Clin Genet 2016: 89: 523–530Printed in Singapore. All rights reserved

© 2015 John Wiley & Sons A/S.Published by John Wiley & Sons Ltd

CLINICAL GENETICSdoi: 10.1111/cge.12598

Review

Clinical implementation of NIPT – technicaland biological challenges

Brady P., Brison N., Van Den Bogaert K., de Ravel T., Peeters H., Van EschH., Devriendt K., Legius E., Vermeesch J.R. Clinical implementation ofNIPT – technical and biological challenges.Clin Genet 2016: 89: 523–530. © John Wiley & Sons A/S. Published byJohn Wiley & Sons Ltd, 2015

Non-invasive prenatal testing (NIPT) for fetal aneuploidy detection isincreasingly being offered in the clinical setting. Whereas the majority oftests only report fetal trisomies 21, 18 and 13, genome-wide analyses havethe potential to detect other fetal, as well as maternal, aneuploidies. In thisreview, we discuss the technical and clinical advantages and challengesassociated with genome-wide cell-free fetal DNA profiling.

Conflict of interest

JRV is founder and stockholder of Cartagenia.

P. Brady, N. Brison, K. Van DenBogaert, T. de Ravel,H. Peeters, H. Van Esch,K. Devriendt, E. Legius andJ.R. Vermeesch

Centre for Human Genetics, KU Leuven,University Hospital Leuven, Leuven,BelgiumKey words: cell-free DNA, (cfDNA) –cell-free fetal DNA, (cffDNA) – copynumber variation, (CNV) – genomesequencing – mosaicism –non-invasive prenatal testing, (NIPT) –prenatal diagnosis

Corresponding author: Prof. JorisVermeesch, Centre for HumanGenetics, KU Leuven, UniversityHospital Leuven, Leuven 3000,Belgium.Tel.: +32 (0)16 345941/345904;fax: +32 (0)16 346060;e-mail: joris.vermeesch@uzleuven.be

Received 5 January 2015, revised andaccepted for publication 9 April 2015

During the past 3 years, non-invasive prenatal testing(NIPT) for fetal aneuploidy detection has become aclinical reality. The clinical introduction succeeded themany clinical validation studies which have applied mas-sively parallel sequencing of maternal plasma cell-freeDNA (cfDNA) using either whole-genome sequencing ortargeted-sequencing methods (either chromosome selec-tive or Single nucleotide polymorphism (SNP) based)(1–27). A meta-analysis has been undertaken on recentclinical validation and implementation of NIPT stud-ies (28), which found high sensitivity and specificityfor trisomies 21, 18 and 13: 99% sensitivity and of99.92% specificity for trisomy 21; 96.8% sensitivity and99.85% specificity for trisomy 18; and 92.1% sensitiv-ity and 99.80% specificity for trisomy 13. False-positiverates for trisomy 21, 18 and 13 were 0.08%, 0.15% and0.20%, respectively. A number of clinical studies wereexcluded from this analysis because pregnancy outcomedata were unavailable (29–33), and other studies havebeen published since (19, 26), all report high rates ofsensitivity and specificity.

A number of professional societies including theInternational Society for Prenatal Diagnosis (ISPD), the

American College of Obstetricians and Gynecologists(ACOG) and the Royal College of Obstetricians andGynecologists (RCOG) have issued position statementsprimarily not only on the use of NIPT for trisomy 21detection but also for the other common autosomal aneu-ploidies (trisomy 18 and 13) (34–38). The general opin-ion is that NIPT is a promising new technology whichhas great potential as a screening tool for those pregnantwomen with an increased risk of fetal aneuploidy. How-ever, it is important to stress that NIPT is not a diagnostictest for fetal aneuploidy, and therefore, a positive NIPTresult requires an invasive test to confirm the findings.Furthermore, these societies considered there to be insuf-ficient evidence to support the use of NIPT for screeningin the general population.

Several recent clinical studies have now demonstratedsimilar sensitivity and specificity for aneuploidy detec-tion (primarily trisomy 21 detection) between patientswith prior high or low risks of aneuploidy (16, 21, 23,31, 33, 39). Furthermore, NIPT also resulted in less falsepositives than with conventional methods of aneuploidyrisk assessment. The fetal fraction, i.e. the percentageof cell-free fetal DNA (cffDNA) within the total cfDNA

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sample obtained from maternal plasma, has been shownto be similar in high- and low-risk groups (21, 40). How-ever, the positive predictive values are expected to belower in the group of women at low risk compared withthose at increased risk of aneuploidy. The positive predic-tive values for the general population will also be depen-dent upon the specific test characteristics and furtherstudies are warranted before NIPT is routinely offeredto all women.

While the implementation of NIPT was warrantedbecause of the high accuracy for fetal trisomy 21, 18 and13 detection, several studies have now demonstrated thatgenome-wide analysis enables the detection of other fetaland also possibly maternal (mosaic) aneuploidies (27,41–47). Such genome-wide analysis has the potential toimprove overall pregnancy management and reduce tech-nical and biological factors which could adversely affecttest accuracies. In this review, we discuss the clinicaladvantages and challenges associated with genome-widecirculating free fetal DNA profiling.

Beyond common aneuploidy screening

Several groups have demonstrated the feasibility todetect all fetal chromosomal aneuploidies, segmentalimbalances, and even submicroscopic copy number vari-ations (CNVs) by sequencing cffDNA from maternalplasma (4, 6, 26, 24, 27, 41, 43–45, 48–52). Despitethe promise of detecting other fetal aneuploidies whichmight aid in the interpretation of fetal development, clin-ical implementation of genome-wide NIPT analysis inthe routine clinical setting remains limited (26, 49, 50).

In their prospective series of 1982 clinical cases, Lauet al. detected seven cases of full aneuploidy for chro-mosomes other than 21, 18, 13, X or Y (50). Follow-uptesting and pregnancy outcome were provided for fiveof these, and confined placental mosaicism (CPM) wasconfirmed in four of the five cases. Two pregnancies(including the case unconfirmed) were complicated byfetal growth restriction and delivered at 33–34 weeksgestation. In the two cases without any follow-up inves-tigations, ultrasound was normal, the pregnancies werecontinued, and no abnormalities were reported dur-ing birth. Another recent clinical study of 100,000patients acknowledged that these types of events weredetected, but did not give specific numbers or details offollow-up/outcome for individual cases (19).

Table 1 provides an overview of the different aneu-ploidies we have detected from genome-wide NIPT ina series of 4000 pregnancies [updated from 1350 preg-nancies reported in (26)].

The clinical utility of reporting all aneuploidiesdetected using NIPT, particularly in low-risk preg-nancies, is controversial because this could lead to anincrease in invasive procedures in women for whomthis would not normally be considered. Already, thereporting of trisomy 13 following NIPT in the groupof low-risk women has been questioned because of thelow-positive predicted values expected and a possibleincrease in invasive procedures because of false-positive

NIPT results (53). In an important subset of fetal ane-uploidies detected using NIPT, the aneuploidy may beconfined to the placenta (see below), and therefore, maynot adversely affect fetal development. However, thereis the risk of placental insufficiency and fetal growthrestriction because of the abnormal placental karyotype(54–56), as well as a risk of mosaic fetal aneuploidyand/or fetal uniparental disomy (UPD) because of atrisomy rescue event (57).

Similarly, routine screening for sex chromosome ane-uploidies (SCA) may be of questionable clinical util-ity, given the variability in phenotype of, for example,monosomy X (Turners), XXY (Klinefelter), XYY andXXX individuals, with some only identified as adultsdue to fertility problems (58–61). Moreover, the accu-racy of SCA detection is substantially lower for trisomy21 detection (6, 16, 25). It has been suggested that thephysical anomalies, developmental delay and/or infer-tility warrant analysis of SCA (62, 63). National poli-cies relating to screening for SCA differ from countryto country, and how these are subsequently counseledfor and managed also differ. Cultural and societal factorsplay a role in parental decision to continue or to terminatea pregnancy because of a SCA for which the phenotypicoutcome cannot be predicted (64).

We believe that a genome-wide analysis can lead to abetter clinical management. However, careful considera-tion of reporting policy is essential in order to minimizeincreases in invasive tests because of aneuploidies whichare confined to the placenta. We envision that the detec-tion of an aneuploidy would trigger careful follow-up byboth the obstetricians and the clinical geneticists. In thecase of normal development upon US examination, fur-ther invasive actions may not be warranted. In case ofabnormal growth patterns, those can be evaluated in lightof the NIPT results and the US findings. Further studiesare warranted to evaluate the outcomes of such pregnan-cies which will eventually allow the development of rulesfor the best clinical follow-up actions to be taken.

Fetal CNV detection

Currently, invasive prenatal genetic diagnosis using con-ventional karyotyping is increasingly being replaced bychromosomal microarray analysis enabling detection ofsubmicroscopic CNVs. Several prenatal chromosomalmicroarray studies have estimated the residual risk for apathogenic CNV in the absence of any major ultrasoundanomalies at between 0.5% and 1.7% (65–67). Sub-microscopic CNVs can be a cause of severe childhooddevelopmental disorders which may not present anyfeatures upon the routine US examination. The currentmainstream approaches to NIPT focusing only uponthe detection of common autosomal and SCA willnot detect other imbalances present on the remainingchromosomes. The frequency of chromosome abnor-malities, which would be missed if invasive prenatalchromosomal microarray-based analysis would bereplaced by targeted NIPT (68–70), has been estimatedto be 16.9%, including 2% of those pregnancies deemed

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Table 1. Different aneuploidies detected during 4000 NIPT

Risk group Follow-up invasive testing

Aneuploidy type Total number observed High risk Low risk Confirmed Not confirmed No follow-up

Trisomy 21 38 22 16 29 9Trisomy 18 9 4 5 6 a 1 (AF) 2Trisomy 13 3 1 2 1 b 2Trisomy 1 1 1 1Trisomy 7 3 2 1 2 (AF) 1Trisomy 8 1 1 1 (AF)Trisomy 9 1 1 1 (miscarriage)Trisomy 10 1 1 1Trisomy 15 1 1 1 c

Trisomy 16 1 1 1 (fetal mosaicism)Trisomy 20 1 1 1Trisomy 22 1 1 1 (AF)Partial trisomy 18 2 2 1 1 (AF)Monosomy 20 1 1 1 (AF)Monosomy 22 1 1 1 (AF)Total (21,18,13) 50 27 (54%) 23 (46%) 36 1 13Total (other chromosomes) 15 4 (27%) 11 (73%) 3 7 5Total 65 31 (48%) 34 (52%) 39 8 18

AF, amniotic fluid; NIPT, non-invasive prenatal testing.High risk is classified as those with an increased risk of aneuploidy from the combined test (<1/300) (21%). Low risk (total 79%)includes those with an increased risk of aneuploidy because of advanced maternal age alone (31%) and those referrals because ofmaternal anxiety (46%) or prior family history (2%).aOne of those six samples was shown to be normal on AF and 77% mosaic on CVS analysis for trisomy 18.bA total of 70% mosaic on CVS analysis.cMosaicism for trisomy 15 and UPD 15.

at high risk of aneuploidy based upon abnormal serumscreening results.

Because of the clinical importance of segmentalaneuploidies, several groups are exploring analyticalmethods to increase the resolution achievable usingNIPT for which proof-of-concept has been provided(27, 71). Some commercial NIPT providers [includ-ing Sequenom (San Diego, CA, USA) and Natera(San Carlos, CA, USA)] have started offering theoption of additional testing for a small number ofmicrodeletion syndromes and other autosomal ane-uploidies. One commercial provider (Natera) hasrecently demonstrated proof-of-principle (24) andsubsequent validation study (71) of their targetedSNP-based NIPT assay to include a small number ofmicrodeletion syndromes (including 1p36, Cri-du-Chat,DiGeorge, Wolf–Hirschhorn, Prader–Willi, Angel-man, Miller–Dieker and Phelan–McDermid). Another(Sequenom) has published validation of genome-widefetal CNV detection (27). However, prospective clinicalresults have not yet been published and studies on testaccuracy, specificity and sensitivity are lacking; hence,their commercial offerings are premature.

Biological sources of false-positiveand false-negative NIPT results

Placental mosaicism

The source of cffDNA has been shown to be placentalin origin (72). It is well documented from conventionalcytogenetic examination of Chorion villus sampling

(CVS) tissue that confined placental mosaicism occursin which the placental tissue contains an abnormal cellline which is not present upon subsequent examinationof amniocentesis or other fetal material. This CPM isobserved in around 1% of invasive tests (73). One wouldtherefore expect the rate of discordant results because ofCPM observed from conventional cytogenetic analysisof CVS and amniocentesis to be similar for NIPT andamniocentesis results. The origins and types of CPMare detailed further below because this is an importantbiological source of ‘false-positive’ NIPT results whichcannot be overcome by technical improvements [we referto Chromosome Abnormalities and Genetic Counselingby Gardner & Sutherland, for more information on CPM(73)].

Mitotic and meiotic CPMMitotic CPM arises from a normal diploid zygote, fol-lowing a post-zygotic error in a placental cell lineage.This generally leads to localized regions of placentaltrisomy and low levels of mosaicism upon cytogeneticinvestigation. In contrast, meiotic CPM arises from atrisomic zygote in which a trisomy rescue event hasoccurred during early fetal development. Generally, thefetus is diploid, and the placenta shows high levels ofmosaicism or full aneuploidy. However, there is a riskof mosaicism in the fetus dependent upon when loss ofthe trisomic chromosome occurs and in which embry-onic cell lineage. Additionally, there is a risk for fetalUPD following a trisomy rescue event dependent upon

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which chromosome is lost. The adverse effects of UPDon fetal development may be because of the presence ofimprinted genes or homozygous mutations for the chro-mosome of interest.

Different types of CPM: types I, II and IIICPM is further categorized into one of three typesaccording to the specific cell lineage affected by theabnormal cell line. The abnormal cell line may beconfined to the trophoblast (type I, observed upondirect CVS and short-term culture), the chorionicstroma/mesenchymal core (type II, observed uponlong-term culture), or both these placental lineages(type III). Types I and II are generally of mitotic origin,whereas, type III is primarily meiotic in origin. CPMtype I is associated with an increased risk of spontaneousabortion, intrauterine fetal death, intra uterine growthretardation (IUGR) and perinatal morbidity. CPM typeII is generally associated with a normal pregnancy out-come, with IUGR and pregnancy loss being infrequent.CPM type III is commonly associated with intrauterinefetal death or IUGR, with a large proportion being dueto mosaic trisomy 16 (56, 74–78). Note that cffDNA isderived from trophoblasts, and hence, only types I andIII errors are expected upon NIPT. Large-scale Europeanand US studies of CVS have shown that the chromo-somes involved in different types of CPM observed arenot randomly associated (79–81).

False-positive and false-negative NIPT results becauseof CPM

Unsurprisingly, numerous cases are now reported of‘false-positive’ NIPT results because of CPM (26,39, 50, 82–90), and these findings may be of clinicalrelevance being associated with IUGR for example(50). There is also the possibility of UPD in the fetusfollowing a trisomy rescue event early in embryonicdevelopment. The risk of UPD following prenatal detec-tion of mosaicism for trisomy 15 has been estimated at11–25% (57). We have reported a case in which the fetuswas affected with UPD15 (26). A case of uniparentaldisomy 21 because of trisomic rescue has also beenreported following discordant NIPT and fetal karyotyp-ing results (89). Given the potential for UPD, it may beadvisable to follow up discordant NIPT and invasive testresults with UPD testing, in particular for those chro-mosomes with well characterized imprinted syndromes,including: patUPD(6) – transient neonatal diabetes(TND; OMIM #601410); matUPD(7) – Silver–Russelsyndrome (SRS; OMIM#180860); segmentalpatUPD(11) – Beckwith–Wiedemann syndrome(BWS; OMIM #130650); matUPD(14) – Templesyndrome (TS; OMIM *605636 and #176270);patUPD(14) – paternal UPD(14) syndrome (OMIM#608149); matUPD(15) – Prader–Willi syndrome(PWS; OMIM #176270); and patUPD(15) – Angelmansyndrome (AS; OMIM #105830) (57).

The possibility for CPM means that an amniocentesisis the preferred sampling method for a follow-up con-firmatory invasive test after a positive NIPT result in

order to exclude CPM. As circulating cffDNA is derivedfrom the placental tissue, NIPT results are expected tobe highly concordant with CVS. However, an amniocen-tesis is more reflective of the true fetal genotype. Thisemphasizes that it is essential for an invasive follow-uptest in order to confirm a positive NIPT test. Any deci-sion for termination of pregnancy should not be basedupon positive NIPT results alone.

Multiple pregnancies

It has been demonstrated that NIPT can be used for detec-tion of aneuploidy in twin pregnancies (19, 91–94). Thequantity of cffDNA has been shown to be higher in twinpregnancies compared with singleton pregnancies (95).Zygosity can be determined from maternal plasma DNAsequencing and thus the fetal fraction from each twin canbe estimated in dizygotic pregnancies (96). However, theaccuracy may not be as high as for singleton pregnancies(94).

One possible biological cause of inaccurate NIPTresults may be the presence of a ‘vanishing twin’. Casesof false-positive NIPT results in which the presence of avanishing twin could be confirmed have been reported(25, 26, 32, 50, 97, 98). Furthermore, Curnow et al.estimated the theoretical incidence of a vanishing twinwith a chromosome abnormality to be around 0.11%,which is in line with the false-positive rates reportedin a meta-analysis of NIPT (28). This emphasizes theimportance of detailed ultrasound examination, particu-larly following discordant NIPT results.

Maternal CNVs

NIPT relies upon the analysis of cfDNA derived from thematernal plasma, and the majority of cfDNA is maternalin origin which can complicate the analysis and interpre-tation of NIPT results. The counting statistics of conven-tional Z-score chromosome-wide analysis methods canbe affected by maternal CNVs leading to false-positiveand false-negative NIPT results. The development andclinical application of analysis pipelines which allows fordifferentiation between localized and chromosome-wideevents, including for example maternal CNVs may avoidsuch errors (26). Wang et al. reported 2 of 25 sex chro-mosome anomalies detected by NIPT were determinedto be false positives because of maternal X chromosomeCNVs (99). Another recent study found that maternalCNVs were a major factor contributing to false-positiveand false-negative NIPT results, as well as fetal/placentalmosaicism (39). These findings demonstrate the impor-tance of differentiating between whole chromosome andsub-chromosomal events as well as those which are ofmaternal origin.

Maternal mosaicism

Maternal mosaicism can also be a source of false-positiveresults. Cytogenetic investigations have shown that lossof an X chromosome in blood cells occurs with increas-ing frequency as female age increases (100). This can

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Fig. 1. Mosaic segmental maternal aneuploidy of chromosome 13. (a) Non-invasive prenatal testing (NIPT) for chromosome 13. Using conventionalZ-score analysis, this would be called as a monosomy 13. Genome-wide analysis identifies this as a partial imbalance. The read depth analysis suggeststhis imbalance to be a maternal event. Refer to our previous manuscript for full details of our analysis pipeline. (b) Chromosome 13 array CGH plotderived from DNA extracted from maternal white blood cells. Chromosome 13 is shown at the top, the array result below plotted using genomic locationon the x-axis and signal log ratio on the y-axis, and the deleted region highlighted. The average deviation of signal intensity ratio observed is −0.17,which is consistent with mosaicism at a level of ∼17%. Microarray analysis of DNA from amniotic fluid revealed the fetus to be normal (data notshown). (c) Shows a metaphase fluorescence in situ hybridization (FISH) result using probes for 13q telomere in red [TelVysion 13q (Vysis)] and13q14 in green [LSI 13q14 (Vysis)] showing an abnormal cell which confirms the presence of the deletion in the mother. This deletion was observed in22% of cells analyzed (66/300 cells) confirming mosaicism for the deleted region. FISH analysis of amniotic fluid cells in the fetus was normal (datanot shown).

Fig. 2. Proposed clinical workflow following non-invasive prenatal testing (NIPT). For those women undergoing NIPT because of increased risk ofaneuploidy, in the absence of US anomalies, a normal NIPT result implies that an invasive test is not necessary to exclude fetal aneuploidy. WhereUS anomalies indicative of a chromosomal disorder are observed, invasive testing and microarray analysis (or mutation analysis if appropriate)is offered/recommended even after a normal NIPT result. Following an abnormal NIPT result (for chr 21, 18, 13) invasive testing (preferably byamniocentesis) is required to confirm the finding. A concordant abnormal result on amniocentesis will confirm the fetal abnormality in most cases.However, in some cases the amniocentesis will return a normal fetal result after an abnormal NIPT result, and this may be suspected to be as a result ofconfined placental mosaicism (CPM). In such cases, a maternal karyotype or microarray should be performed to exclude a maternal cause, e.g. maternalcopy number variation (CNV), maternal mosaicism, or in rare cases a maternal tumor. In order to resolve such discordant cases, the sampling of placentaltissue at birth can aid follow-up. Where other autosomal aneuploidies are observed by genome-wide analysis, a detailed US examination along withgenetic counseling will support the patient in deciding to continue with the pregnancy or opt for invasive testing. Furthermore, where CPM is suspected,there is a risk for UPD because of trisomy rescue and it is therefore advisable to undertake UPD analysis, in particular where chromosomes 6, 7, 11,14, 15 or 20 are involved because of the presence of known imprinting disorders. Again, the sampling of placental tissue at birth can aid follow-up ofdiscordant cases or those not undergoing invasive testing. Finally, in the case of (segmental) aneuploidies on >2 chromosomes and identical genomerepresentation profiles upon repeat sampling a maternal tumor may be suspected as a biological cause and referral to oncology for whole body magneticresonance imaging (MRI) is warranted.

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be a source of false-positive monosomy X results fromNIPT. Wang et al. found that 8.6% of NIPT resultspositive for SCA were due to maternal mosaicism (85).Unidentified mosaicism in the mother for other chromo-somes (or chromosome segments) may also be a sourceof false-positive NIPT results. We detected an apparentsegmental loss of chromosome 13 via NIPT, but whichwas subsequently demonstrated to be present in mosaicform in maternal white blood cells and absent in thefetus (Fig. 1).

It is debatable whether cytogenetic analysis shouldbe performed upon maternal cells for all positive NIPTcases, but may be advisable in some circumstances toavoid an unnecessary invasive procedure, and is certainlyadvised in the follow-up of discordant NIPT and invasivetests.

Maternal tumor

Another potential source of abnormal chromosomecomplements in maternal-derived cfDNA is fromapoptosis of tumor cells of maternal origin. Casesof erroneous NIPT results were subsequently shownto be resulting from malignancies (19, 32, 101).The use of genome-wide profiling has the poten-tial to spot ‘tumor like’ genome-wide aneuploidyprofiles and hence identify presymptomatic can-cers in pregnant women (102). Maternal causes forfalse-positive NIPT results also highlight the impor-tance of pre- and posttest genetic counseling. Itshould be made clear to patients beforehand whetherthey will later be informed of incidental findingsobserved.

Future perspective

The next few years will see further widespread adoptionof NIPT leading to a dramatic reduction in inva-sive sampling for prenatal genetic diagnostic tests inmany countries. With reduction in costs and tech-nological and algorithmic advances, the resolutionand scope of analysis will increase in genomic con-tent and resolution. We envision that genome-wideprofiling will improve the overall pregnancymanagement. We propose a workflow for follow-upof NIPT results in Figure 2.

With dropping sequencing costs, increased sequencingdepth will be achieved. The accuracy for the detectionof smaller imbalances will thus further increase whichwill eventually allow a resolution of current array ana-lyzes to be achieved. Several groups have demonstratedproof-of-principle for non-invasive analysis of the totalfetal genome although the cost will remain prohibitivelyhigh for the near future (103–105). This raises the ques-tion of which resolution is (i) achievable (while main-taining a high accuracy) and (ii) desirable (patient andclinician). Which regions to enrich for, to interrogate,or to report raises the same issues as has been seenwith the introduction of prenatal microarray analysis andexome/genome sequencing (106).

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