First-Trimester Ultrasound: A Comprehensive Guide

8. Aneuploidy Screening: The Ongoing Role of First-Trimester Ultrasound

Kristen M. Rauch Melissa A. Hicks1Henry Adekola2 and Jacques S. Abramowicz 


Department of Obstetrics and Gynecology, Center for Fetal Diagnosis and Therapy, Wayne State University Physician Group, 3980 John R –, Box 160, Detroit, MI 48201, USA


Department of Obstetrics and Gynecology, Detroit Medical Center/Hutzel Women’s Hospital, Detroit, MI, USA


Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 3990 John R. Street, Detroit, MI 48201, USA

Kristen M. Rauch (Corresponding author)


Jacques S. Abramowicz



AneuploidyCirculating cell free fetal DNA (ccffDNA)Genetic counselingMaternal serum screeningMicroarrayNasal boneNuchal translucencyPrenatal screeningSerum markersTrisomy 13Trisomy 18Trisomy 21


The concept of prenatal screening for aneuploidy began with the discovery that fetal Down syndrome risk correlated with maternal age [1]. Maternal serum screening (MSS) utilizing feto-placental proteins was developed in an attempt to provide more pregnancy specific risk assessment [2]. While studying increased maternal serum alpha-fetoprotein for the detection of open neural tube defects it was also noted that this marker was decreased in pregnancies with Down syndrome [3]. Other markers including, human chorionic gonadotropin, unconjugated estriol, and dimeric inhibin A, were then also found to display a characteristic pattern in pregnancies with Down syndrome leading to the development of second-trimester double, triple, and quadruple screening, respectively [4]. The accuracy of these maternal serum screens is heavily dependent on the clinical information entered into the algorithm. Incorporation of just one incorrect parameter (i.e., gestational age) can provide a false-positive or false-negative result. An additional drawback to these screens is the delay in performance until the second trimester, excluding the option for early termination in the case of an affected pregnancy.

First-trimester screening including incorporation of fetal nuchal translucency (NT), pregnancy-associated plasma protein A (PAPP-A), and the beta subunit of human chorionic gonadotropin (β-hCG), soon emerged as a superior screening method [5]. This approach eliminates the error due to inaccurate gestational dating, since ultrasound measurement of the fetal crown-rump length is part of the algorithm. First-trimester screening achieves a high detection rate for Down syndrome (85 %–90 %) and trisomy 18 (90 %–95 %) with a 5 % false-positive rate, and provides earlier prenatal diagnosis and the option of termination in the case of an affected pregnancy [67].

Several screening modalities that incorporate elements in both the first and second trimesters were then created to further increase the detection rate and decrease the false-positive rate. There are various strategies to performing this type of combined screening, including those which incorporate only serum feto-placental protein markers (i.e., serum integrated) as well as those incorporating ultrasound and serum markers (i.e., integrated, sequential, and contingent) [8]. The type of strategy utilized depends on patient preference as well as availability of certified NT providers.

Second-trimester ultrasound for evaluation of structural malformations and “soft markers” is also utilized as a screening for fetal aneuploidy. Some centers will perform “genetic sonograms” by incorporating likelihood ratios for various ultrasound markers to produce a risk for aneuploidy, mainly Down syndrome. This information is often interpreted in the context of the patient’s other risk factors, including age and MSS results. There is a wealth of literature regarding the utility of second-trimester ultrasound screening for aneuploidy, which is outside the scope of this chapter [912].

In 1997 Lo et al. first discovered circulating cell-free fetal DNA (ccffDNA) in the plasma of pregnant women initiating efforts to create a reliable noninvasive method for detecting fetal aneuploidy [13]. More than a decade later massively parallel sequencing (MPS) of cell free fetal DNA was shown to detect an overrepresentation of chromosome 21 material in pregnancies affected with trisomy 21 [1415]. This led to a number of clinical trials validating MPS as a highly sensitive and specific noninvasive tool to detect common chromosomal aneuploidies [1618] in a high-risk patient population. This technology became clinically available in late 2011 and has significantly shifted the paradigm of prenatal screening and diagnosis in many centers throughout the world and resulted in a decrease in the number of invasive procedures performed for aneuploidy testing [19]. Screening for aneuploidy through cell free fetal DNA analysis has caused us to reevaluate the way we think of screening, even in low-risk populations [20]. The traditional definition of screening, in which the majority of individuals with a positive result do not have the disease of interest, which is true of standard maternal serum screen modalities, does not apply because of the high positive predictive value of the results [21].

Biochemical and Ultrasound Screening for Aneuploidy

Historical Approaches

As early as the 1960s, maternal age was recognized as a risk factor for fetal aneuploidy. Rates of chromosome abnormalities at different maternal ages (Table 8.1) were published in the 1980s to aid genetic counseling for these conditions, especially given the uptake in prenatal diagnosis due to improved safety and efficacy of invasive prenatal diagnosis [122].

Table 8.1

Estimates of rates of chromosome abnormalities in live-born infantsa

Age of mother at term (years)

Risk for trisomy 21 (Down syndrome) (%)

Total risk for any chromosome abnormalityb (%)























































































































































aAdapted from Hook 1981 and Hook et al. 1983

bIncludes trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), and sex chromosome aneuploidies (XYY and XXY). Monosomy X (Turner syndrome) is excluded as it is not significantly correlated to maternal age. Trisomy X (XXX) is excluded as the clinical significance of this aneuploidy was in question at time of calculation of these estimates

cNo risk range may be constructed for women less than 20 years [122]

Historically, women over 35 years of age at term were considered “high risk” for chromosome aneuploidy and were offered invasive prenatal diagnosis via fetal karyotyping following amniocentesis or chorionic villus sampling. Age 35 was selected as the cut-off, in part, due to the often-quoted 1 in 200 risk of complications with amniocentesis; the risk of aneuploidy was felt to be approximately equal to or greater than the procedural risk. The cost-effectiveness and utility of this approach was questioned, particularly in light of the procedure-related pregnancy loss rate [23].

Second-Trimester Maternal Serum Biochemical Screening

Second-trimester biochemical maternal serum screening started with the finding that low maternal serum alpha-fetoprotein (MS-AFP) was associated with an increased risk of Down syndrome [3]. Maternal serum screening for aneuploidy then expanded to include multiple additional markers: hCG, unconjugated estriol (uE3), dimeric inhibin A (DIA), and in some laboratories, invasive trophoblast antigen (ITA) [4]. These quadruple (“Quad”) or “Penta” screens provided additional parameters by which to estimate a woman’s risk of fetal aneuploidy in a given pregnancy, with higher sensitivity than age alone (approximately 80 % for Down syndrome at a 5 % false-positive rate). The “pattern” of high or low levels (as calculated by MoM) of these analytes, combined with maternal age, weight, gestational age (ideally confirmed by ultrasound biometry), and race are used to calculate risk for fetal Down syndrome, trisomy 18, and ONTD; see Table 8.2 for a summary. In some centers, a risk for Smith–Lemli–Opitz syndrome (SLOS, an autosomal recessive condition characterized by a range of intellectual disability, growth restriction, and structural anomalies) is also calculated based on low serum estriol. Although no uniformly accepted practice exists, it has been suggested in multiple studies that unexplained extreme values of second-trimester analytes should prompt further investigation and increased maternal-fetal monitoring [2425].

Table 8.2

Patterns of second-trimester maternal serum analytes and risk assessment for screened conditions [2426]


Trisomy 21

Trisomy 18


Other risks with high levels (>2.0 MoM)

Other risks with low levels (<0.5 MoM)


Birth defects (not limited to ONTD or OAWD), fetal death, placental abnormality, IUGR, fetal distress

Fetal death, preterm birtha


Birth defects (not limited to trisomy 21), IUGR, fetal distressb

IUGR, birth defectsb


No significant risks

IUGR, fetal death; certain single-gene conditions (X-linked ichthyosis, congenital adrenal hyperplasia)


Preeclampsia, fetal death, preterm birth, IUGR

No significant risks

Abbreviations: IUGR, intrauterine growth restriction (birth weight <10th percentile for gestational age) [25]; OAWD, open abdominal wall defects; ONTD, open neural tube defects

aMS-AFP levels considered low at <0.25 MoM [27]

bhCG levels considered high at >2.5 MoM and low at <0.4 MoM [25]

First-Trimester Maternal Serum Biochemical Screening

In an effort to perform risk assessment for aneuploidy at an earlier gestational age, first-trimester markers for aneuploidy were investigated. Previously used second-trimester markers were explored for utility in the first trimester, but only hCG was found to be informative; elevated hCG is associated with an increased risk for Down syndrome. PAPP-A, another product of the placenta, is found in low levels in maternal serum of affected pregnancies. Low levels of both analytes are concerning for trisomy 18. Taken together, these two markers had a 65 % detection rate at a 5 % false-positive rate for Down syndrome; however, second-trimester maternal serum screening had a higher detection rate. Therefore, additional markers were needed to improve first-trimester screening [5].

First-Trimester Ultrasound Markers for Chromosomal and Genetic Anomalies

Nuchal translucency (NT), a measurement of the thickness of the subcutaneous fluid at the back of the neck of the fetus in the late first trimester, was observed to be increased in fetuses with Down syndrome. Stringent, efficacious and standardized methods for measurement of the fetal NT were developed in the early 1990s [28]. NT alone was found to have a ~75 % detection rate and ~5 % false-positive rate for Down syndrome [29]. Kagan and colleagues found that 19.2 % of pregnancies with NT >3.4 mm had an abnormal karyotype [30]. Furthermore, the incidence of chromosomal defects increased with NT thickness, from approximately 7 % with an NT of 3.4 mm (95th percentile for crown-rump length) to 75 % for an NT of ≥8.5 mm. The majority of fetuses with Down syndrome had an NT of <4.5 mm, whereas in the majority of fetuses with trisomies 13 or 18, NT measurement was 4.5 to 8.4 mm. Fetuses with Turner syndrome tended to have an NT of 8.5 mm or more (Table 8.3).

Table 8.3

Common genetic conditions associated with increased first-trimester nuchal translucency [3133]


Incidence (% of fetuses with increased NT)




Testing considerations

Turner syndrome (Monosomy X)

1 in 2000 females (~6.7 %; associated with larger NTs compared to Down syndrome. Highest incidence (12.3 %) if NT is 5.5–6.5 mm [30]

Presence of a 45,X cell line


Prenatal: cardiac defects and cystic hygroma (resulting in a webbed neck in the postnatal period); associated high risk of IUFD

Postnatal: short stature, cardiac and kidney malformations, webbed neck, lymphedema, reduced fertility, and risk for mild developmental and learning delays [35]

May be diagnosed incidental to prenatal screening or testing for fetal trisomy. Low-level mosaicism may be undetectable by standard fetal karyotype and chromosome microarray. FISHa of additional interphase cells may improve detection and identify Y chromosome material, which confers a risk of postnatal gonadoblastoma [34]

Noonan syndrome

1 in 1000–2500 (2–5 %) [3637]

Mutation in genes encoding proteins in Ras/MAPK signaling pathway (most commonly PTPN11, but also SOS1RAF1BRAFMAP2K1MAP2K2NRASSHOC2CBLHRAS, and KRAS) [38]

Autosomal dominant; de novo in 25–70 % of cases [39]

Prenatal: hydrops fetalis, cardiac anomalies; polyhydramnios, bilateral pyelectasis and ventriculomegaly have been reported [40]

Prenatal molecular genetic diagnosis on chorionic villi or amniocytes via a panel of genes is clinically available, though detection rate is only ~70 % [33]

Postnatal: wide variability; short stature, congenital heart defect, and developmental delay of variable degree. Other findings can include broad or webbed neck, pectus, cryptorchidism, varied coagulation defects, lymphatic dysplasias, and ocular abnormalities. Apparently asymptomatic individuals exist. Part of a spectrum of disorders (including Leopard syndrome, cardiofaciocutaneous (CFC) syndrome, and Costello syndrome) [38]

22q11.2 deletion syndrome (DiGeorge syndrome, velocardiofacial syndrome [VCFS])

1 in 4000 (~3 % if prenatal heart defects noted [41]

Microdeletion of chromosome 22 at band q11.2; size of deletion may vary

Autosomal dominant; de novo in ~90 % of cases [4243]

Prenatal: conotruncal heart defects; hypoplastic/absent thymus, bilateral club feet, and renal cystic dysplasia have also been reported [4445]

Prenatal testing (FISH or chromosome microarray) for 22q11.2 deletions may be considered in fetuses with congenital heart defects and concomitant increased NT; the value of routine testing in the absence of heart defects is not clear [364648]

Postnatal: conotruncal heart defects, neonatal hypocalcemia, hypothyroidism, palatal defects, immune deficiency, short stature, gastrointestinal, genitourinary and ophthalmologic disease; normal IQ to mild intellectual disability, autism spectrum disorder, increased risk for psychiatric disorders in adulthood [43]

Skeletal dysplasias

Estimated at ~1 in 2200 to 4400 live births [49]

Heterogeneous group of genetic conditions

May be autosomal dominant, autosomal recessive or X-linked; many de novo [49]

Prenatal: hydrops, short femurs, abnormal skull shape and mineralization, abnormal profile, abnormal chest. Second-trimester anatomy scan may reveal additional findings. Risk of IUFD, particularly where chest anomalies increase likelihood of pulmonary insufficiency. Some not compatible with long-term survival [49]

Given the large number of genes associated with skeletal anomalies, gene “panels” that simultaneously test multiple genes may assist in determining the correct diagnosis and the recurrence risk of the condition for future pregnancies [3350]. If possible, postnatal evaluation by a pediatric geneticist may be beneficial in establishing a diagnosis

Postnatal: short stature, limb shortening; varies greatly depending on the underlying diagnosis

aFISH, or fluorescent in-situ hybridization, a method for rapid enumeration of chromosomes 21, 13, 18, X and Y on uncultured chorionic villi and amniocytes. For additional information, please refer to the 2001 review by Tepperberg and colleagues [51]

While the risk of numerical chromosome anomalies can be clarified by fetal karyotype, microdeletion and microduplication syndromes have been increasingly associated with thickened fetal NT. Chromosomal microarray (CMA, also called array comparative genomic hybridization or array CGH) can interrogate fetal DNA obtained by CVS or amniocentesis and detect small (~1–3 megabases [Mb]) genome-wide deletions or duplications of DNA (copy number variations [CNVs]). This is performed by comparing fetal DNA against a reference genome via a microchip-based reaction. For additional information about this tool, the reader is referred to ACOG Committee Opinion No. 581: the use of chromosomal microarray analysis in prenatal diagnosis [52].

In a 2015 study by Lund and colleagues, in fetuses with isolated NT ≥3.5 mm, clinically significant CNVs were detected in 12.8 % of cases with normal karyotype; an additional 3.2 % had CNVs of uncertain clinical significance [53]. Clinically significant CNVs were detected in 14.3 % of fetuses with an NT of 3.5 to 4 mm, structural anomalies in other systems, and a normal karyotype; detection rate was 16.7 % in similar cases where NT measured ≥4 mm [54]. Notably, chromosomal microarray can detect 22q11.2 deletion syndrome, the CNV most commonly associated with increased NT and cardiac defects (see Table 8.3).

It should be noted that, while the clinical significance of thousands of pathologic and benign CNVs has been well documented, there remain regions of the genome for which the significance of a microduplication or microdeletion is not known. Furthermore, chromosomal microarray may incidentally detect consanguinity (including incest), non-paternity, and genetic abnormalities associated with adult-onset disorders that may be inherited from an asymptomatic parent. Therefore, it is recommended that genetic counseling with informed consent be obtained prior to performing microarray in cases with increased fetal NT [52].

In the event that fetal chromosome abnormality has been ruled out, an increased NT may be indicative of many other genetic and nongenetic conditions. Of the single gene conditions, the most common is Noonan syndrome (see Table 8.3). A link between increased NT and several other single-gene conditions and have been suggested. However, due to the rarity of most of these conditions (most have an incidence of <1 in 10,000), a definitive association between increased NT and these conditions cannot be statistically proven. Furthermore, the single-gene and often de novo molecular cause of these conditions is not amenable to comprehensive prenatal genetic diagnosis [3132]. As can be surmised from Table 8.3, many conditions have sonographically diagnosable fetal anomalies, in addition to an increased NT, albeit, not always in the first or early second trimesters.

In addition to nuchal translucency, evaluation of the fetal nasal bone is a benefit of first-trimester ultrasound. Hypoplastic or absent nasal bone has been associated with fetal Down syndrome. Nasal bone evaluation between 11 0/7 to 13 6/7 weeks is included in first-trimester screening for Down syndrome in some centers, as it is independent of other first-trimester markers (free β-hCG, PAPP-A, and NT). In one series, the nasal bone was absent in 2.6 % of the euploid fetuses (though this may vary with ethnicity); it was absent in 59.8 % of the fetuses with trisomy 21, 52.8 % with trisomy 18, 45.0 % with trisomy 13 and in none of the fetuses with Turner syndrome [55]. At a false-positive rate of 5 %, nasal bone evaluation in addition to NT and serum analytes was estimated to achieve a sensitivity of >95 % for trisomy 21, 18 and 13, and is not thought to significantly prolong ultrasound examination time [5556]. Much like nuchal translucency, rigorous guidelines have been established for measurement of this feature, which is outside the scope of this chapter (see Chap. 9).

More recently, first-trimester evaluation of flow in the ductus venosus and across the tricuspid valve via Doppler has been proposed to aid in risk assessment and improve detection rate for fetal aneuploidy. In one prospective study, a ductus venosus pulsatility index for veins (DV-PIV) demonstrating a reversed a-wave was estimated to detect 96 %, 92 %, 100 %, and 100 % of trisomies 21, 18, and 13 and Turner syndrome, respectively, at a false-positive rate of 3 %, when combined with maternal age, NT, fetal heart rate, and β-hCH and PAPP-A [57]. In the same cohort of patients, assessment of tricuspid flow for regurgitation demonstrated the same detection rates for chromosomal anomalies as DV-PIV [58].

Benefits of First-Trimester Ultrasound in Detection of Non-chromosomal Fetal Anomalies

Despite increased utilization of noninvasive DNA screening (NIDS) for fetal aneuploidy, first-trimester ultrasound remains a useful screening tool for non-chromosomal conditions that may have a significant impact on prenatal and postnatal outcome. Increased NT is a risk factor for fetal congenital heart defects (CHD), although no pattern of specific CHD has been described. An NT >95th percentile for crown-rump length has been associated with a significantly increased risk for CHD in fetuses with a normal karyotype, with a detection rate for major CHD of ~44 % for a 5.5 % false-positive rate [59]. Doppler evaluation of the tricuspid valve and ductus venosus at first-trimester ultrasound can also improve the detection rate; 32.9 % of euploid fetuses with major CHD had tricuspid valve regurgitation, and 28 % had an abnormal a-wave in the ductus venosus (compared to 1.3 % and 2.1 % of those without CHD, respectively) [60]. The current consensus is that fetal echocardiogram, performed as early as the late first trimester, should be considered in pregnancies with increased NT (>3.5 mm), particularly as some studies have showed improved neonatal outcome in ductal dependent CHD after being identified via NT measurement [316162]. Additional discussion on first-trimester detection of CHD is found in Chap. 11.

First-trimester increased NT has also been associated with fetal death, with risk appearing to directly correlate with NT measurement; overall risk is ~4 %, ranging from ~2 % at 3.5 mm to ~17 % at >6.5 mm [3363]. It may be an early indicator of structural anomalies, including but not limited to body stalk anomaly, diaphragmatic hernia, omphalocele, orofacial clefts, fetal akinesia sequence and megacystis. The overall incidence of structural anomaly with NT >3.5 mm is estimated at ~12 % in the presence of a normal fetal karyotype [323363]. Fetal infection is often cited as a possible cause for increased NT. Parvovirus B19 infection is the only specific pathogen associated with increased NT, most likely secondary to myocardial dysfunction or fetal anemia [64].

In twin gestations, first-trimester markers (including NT, nasal bone, tricuspid valve flow, and DV-PIV) may be helpful in risk assessment for aneuploidy as they are independent measurements for each fetus, regardless of chorionicity; however, NT is also helpful in assessing risk of twin-to-twin transfusion syndrome for monochorionic twins [65].

Current Methods of First-Trimester Maternal Serum Aneuploidy Screening

In 2007, the American College of Obstetrics and Gynecology published a practice bulletin stating that “first-trimester screening using both nuchal translucency measurement and biochemical markers is an effective screening test for Down syndrome in the general population… Screening and invasive diagnostic testing for aneuploidy should be available to all women who present for prenatal care before 20 weeks of gestation regardless of maternal age”[4].

There are many current screening methodologies to address these recommendations, using different combinations of first-trimester ultrasound, first-trimester biochemical markers, and second-trimester biochemical markers to generate a risk assessment for aneuploidy. First-trimester analyte screening uses PAPP-A and hCG analytes; first-trimester combined screening adds nuchal translucency with or without nasal bone measurement. Similarly, integrated and serum integrated screening combine first-trimester PAPP-A measurement with second-trimester analytes, with our without first-trimester ultrasound parameters, respectively. Stepwise sequential and contingency screening allow for women defined as “high-risk” to be notified of increased risk after first-trimester screening, with the option of invasive prenatal testing; women in a “moderate-risk” category may receive second-trimester analyte screening to give an aneuploidy and ONTD risk assessment with the highest detection rate. Women identified as “low-risk” after first-trimester methods are not offered second-trimester analyte measurement in contingency screening [8].

The wide array of options may appear complicated to health care providers and patients alike; the benefits, limitations and possible scenarios for use are summarized in Table 8.4.

Table 8.4

Summary of first- and second-trimester maternal serum screening options [458]

Screening Method

Detection rate (DR)




Clinician likely to utilize test

Trisomy 21a (%)

Trisomy 18b (%)

First-trimester analyte screening



Free beta-hCG and PAPP-A drawn at 9–13 6/7 weeks

First-trimester result, NT not required, one visit, CVS an option if screen positive

Lower DR compared to options utilizing NT; no screening for ONTD

Clinician with early-to-care population with no access to certified NT provider, but does have access to CVS, and prefers one visit for screening

Combined first-trimester screening



Nuchal translucency ± nasal bone measurement and trimester analyte screening at ~10–13 6/7 weeks

First-trimester result, one visit, CVS an option if screen positive

NT required, lower DR compared to integrated screen; no screening for ONTD

Clinician with early-to-care population who has access to certified NT provider and CVS, and prefers one visit for screening

Integrated screening



PAPP-A and NT measurement at ~10–14 weeks; AFP, hCG, uE3 and DIA drawn at 15–21 6/7 weeks

Highest DR of all maternal serum screening tests

Two visits and NT required; results given in second trimester

Clinician with early-to-care patient population who has access to a certified NT provider, but does not have access to CVS

Serum integrated screening



PAPP-A only at ~10–14 weeks; AFP, hCG, uE3 and DIA drawn at 15–21 6/7 weeks

Highest DR for screening when NT is not available; NT not required

Two visits, results given in second trimester, lower DR compared to screens that include NT

Clinician with early-to-care population who does not have access to certified NT provider or CVS

Stepwise sequential screening



PAPP-A, b-hCG, and NT in first trimester; risk reported if elevated. If low risk, AFP, hCG, uE3, and DIA in second trimester

First-trimester result for highest risk patients allows option of CVS; DR higher than combined FTS while allowing for some first-trimester results

Two visits for most patients; NT required; lower DR compared to integrated screen

Clinician with early-to-care population and high follow-up compliance, with access to a certified NT provider and CVS, who wants information early enough to offer CVS if risk is high, and wants to avoid moderate risk group created by contingency screening

Contingency screening



PAPP-A, b-hCG, and NT in first trimester; results reported; high risk offered diagnostic testing, low-risk screening complete; moderate risk group receives AFP, hCG, uE3, and DIA in second trimester

First-trimester results for high- and low-risk patients, minimizing number of patients needing a second visit

Two visits for moderate risk group; NT required; Initial moderate risk group may not feel as reassured with second trimester negative screen result as initial low-risk group; no screening for ONTD in low-risk group

Clinician with an early-to-care population and high follow-up compliance who has access to a certified NT provider and to CVS, and who feels the benefit of a one visit screen for most patients outweighs the anxiety caused for patients who fall into the moderate-risk group and are later re-stratified to a low-risk group

Multiple marker serum screening



AFP, hCG, uE3, DIA (± ITA) drawn between 15 and 21 6/7 weeks

Allows for screening in women presenting for care after first trimester; one visit

Results given in second trimester; lower DR compared to screens involving first-trimester elements; risk of conflicting assessments if performed without knowledge of prior combined first-trimester screeningc

Clinician with patients who present primarily in the second trimester for screening or patients whose insurance does not cover NT screening.

Noninvasive DNA screening (NIDS)d



Maternal blood sample drawn between 10 and 21 6/7 weekse

First- or second-trimester result in one visit, NT not required, highest DR

New technology with shorter publication history and less information on payer coverage

Clinician comfortable with new technology who wants a screening test with high DR and low FPR that can be applied in first and second trimesters

aTypically at a 5 % false-positive rate (FPR) with a 1/270 cut-off for screen positive

bTypically at a 0.5 % FPR with a 1/100 cut-off for screen positive

c Per ACOG’s screening guidelines, second-trimester multiple marker serum screening should not be performed following first-trimester analyte or combined screening; MS-AFP only for ONTD risk assessment is recommended [4]

dSome practitioners endorse combining measures from NIDS and first-trimester biochemical markers using Bayes’ theorem to provide more accurate patient-specific risks and improved NIDS performance (particularly when fetal fraction is <4 %); however, the information necessary for this calculation (depth of sequencing, Z-score, and fetal fraction) are often not readily available from the suppliers of NIDS [66]

eIn theory, maternal blood can be drawn any time after 10 weeks gestation; gestational ages at which NIDS has been validated may vary by laboratory

Other Outcomes

It is important to note that, while first-trimester screening provides a risk assessment for Down syndrome and trisomy 18, positive screens may lead to incidental diagnoses of other conditions. In one 10-year study, of 97 screen-positive pregnancies with abnormal fetal karyotypes, ~30 % had chromosome abnormalities other than trisomy 13, 18 or 21. Such findings included trisomy 16, triploidy, sex chromosome aneuploidies (45,X or Turner syndrome and 47,XYY), unbalanced translocations, and marker chromosomes. These clinical outcomes are expected to range from likely benign to lethal, underlining the importance of pretest and posttest counseling for the possibility of a chromosome anomaly other than the most common trisomies [46].

Noninvasive DNA Screening for Aneuploidy

The method of testing that utilizes cell free fetal DNA from the plasma of a pregnant woman to screen for aneuploidy in the fetus was previously known as noninvasive prenatal diagnosis (NIPD) or noninvasive prenatal testing (NIPT). Over time this terminology fell out of favor given that a result is not considered diagnostic. The term noninvasive prenatal screening (NIPS) then became the preferred designation [67]. We suggest the term noninvasive DNAscreening (NIDS) as an alternative. Indeed, “prenatal screening” applies to any type of noninvasive technology including ultrasound and maternal serum screening. Hence we will utilize the term NIDS as this better distinguishes this technology from more traditional screening modalities.

Circulating cell free fetal DNA (ccffDNA) comprises approximately 3 % to 13 % of the total cell free maternal DNA [13]. The percentage of the total cell free maternal DNA that is fetal in origin is termed the fetal fraction. It is important to note that this “fetal DNA” is thought to be derived primarily from placental trophoblasts [68], and it is well known that chromosomal abnormalities may exist in the placenta that are not present in the fetus [69]. The ccffDNA is cleared from the maternal blood within hours after childbirth [70]. The fetal fraction varies among women and several factors including gestational age, multiple gestations, and BMI are known to affect the magnitude of this fraction [7173]. The quantity of ccffDNA increases during the first trimester and is felt to be at a sufficient quantity to perform NIDS by approximately 9 weeks gestation [74]. Therefore most clinically available tests are available starting at 10 weeks gestation. While evidence suggests that high maternal BMI is associated with a lower fetal fraction, no clear BMI cut-off has been set to guide practitioners as to when a patient’s BMI may result in an inability to perform this testing [73]. Because gestational age and multiple gestations affect fetal fraction and subsequently, interpretation of NIDS results, ACOG recommends performing a baseline ultrasound examination for any patient considering NIDS, prior to testing [75].

Several studies have attempted to delineate the accuracy of NIDS in samples of varying fetal fraction. These studies suggest that the methodology and bioinformatics utilized will affect the fetal fraction required to achieve high sensitivity and specificity [76]. The fetal aneuploidy status itself can also affect the fetal fraction; for example, trisomy 13 is associated with reduced fetal fraction due to a smaller placenta mass associated with this aneuploidy [77]. Brar et al. demonstrated that there was no significant difference in fetal fraction between those at low and high a priori risk for aneuploidy [78].

Test Methodologies

Two main methodologies for NIDS have been validated and introduced into clinical practice. The more common approach, known as massively parallel shotgun sequencing (MPSS), is accomplished through quantifying millions of cell free DNA fragments. Each cell free fragment is assigned to its chromosome of origin. The total quantity of cell free fragments from the patient’s specimen is then compared to a reference genome. The result is positive if there is an overrepresentation of the chromosome in the patient’s specimen compared to the reference. A large number of chromosome fragments must be counted in order to detect aneuploidy; this is especially important when the fetal fraction is low as the difference between aneuploidy and euploidy will be small. Sequencing biases depending on the guanine and cytosine (GC) base pair content of the DNA fragments necessitates adjustments to allow for DNA base composition [7980]. The MPSS approach could be used for detection of all aneuploidies, although clinical trials have only yet validated testing for non-mosaic chromosomes 21, 18, 13, and monosomy X. A related strategy known as targeted massively parallel sequencing (t-MPS) differs in that it selectively amplifies only the chromosomal regions of interest (i.e., 21, 13, 18) and then determines whether there is an excess of one chromosome relative to another [81].

Clinical laboratories that utilize massively parallel sequencing employ different interpretation strategies. A z-score may be calculated to determine the ratio of observed sequences from a given chromosome of interest versus a reference chromosome; an elevated z-score is suggestive of trisomy for the chromosome of interest [18]. Some laboratories may determine a positive result using a single z-score threshold (i.e., z-score greater than or equal to 3) [18], while others utilize a dual threshold model in which risk is stratified into categories such as “aneuploidy suspected” (z-score between 2.5 and 4) and “positive” (z-score greater than 4) [16]. Results may be presented categorically or as a risk score (i.e.,1 in 10,000).

Another approach to NIDS is the targeted counting of specific DNA sequences. With this methodology only selected loci from the chromosomes of interest are sequenced. This methodology is also known as single nucleotide polymorphism (SNP) based NIDS. Several thousand SNPs are sequenced. Each specimen is then evaluated based on the hypothesis that the fetus is monosomic, disomic, or trisomic. The position of the SNPs on the chromosomes and the possibility that recombination may have occurred must be considered. Likelihood is then calculated that the fetus is either diploid (“normal”), aneuploid, or triploid. The advantage of this technology is distinguishing between maternal and fetal SNPs, which allows for the detection of triploidy and may identify regions of fetal chromosome homology that could indicate consanguinity or uniparental disomy [82]. However, there must be a sufficient quantity of informative SNPs to provide an accurate result. A risk score is generated for each chromosomal abnormality evaluated.

Test Performance

Initially NIDS included common autosomal aneuploidies, Down syndrome, trisomy 18, and trisomy 13. Several validation studies have been completed to assess test performance for these conditions. Although the achieved detection rates and false-positive rates vary slightly among the various studies, overall NIDS utilizing the MPSS approach (based on outcomes from eight studies) achieved detection rates of approximately 99 %, 97.6 %, and 89.2 % for Down syndrome, trisomy 18 and trisomy 13, respectively. Validation studies which utilized the t-MPS methodology (including outcomes from six studies) achieved detection rates of 99.4 %, 97.9 %, and 81.8 % for Down syndrome, trisomy 18, and trisomy 13, respectively. The SNP-based approach (based on outcomes of two studies) achieved detection rates of 100 %, 96.4 %, and 100 % for Down syndrome, trisomy 18, and trisomy 13, respectively. However, the total number of aneuploidy cases included in the SNP-based studies was smaller (124 total cases) compared to that of the studies using t-MPS (274 total cases) or MPSS (680 total cases) approach. All three methodologies achieved low false-positive rates for the common aneuploidies, ranging from 0 to 0.32 % [81]. The positive predictive value (PPV) of a high-risk NIDS result was evaluated as part of the Comparison of Aneuploidy Risk Evaluations (CARE) study, a prospective, blinded, multicenter observational study comparing results of NIDS (performed by MPSS) with those of conventional screening for trisomy 21 and 18 in a general obstetrical population . The PPV with NIDS was 45.5 % for trisomy 21 versus 4.2 % with standard screening. The PPV with NIDS was 40 % for trisomy 18 versus 8.3 % with standard screening [20]. In a Chinese cohort of women younger than 35 years old 1741 samples were analyzed with NIDS (performed by MPSS) and an overall PPV of 86.67 % was calculated for the aneuploidy samples of all five chromosomes evaluated (21, 18, 13, X, and Y). This was compared to a PPV of 2.41 % with standard serum screening [21].

Testing for non-mosaic 45,X was subsequently evaluated by several groups. However, sample sizes in these studies were small and may have included ascertainment bias through preferential inclusion of nonviable cases and those with abnormal serum and/or ultrasound findings [81]. Observed detection rates for 45,X ranged from 75 % [16] to 91.5 % [83] using MPS methodology. Using a SNP-based methodology a detection rate of 92 % was achieved [84].

More recently, testing for sex chromosome aneuploidies and select microdeletion syndromes have been added to the test panels available through some commercial laboratories. However, robust estimates of the efficacy of testing for these conditions are not yet available and given the rarity of these disorders, positive predictive values are expected to be low [85]. When considering sex aneuploidies and microdeletions, it is also important to keep in mind that these conditions are more likely than the traditional aneuploidies to be present in the mother. For example, age-related loss of an X-chromosome can lead to somatic mosaicism for 45,X cells [86].

Initially clinical validation studies of NIDS primarily focused on women identified as being at high risk for aneuploidy either by maternal age, abnormal serum screening, or abnormal ultrasound findings. More recently, studies have attempted to determine whether the performance of NIDS would be similar in a general obstetrical population. In a cohort of over 2000 women undergoing routine first-trimester aneuploidy screening a detection rate of over 99 % and false-positive rate of <1 % was found for trisomies 21 and 18, a similar performance to that observed in high-risk cohorts [87]. Pergament et al. evaluated the performance of SNP-based NIDS in samples from 1052 women, 49 % of which were low risk for aneuploidy. The study found that sensitivity and specificity for trisomy 21, 18, 13, and monosomy X did not differ between low-risk and high-risk populations [88]. The question, therefore, has been raised whether this should be applied to the entire population [89].

Some laboratories that perform NIDS with MPS methodology offer testing for twin gestations. Testing for monozygotic twins is expected to perform similarly to a singleton gestation. Testing in dizygotic twin and higher-order multiple gestations is complicated by the fact that the per-fetus fetal fraction may be lower. In fact, the non-reportable rate is higher (7.4 %) than that for singleton pregnancies (2 %) [90]. Additionally, if one fetus is euploid while the other is aneuploid, there is a dilution of the ccffDNA from the aneuploidy fetus resulting in decreased detection rates compared to singleton gestations [91].

Failure to obtain a NIDS result occurs in a small proportion of patients and may occur for a variety of reasons. The most common cause for test failure is insufficient fetal fraction in the maternal plasma specimen. This occurs in 2 % or less of patients undergoing NIDS with MPS methodology [90] and approximately 8 % of patients undergoing the SNP-based method, although the no-call rate of SNP-based methodology may be reduced by inclusion of a paternal specimen [88]. A low fetal fraction obtained in the initial sample is associated with a relatively high chance of failure with a second sample. Wang et al. evaluated 135 cases of test failure due to insufficient fetal DNA which were re-drawn, and found that 44 % of these patients had insufficient fetal DNA in their second specimen [73]. A study by Pergament et al. that evaluated the performance of SNP-based NIDS in both high and low risk cohorts, found that a non-reportable sample was 2.5 times more likely to be aneuploid [88]. Therefore, it is appropriate to consider invasive diagnosis rather than re-draw of NIDS in women with low fetal fraction, especially in cases where there is high risk of aneuploidy and gestational age is advanced.

Test Interpretation

Negative (low-risk) NIDS results should be carefully interpreted in the context of the patient’s other clinical information including age, MSS results, family history and fetal ultrasound findings. Patients 35 years of age or older (advanced maternal age) should be informed that NIDS covers only the most common fetal aneuploidies seen in the advanced maternal age population. A study by Grati et al. investigated the proportion of clinically relevant chromosome abnormalities which are part of traditional prenatal screening (i.e., trisomies 21, 18, 13, monosomy X, triploidy). As part of the study 1, 178 abnormal karyotypes were identified among patients undergoing CVS or amniocentesis due to advanced maternal age. Approximately 24 % (280/1178) of these abnormal karyotypes (including autosomal aneuploidies, unbalanced structural rearrangements, supernumerary marker chromosomes containing euchromatic material, fetal mosaicism, and apparently balanced de novo reciprocal translocations) were not covered by traditional prenatal screenings [92]. Patients who have an abnormal MSS result should be informed that although those results indicate a risk for a specific aneuploidy, other aneuploidies and fetal conditions that are not covered by NIDS can present with an abnormal MSS [46].

It is recommended that patients who have other factors suggestive of a fetal aneuploidy have genetic counseling and be presented with the option of invasive prenatal diagnosis regardless of NIDS results. This is important because NIDS does not screen for all chromosomal or genetic conditions. Indications for invasive prenatal diagnosis regardless of NIDS results include the presence of ultrasound abnormalities and personal or family history of a chromosome abnormality. Patients with other risk factors such as AMA, abnormal MSS, and ultrasonographic soft markers may also benefit from detailed counseling regardless of their NIDS results.

Positive (high-risk) NIDS results should be followed up with confirmatory invasive prenatal diagnosis for definitive fetal karyotyping. While some patients may decline confirmatory testing due to the procedural risk for miscarriage, it is not recommended that patients make decisions regarding pregnancy termination without confirmatory testing. This is important because false-positive results can occur. Several false-positive results have been reported with biological basis, such as confined placental mosaicism (CPM), maternal chromosome abnormality, vanishing twin, maternal cancer [93], and chromosomal microduplication/microdeletion syndrome [9495]. Concern for CPM makes amniocentesis, rather than CVS, the preferred choice for invasive prenatal diagnosis to clarify a positive NIDS result. A flow-chart summarizing our recommendations in cases of positive NIDS is outlined in Fig. 8.1.


Fig. 8.1

Recommended follow-up of a positive NIDS result

Given the complexities of NIDS it is important that women receive adequate pre and post-test counseling with a qualified healthcare provider. The American College of Obstetrics and Gynecology, American College of Medical Genetics, International Society for Prenatal Diagnosis and National Society of Genetic Counselors have all released statements which outline the appropriate utilization of NIDS, including information which should be provided to the patient in order to obtain informed consent. A summary of these recommendations is included in Box 8.1.

Box 8.1 Key Points Required in Pretest and Posttest Counseling Regarding NIDS

·               NIDS provides highly sensitive and specific screen results for the common autosomal aneuploidies (trisomy 21, 18 and 13) in singleton gestations after 9 weeks gestation. However, results are not considered diagnostic as false-positives and false-negative results do occur.

·               The clinical validity of NIDS has been largely studied in a high-risk population (i.e., AMA, abnormal ultrasound or MSS), although limited evidence has suggested performance is similar in a low-risk population.

·               Some clinically available NIDS includes evaluation for select chromosomal microdeletions; however, the sensitivity and specificities for these conditions is still being elucidated and depends on size of the deletion.

·               A positive NIDS result requires genetic counseling and confirmation by invasive prenatal diagnosis (preferably amniocentesis) for fetal karyotyping.

·               NIDS should not be considered a replacement of invasive prenatal diagnosis as results are not definitive and screening is performed only for a select number of aneuploidies. A family historyshould be reviewed to determine if the patient should be offered other forms of screening or prenatal diagnosis for a particular disorder.

·               Although there are studies suggesting the efficacy of NIDS in multiple gestations, these studies included a small number of patients.

·               In a proportion of cases there is insufficient fetal fraction or test failure for some other reason. There is some evidence to suggest that the risk of aneuploidy may be higher among women receiving a failed or un-interpretable result.

·               In cases where mosaicism is present or there has been early demise of a co-twin, results may be inaccurate.

Other Considerations

The high sensitivity and specificity achieved by NIDS has the potential to reduce the number of procedure related losses of euploid fetuses. Garfield and colleagues used the results of the “MatErnal bLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA)” Study Group to develop a model for evaluating the impact of NIDS if incorporated into routine prenatal care for all high-risk women. This model estimated that use of NIDS would result in a 66 % reduction in miscarriages related to invasive prenatal diagnosis and lead to 38 % more women receiving a prenatal diagnosis of Down syndrome. In addition the model estimated that total costs for prenatal screening and invasive diagnosis would be slightly decreased (by 1 %) annually [96].

Which Screening Test to Choose?

As with traditional maternal serum screening, there are distinct advantages and disadvantages to each of the NIDS testing methodologies. Although each laboratory includes an assessment for common aneuploidies (i.e., trisomies 21, 18, and 13), there are differences among laboratories in the inclusion of sex aneuploidies and microdeletion/duplications. Some methods may not be optimal or available for certain patients or groups of patients; therefore, test selection should be made by a qualified provider. Table 8.4 summarizes advantages, disadvantages, and limitations of various screening modalities.

The Future of First-Trimester Aneuploidy Screening

Given the wealth of literature supporting the sensitivity and specificity of NIDS as well as the rapid incorporation of this screening tool into clinical practice, it is clear that NIDS is here to stay. The question has been raised about the role of first-trimester ultrasound with NT measurement and nasal bone evaluation in the setting of routine NIDS. First-trimester ultrasound remains a valuable and potentially cost-effective means of early detection of fetal structural anomalies (such as cardiac defects), accurate determination of gestational age, assessment of multiple gestations, comprehensive evaluation of the uterus and adnexa, and prediction of adverse maternal-fetal complications, in addition to being a screening tool for fetal aneuploidy and clarifying NIDS results [97].

While some NIDS platforms evaluate for a limited number of microdeletion/duplication syndromes, currently they do not report genome-wide findings of CNVs; this highlights the importance of first-trimester NT measurement in increasing suspicion for these conditions in the fetus. The clinical utility of traditional first-trimester screening modalities including ultrasound and serum biochemical screening is still compelling.

The future of aneuploidy screening will likely focus on how to incorporate these various components to achieve the highest sensitive/specific and cost-effective screening strategy. For example, based on a modeled analysis, Kagan and colleagues propose a contingent screening policy incorporating ductus venosus pulsatility index for veins (DV-PIV) along with nuchal translucency and maternal age. Those with an “intermediate” risk of aneuploidy (>1:3000) would undergo NIDS; those at “high risk” (>1:10) would undergo invasive prenatal diagnosis and fetal karyotyping. This model, based on data for population-based screening, improves cost effectiveness as compared to NIDS only, while maintaining a similar detection rate (96 % for Down syndrome, 95 % for trisomy 18, and 91 % for trisomy 13) [98]. This highlights the importance of first-trimester ultrasound as part of newer screening protocols incorporating NIDS. With the continued advancements in NIDS technology these strategies will need to be continually reevaluated and developed over time.

Patient Considerations and Perspectives

There is a paucity of literature addressing patients’ attitudes towards prenatal screening, and the perceived “value” of these tests. While some studies attempt to estimate the value of screening in terms of “average cost per Down syndrome birth avoided” [9799], the psychosocial, ethical, and emotional aspects of screening are more difficult to quantify.

Some studies have examined the preferences of pregnant women with regard to the methods of screening. In one such study, 19.2 % of women opted out of screening; of those that opted in, 97.8 % preferred first rather than second-trimester screening [100]. A criticism of first-trimester screening is that some women with increased fetal NT will experience pregnancy loss, and therefore are exposed to “unnecessary” decisions about invasive testing and pregnancy termination. It should be noted, however, that in another study 69 % of women stated they would still choose NT screening regardless of risk of miscarriage, citing value in the knowledge of an underlying reason for a pregnancy loss, should one occur [101]. Early knowledge of fetal disorders has been shown to improve preparation of parents, and reduces physical and psychological trauma in cases of elective termination; therefore the provision of first-trimester screening is felt by some to significantly enhance the autonomy of pregnant women [102104].

Lenhard et al. found that mothers of children with Down syndrome, diagnosed prenatally, felt discriminated against, as if others implied that the birth of their child “should have been ‘avoided’” via prenatal diagnosis and selective termination of pregnancy [105]. Accordingly, they found that mothers of children with Down syndrome tended to experience the availability of prenatal screening and diagnosis as an emotional burden. However, the improved medical care and psychosocial support for children with Down syndrome and their families may outweigh the emotional stress caused by the option of prenatal screening and diagnosis [106].

Skotko collected opinions regarding prenatal screening and diagnosis from 141 mothers who received a prenatal diagnosis of Down syndrome for their children via amniocentesis. He found that most respondents reported that their birthing experience was positive following a prenatal diagnosis of Down syndrome; by contrast, mothers who learned about the diagnosis after birth labeled their experience as negative [107]. This may be because mothers who receive a prenatal diagnosis may have the chance to resolve any grief prior to their child’s birth. Mothers surveyed felt that results of maternal serum screening should be explained as a risk assessment, with Down syndrome being first explained after the results of a screening test, before invasive prenatal diagnosis is undertaken. Sensitive, accurate, and consistent messages about the range of ability in Down syndrome should be included.

As no therapeutic or curative intervention yet exists for Down syndrome or other chromosomal anomalies, prenatal screening exists to allow women to prepare for the birth and care of a child with chromosome anomalies, create a plan for special-needs adoption, or to pursue the option of pregnancy termination. Based on the results of the survey by Skotko, it is important for healthcare providers to note that not all women who consent to screening believe that having a child with a chromosome anomaly would be an undesired outcome, or would ultimately terminate a pregnancy for this reason. Mothers are more likely to want to consider all options and understand the known prognosis for children with Down syndrome, and gather as much information as possible before making a decision [108]. All reasons for prenatal screening and diagnosis, including reassurance, advance awareness before delivery of a child with a chromosome anomaly, adoption, and pregnancy termination should be discussed with patients in a nondirective manner. Up-to-date information and contact with local support groups for chromosome abnormalities such as Down syndrome should be provided [107].

Teaching Points

·               Multiple methods of aneuploidy screening exist, with varying detection rates, benefits and limitations. These differences may help guide a practitioner to choose a method that is best suited to their patient population based on geography, costs, and socioeconomic factors.

·               First-trimester ultrasound is beneficial, including NT assessment when available, even if maternal biochemical screening is not pursued, as it can provide dating information and risk assessment for nongenetic anomalies and pregnancy outcome.

·               Ideally, informed consent via pretest counseling should be obtained prior to screening to ensure patients’ understanding of the non-diagnostic, risk assessment nature of first-trimester screening and the possibility of identifying a high risk for conditions other than trisomies 13, 18 and 21.

·               Based on the available literature, first-trimester screening is preferred over second-trimester screening by patients, and may serve to increase patient autonomy and improve outcomes for families delivering a child with a prenatally diagnosed chromosome anomaly.

·               Noninvasive DNA screening (NIDS) via cell free fetal DNA analysis provides highly sensitive and specific screen results for the common autosomal aneuploidies (trisomy 21, 18, and 13) in singleton gestations after 9 weeks gestation. However, results are not considered diagnostic as false-positives and false-negative results do occur.

·               Because gestational age and multiple gestations affect fetal fraction and subsequently, interpretation of NIDS results, ACOG recommends performing a baseline ultrasound examination for any patient considering NIDS, prior to testing.

·               The clinical validity of NIDS has been largely studied in a high-risk population (i.e., AMA, abnormal ultrasound or MSS) although limited evidence has suggested performance is similar in a low-risk population.

·               A positive NIDS result requires genetic counseling and confirmation by invasive prenatal diagnosis (preferably amniocentesis) for fetal karyotyping.

NIDS should not be considered a replacement of invasive prenatal diagnosis as results are not definitive and screening is performed only for a select number of aneuploidies. A family history should be reviewed to determine if the patient should be offered other forms of screening or prenatal diagnosis for a particular disorder.



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