal specific methylation enrichment, which was the key to their study. They selected a subset of DMRs on chromosome 21 and after performing real-time quantitative PCR they were able to discriminate normal from trisomy 21 cases [37]. In addition to the evaluation of the fetal specific methylation ratio values at each site, they provided an equation by linear regression statistical analysis that incorporates the best eight DMRs and therefore increasing the discriminating power (Figure 2):

Figure 2:

Schematic illustration of the fetal-specific DNA methylation ratio approach (reprinted after permission from [37]).

D=−6.331+0.959X_EP4+1.188X_EP5+0.424X_EP6+0.621X_EP7+ 0.028X_EP8+0.387X_EP10−0.683X_EP11+0.897X_EP12 (where: D is the discriminating value, X is the methylation ratio value arising from real-time qPCR C_T raw data_, Ep the 8 DMRs that can efficiently discriminate normal cases from trisomy 21). When D>0 the case is assigned as trisomy, when D≤0 the case is assigned as normal [37]. Then they studied 100 cases of pregnancy, which included 25 trisomy 21 pregnancies and the results demonstrated 100% sensitivity and 99.2% specificity [50].

The MeDIP assay can tolerate sample impurities – and thus, no prior sample purification is required – and is not affected by the amount of ccffDNA or fetal gender or the presence of informative polymorphic sites as it may happen to SNP-related methods [50, 51]. It can be applicable for low starting DNA templates, generating sufficiently enriched outputs, a development that renders possible its implementation with plasma samples [52]. Moreover, it is a technically robust methodology, available in most diagnostic labs, easy to use and affordable.

Nevertheless, the efficiency and performance of MeDIP greatly depends on determining the ideal combination of affinity reagents. This is very important, especially in regions with varying methylcytosine density such as the DMRs identified for the NIPT of common chromosomal aneuploidies [53]. However, an appropriate combination of DMRs should provide an accurate NIPT of normal and trisomy cases. Further refinements are anticipated with the inclusion of new DMRs in the algorithm, or removal of others due to the presence of copy number variations (CNVs) in their locus. In the future, it should be possible to expand this method for NIPT of aneuploidies of chromosomes 13, 18, 21, X. However, technical concerns have been raised already by other groups regarding the validity of this method [54] while others have suggestions to improve it [55].

A very recent interesting development in using DNA methylation for NIPT has been the implementation of sodium bisulfite DNA treatment in combination with NGS [35]. This chemical treatment of the DNA is associated with a high degree of DNA degradation in >90% of the template DNA [34]. So the accuracy and sensitivity of the test will be reduced because during pregnancy, the amount of fetal DNA in maternal plasma is already very low and further degradation will result in even fewer fetal DNA available for quantification [34, 35]. On the other hand, manipulations for removal of sodium bisulfite will bias the correct interpretation of the results. Additionally, the assay can only evaluate the methylation status of a specific and very limited number of genomic sequences; only those that include recognition site of a methylation-dependent restriction enzyme.

Non-invasive prenatal test in clinical practice

The American College of Obstetricians and Gynecologists Commitee on Genetics and the Society for Maternal-Fetal Medicine Publications Committee, have recommended that women be offered prenatal assessment for aneuploidy either by screening or invasive prenatal diagnosis regardless of maternal age; testing for ccffDNA is an option that can be considered as a primary screening test in women at increased risk of aneuploidy with the following criteria [56]:

• Maternal age 35 years or older at delivery

• Fetal ultrasonographic findings indicating an increased risk of aneuploidy

• History of a prior pregnancy with a trisomy

• Any positive test result for aneuploidy, including first trimester biochemical combined or sequential, integrated or quadruple screen, e.g., in combination with 2nd trimester AFP, uE3 and HCG α-test results [14]

• Parental balanced Robertsonian translocation with increased risk of fetal trisomy 13 or trisomy 21

• Follow-up confirmatory test for women with a positive first-trimester or second-trimester screening test result.

Pre-test counseling regarding the limitations of ccffDNA testing is recommended and should include a discussion with a clear statement that this test provides information solely for trisomy 21 and trisomy 18 and, in some laboratories, trisomy 13. The use of a non-invasive test should be an active, informed choice and not part of a routine prenatal laboratory testing. The family history should be reviewed to determine if the pregnant woman should be offered other forms of screening or prenatal diagnosis for a particular disorder. A baseline ultrasound examination may be useful to confirm viability, a singleton gestation, gestational dating, as well as to rule out obvious anomalies.

This technology can be expected to identify approximately 98% of cases of Down syndrome with a false-positive rate of <0.5%. Because false-positive test results can still occur, confirmation with amniocentesis or CVS is recommended. In general, referral for genetic counseling is suggested for pregnant women with NIPT positive test results. Patients also need to be aware that a negative test result does not ensure an unaffected pregnancy; false-negative test results can occur as well. In the high-risk population, a second-trimester ultrasound examination is suggested to evaluate pregnancies for structural anomalies. In patients in whom a structural fetal anomaly is identified, invasive diagnostic testing should be offered because a ccffDNA test can only detect major trisomies at the moment. Maternal serum α-fetoprotein screening or ultrasonographic evaluation for open fetal defects should continue to be offered.

At the moment, it should not be offered to low-risk women or women with multiple gestations because it has not been sufficiently evaluated in these groups. CcffDNA testing does not replace the accuracy and diagnostic precision of prenatal diagnosis with karyotyping of either CVS or amniocentesis material, which remain an option for women. Analogous guidelines exist from the American College of Medical Genetics and Genomics [47] and the International Society for Prenatal Diagnosis [57].

Available commercial NIPT testing

CcffDNA-based NIPT of aneuploidy by using NGS approaches is the first NIPT method that appeared in clinical practice and detects trisomy with high accuracy. Through the continuous development and improvement of algorithms for data sequencing and analysis, many commercial tests are now available and are presented in chronological order in the following text:

Pre-analytical conditions

All tests are usually performed on maternal whole blood drawn in specific tubes: ccffDNA BCT™ tubes (Streck), since they have been proved to minimize increases in background DNA levels caused by temperature fluctuations or agitation that can occur during blood sample storage and shipping [55].

The risks, benefits, alternatives and limitations of the testing as well as a description of the population used for clinical validation should be explained to the pregnant woman after counseling and a signed informed consent for NIPT testing should accompany the sample. The family history should be reviewed to determine if the patient should be offered other forms of screening or prenatal diagnosis for a particular disorder. Gestational age (GA) at the time of blood draw should be noted only in whole weeks as this is the kind of values employed from the databases for this analysis [64].

Quality assurance

The clinical use of non-invasive prenatal testing to screen high-risk patients for fetal aneuploidy is becoming increasingly common. Guidelines on the use of this testing in clinical practice have been published; however, data on actual test performance in a clinical setting are lacking, and there are no guidelines on quality control and assurance. The different non-invasive prenatal tests employ complex methodologies, which may be challenging for health-care providers to understand and utilize in counseling patients, particularly as the field continues to evolve. How these new tests should be integrated into current screening programs and their effect on health-care costs remain uncertain.

Most of the described technologies for genetic analysis of cffDNA have been patented or exclusively licensed to a small number of companies in the US. Patenting can have both positive and negative effects on the availability of tests, clinical adoption and patient access to NIPT. Perceived or real intellectual property barriers could reduce market competition, limit quality assurance and improvement mechanisms, decrease availability of alternative or cheaper tests and reduce eventually the cost effectiveness of NIPT. However, it is assumed that soon these technologies will be offered by a plethora of academic and medical institutions around the world using in-house NGS or other NIPT methodologies or as reagents to be sold separately (e.g., the recent CE-IVD Premaitha Iona test reagent).

American College of Medical Genetics and Genomics (ACMG) recommends compliance with the standards and guidelines for clinical genetics laboratories. Considering the nature of the methods used, NIPT is subject to the same internal and external quality control requirements as those for clinical molecular laboratory tests (accreditation). Quality control should assess the entire test process, including pre-analytical, analytical and post-analytical phases. Appropriate internal quality controls, e.g., stabilized plasmas with different levels of ccffDNA percentages and at different maternal ages should be constructed and analyzed periodically. Until external proficiency testing schemes sponsored by professional or regulatory organizations are available, alternative methods for proficiency testing preferably by using an interlaboratory comparison method should be required. Test performance characteristics should be available to patients and providers assessing testing, e.g., the National Accreditation bodies [47].

Other NIPT purposes

Besides the main use in detection of trisomies 21, 18 13 and monosomy X or other sex chromosomal aneuploidies (XXX, XYY, XXY), NIPT can be used for the determination of other fetal characteristics:

Limitations

The clinical use of NIPT to screen high-risk patients for fetal aneuploidy is becoming increasingly common. Several studies have demonstrated high sensitivity and specificity and there is hope that these tests will result in a reduction of invasive diagnostic procedures as well as their associated risks. Chiu et al. identified that if referrals for amniocentesis or CVS were based on NIPT results, approximately 98% of the invasive diagnostic procedures could be avoided [5]. Guidelines on the use of this testing in clinical practice have been published. The different non-invasive prenatal tests employ complex methodologies, which may be challenging for healthcare providers in counseling patients, particularly as the field continues to evolve. How these new tests should be integrated into current screening programs and their effect on health-care costs, need further study [77].

There is widespread agreement that these tests are not yet ready to replace invasive diagnostic procedures because of many limitations. The main is that they currently detect only a subset of the chromosomal abnormalities that are uncovered by using the standard invasive procedures. The sensitivity and specificity of NIPT varies per tested chromosome and with bioinformatics corrections. This is due, at least in part, to differing GC content. The result of the test does not eliminate the possibility of other anomalies of the tested chromosomes like mosaicism, deletions or duplications. Therefore, when fetal anomalies are detected, invasive diagnostic testing and cytogenomic microarray analysis are more likely to detect these chromosomal imbalances than NIPT and may be a better testing option. Concerning aneuploidy of other chromosomes, molecular or congenital anomalies including neural tube defects are not excluded with NIPT. This is why maternal AFP testing should still be offered at 15–20 weeks gestation in order to screen for open neural tube defects even when NIPT is performed. NIPT does not screen for single-gene mutations at the moment. Sex chromosomal aneuploidies are not reportable usually for known multiple gestations. In some rare cases, false-positive or negative results may occur due to mosaicism in the fetus, placenta or mother, or due to a vanishing twin. It is likely that in real clinical practice the percentages of false-negative or positive may be higher than reported in carefully controlled, small clinical trials. NIPT is less reliable if pregnancy is <12 weeks and so far, it is impossible if pregnancy is <10 weeks. In a small number of pregnancies, due to too little DNA from the fetus in the maternal blood, the NIPT test cannot be performed, neither on pregnancies in which the mothers has had a prior bone marrow transplant. Thus NIPT is not a perfect test and it does not replace the accuracy obtained with diagnostic tests, such as chorionic villus sampling (CVS) or amniocentesis and currently it does not offer other genetic information than regarding the main numerical aneuploidies [66].

Additionally, they have largely been validated in high-risk populations. Indeed, ACOG recommends testing for high-risk women, stating that more research is required to determine how these tests perform in low-risk populations [47]. Perhaps most significantly, the effects of mosaicism (fetal or placental) or other events such as trisomy rescue are unknown, and discordant results have already been reported. There is lack of outcome data for low-risk populations. Studies examining the use of NIPT in screening low risk populations have included between 289 and 2049 patients. So far, ccffDNA testing is not recommended for low-risk women.

Preliminary data available on twins demonstrate accuracy in a very small cohort, but more information is needed before use of this test can be recommended in multiple gestations. Limited data are currently available on the use of NIPT in twins and higher-order pregnancies. When there has been an early demise of a co-twin (“vanishing twin”) results may be inaccurate [57].

Furthermore NIPT is not able to distinguish specific forms of aneuploidy. For example, NIPT cannot determine if Down syndrome is due to the presence of an extra chromosome (trisomy 21), a Robertsonian translocation involving chromosome 21, or a high-level mosaicism. In cases where mosaicism is present (including confined placental mosaicism) results may be inaccurate [78]. Identification of the mechanism of aneuploidy is important for recurrence risk counseling and emphasizes the importance of confirmatory testing following NIPT: in many studies this would be CVS. However, CVS analyzes a small sample of placenta tissue and it is possible that the incidence of placental mosaicism may be higher [77].

The studies on NIPT also assume a normal maternal karyotype. In very rare occasions low-level maternal mosaicism or the presence of a maternal solid tumor will result in variations in the maternal contribution to circulating ccffDNA, which may also impact NIPT results.

Currently, it takes longer for NIPT test results to be returned than for test results on biochemical maternal serum analytes. Providers should keep this in mind when offering patients NIPT if timing is important for reproductive decision making. In most cases, NIPT is offered between 10 and 20 weeks gestation, which allows sufficient time for follow up of positive test results.

Biologic factors such as a mother’s obesity expressed by a high body mass index and early gestational age are associated with reduced available cell-free fetal DNA: in a small percentage of cases, a cell free fetal DNA result will not be able to be obtained. Uninformative test results due to insufficient isolation of cell-free fetal DNA could lead to a delay in diagnosis or eliminate the availability of information for risk assessment [77].

There might be reasons to be concerned about the use of cell-free fetal DNA testing for sex selection, especially in areas where gender imbalance is already widespread. Even if there are laws against sex selection, it would be relatively easy to obtain a blood sample and also relatively easy to send it out of the country and e-mailed a result back. It is certainly worrisome. Companies that offer testing will have to think about how they’re going to determine whether the samples are being used for things that are actually illegal in other countries; it may be their obligation to ensure that they’re not contributing to illegal behavior.

Last but not least, is the fact that the majority of the published literature on the performance of NIPT includes authors affiliated with or funded by commercial laboratories currently offering NIPT on a clinical basis or by companies that are financially investing in the NIPT field [77].

Conclusions

NIPT is one of the most fascinating research areas in molecular medicine of the last decade; however, as with most new technologies, there is room for refinement. The technology will become cheaper, more cost-effective and therefore, even comparable to the biochemical screening tests. Large studies are needed to explore the possibility whether the addition of the cheap PAPP-A 1st trimester screening (without NT) to NIPT could obliterate the need for a definite diagnosis with karyotype in either negative or positive results. NIPT technology is perhaps only a few steps away from an eventual whole genome (or whole exome) fetal sequencing from non-invasively isolated ccffDNA [79]. Where or if it becomes possible to do whole fetal genome analysis in a clinical setting routinely, it will open up the possibility that people can get information about the fetus that is well beyond a handful of fetal conditions, such as trisomies. This raises the concern that people will be faced with a huge amount of information about which there might be a lot of uncertainty and lack of clinical significance. Moving from a limited set of conditions to potentially any kind of human trait that has a major genetic component could really change the way people think about pregnancy and prenatal testing. After having reached the point of diagnosing the fetal diseases in such an early time point, the next step that it could be considered is the prenatal treatment in utero with the least invasive procedures (e.g., with gene editing new available tools).

NIPT should be offered in the context of informed consent, education, and counseling. Patients whose results are abnormal, or who have other factors suggestive of a chromosome abnormality, should receive genetic counseling and be given the option of standard confirmatory diagnostic testing [47].

Developments in proteomics to detect multiple novel biomarkers could also provide a cheaper screening alternative to NGS approaches in NIPT but will most likely display a reduction in sensitivity. However, new biomarkers can only be used for screening purposes, whereas NGS directly identifying fetal DNA can provide – maybe in conjuction with ccffDNA size ratio and epigenetic approaches – real NIPD that could potentially replace current invasive prenatal diagnosis techniques. With the continuous decline in the costs of all the aforementioned tests, NIPD of fetal aneuploidy is an exciting area of research that could become a clinical reality for all pregnancies in the near future.