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 [24, 25].

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].

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).

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 [31, 32]. 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 [55, 56]. 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].

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 [31, 61, 62]. 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 [33, 63]. 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 [32, 33, 63]. 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].

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.