Aneuploidy is the presence of one or more extra chromosomes, usually resulting in trisomy, or loss of a chromosome—monosomy. Data from population-based registries that include births, fetal deaths, and pregnancy terminations indicate an overall prevalence of 4 such abnormalities per 1000 births (Wellesley, 2012). Aneuploidy accounts for more than 50 percent of first-trimester abortions, about 20 percent of second-trimester losses, and 6 to 8 percent of stillbirths and early-childhood deaths (Reddy, 2012; Stevenson, 2004; Wou, 2016). Of recognized pregnancies with chromosomal abnormalities, trisomy 21 composes approximately half of cases; trisomy 18 accounts for 15 percent; trisomy 13, for 5 percent; and the sex chromosomal abnormalities—45,X, 47,XXX, 47,XXY, and 47,XYY—for approximately 12 percent (Wellesley, 2012).
The risk for fetal trisomy increases with maternal age, particularly after age 35 (Fig. 13-2). When counseling, a provider includes specific maternal-age-related aneuploidy risks (Tables 14-1 and 14-2). Other important fetal aneuploidy risk factors include a numerical chromosomal abnormality or structural chromosomal rearrangement in the woman or her partner—such as balanced translocation—or a prior pregnancy with autosomal trisomy or triploidy.
TABLE 14-1Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Singleton Pregnancies ||Download (.pdf) TABLE 14-1 Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Singleton Pregnancies
| ||Down Syndrome ||Any Aneuploidy |
|Age ||Midtrimester ||Term ||Midtrimester ||Term |
|35 ||1/250 ||1/385 ||1/132 ||1/204 |
|36 ||1/192 ||1/303 ||1/105 ||1/167 |
|37 ||1/149 ||1/227 ||1/83 ||1/130 |
|38 ||1/115 ||1/175 ||1/65 ||1/103 |
|39 ||1/89 ||1/137 ||1/53 ||1/81 |
|40 ||1/69 ||1/106 ||1/40 ||1/63 |
|41 ||1/53 ||1/81 ||1/31 ||1/50 |
|42 ||1/41 ||1/64 ||1/25 ||1/39 |
|43 ||1/31 ||1/50 ||1/19 ||1/30 |
|44 ||1/25 ||1/38 ||1/15 ||1/24 |
|45 ||1/19 ||1/30 ||1/12 ||1/19 |
TABLE 14-2Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Dizygotic Twin Pregnanciesa ||Download (.pdf) TABLE 14-2 Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Dizygotic Twin Pregnanciesa
| ||Down Syndrome ||Any Aneuploidy |
|Age ||Midtrimester ||Term ||Midtrimester ||Term |
Broadly speaking, there are two types of aneuploidy screening tests, those that are traditional or analyte-based and those that are cell-free DNA-based. All pregnant women should be offered aneuploidy screening or diagnostic testing early in pregnancy (American College of Obstetricians and Gynecologists, 2016c). Considerations prior to screening are as follows:
Has the patient elected screening? At least 20 percent of women elect not to receive aneuploidy screening, even when financial barriers are removed. Fewer than 40 percent of women with a positive screening result elect prenatal diagnosis (Dar, 2014; Kuppermann, 2014).
Would the patient prefer prenatal diagnosis? Diagnostic testing is safe and effective, and chromosomal microarray analysis provides information about genetic conditions that screening tests and karyotyping alone currently do not (American College of Obstetricians and Gynecologists, 2016b). This is discussed further in Prenatal Diagnostic Procedures and Preimplantation Testing and in Chapter 13 (Chromosomal Microarray Analysis).
Is this a multifetal gestation? All traditional (analyte-based) aneuploidy screening tests are significantly less effective in multifetal gestations, and cell-free DNA screening is not currently recommended with multifetal gestations (Cell-Free DNA for Secondary Screening).
What method will be used for neural-tube defect screening? Whenever a patient elects an aneuploidy screening test that does not include second-trimester serum analytes, screening for neural-tube defects should be performed separately, either with MSAFP assessment or with sonography (American College of Obstetricians and Gynecologists, 2016c).
Does the fetus have a major anomaly? If so, diagnostic testing is recommended rather than screening.
The American College of Obstetricians and Gynecologists (2016c) has affirmed that screening for aneuploidy should be an informed patient choice, with an underlying foundation of shared decision making that fits her clinical circumstances, values, interests, and goals. Elements of counseling prior to aneuploidy screening are listed in Table 14-3.
TABLE 14-3Aneuploidy Screening Counseling Elements ||Download (.pdf) TABLE 14-3 Aneuploidy Screening Counseling Elements
All pregnant women have 3 options: screening, diagnostic testing, and no screening or testing.
The purpose of a screening test is to provide information, not to dictate a course of action.
Diagnostic testing is safe and effective and provides information that screening does not.
The difference between a screening test and a diagnostic test.
Screening evaluates whether the pregnancy is at increased risk and estimates degree of risk.
Detection rate, false-negative rate, and false-positive rate are provided.
Cell-free DNA screening does not always provide a result.
Irreversible management decisions should not be based on screening test results.
With a positive screening result, a diagnostic test is recommended if the patient wants to know whether the fetus is affected.
Basic information is provided about each condition covered by the screening test (prevalence, associated abnormalities, prognosis), and screening test limitations.
A benefit of diagnosis includes earlier identification of associated abnormalities.
In case of trisomy 18 or 13, diagnosis may affect pregnancy management if complications arise such as growth restriction or nonreassuring fetal heart rate.
With sex chromosome aneuploidies, phenotypic expression varies widely. Several are so mild they would otherwise not be diagnosed.
The patient’s a priori risk for fetal aneuploidy may affect her screening test options or election.
Age-related risk information may be found in reference tables.
If a patient has had a prior fetus with autosomal trisomy, robertsonian translocation, or other chromosomal abnormality, additional evaluation and counseling are suggested.
Aneuploidy screening can be challenging because the test characteristics of each option may vary with maternal age and with whether the test is analyte-based or cell-free DNA-based.
The test sensitivity is the detection rate—that is, the proportion of aneuploid fetuses identified by the screening test. Its converse, the false-negative rate, is the percentage of cases that the test is expected to miss. A first-trimester screening test with a sensitivity of 80 percent is expected to miss 1 in 5 cases. The sensitivity of screening tests for Down syndrome has increased steadily during the past 30 years, from just 25 percent with serum AFP alone to more than 90 percent with integrated or sequential screening.
Another key characteristic is the false-positive rate, the percentage of unaffected pregnancies that will “falsely” screen positive. This approximates 5 percent for first-trimester screening, quadruple-marker screening, or integrated screening options (Baer, 2015; Kazerouni, 2011; Malone, 2005b; Norton, 2015). The converse of false-positive rate is specificity— analyte-based screens will be reassuring in approximately 95 percent of unaffected pregnancies. Although test sensitivity has improved, the false-positive rate has been held constant for many different aneuploidy screening tests (Table 14-4). Both statistics are relevant for counseling. An additional consideration is that with all analyte-based screening tests, women 35 and older have higher rates of positive results (Kazerouni, 2011; Malone, 2005b).
TABLE 14-4Characteristics of Screening Tests for Trisomy 21 in Singletons ||Download (.pdf) TABLE 14-4 Characteristics of Screening Tests for Trisomy 21 in Singletons
|Screening Test ||Detection Rate ||False-Positive Rate ||Positive-Predictive Valuea |
|Quadruple screen: |
| AFP, hCG, estriol, inhibin ||80–82% ||5% ||3% |
|First-trimester screen: |
| NT, hCG, PAPP-A ||80–84% ||5% ||3–4% |
| First-trimester NT alone ||64–70% ||5% || |
|Integrated screening ||94–96% ||5% ||5% |
|Sequential screening: |
| Stepwise ||92% ||5.1% ||5% |
| Contingent ||91% ||4.5% ||5% |
|Cell-free DNA screening: |
| Positive result ||99% ||0.1% ||Table 14-5 |
| Low fetal fraction or no result ||— ||4–8% ||4% |
Importantly, neither sensitivity nor false-positive rate conveys individual risk. The statistic that patients and providers usually consider to be the test result is the positive-predictive value, which is the proportion of those with a positive screening result who actually have an aneuploid fetus. It may be expressed as a 1:X ratio or as a percentage. The positive-predictive value is directly affected by disease prevalence, so it is much higher for women aged 35 years and older than for younger women (Table 14-5). Positive-predictive values can also be reported for cohorts of pregnancies. For example, the positive-predictive value reported in a research trial is the proportion of women with positive screening results who have affected fetuses (see Table 14-4). Negative-predictive value is the proportion of those with a negative screening test result who have unaffected (euploid) fetuses. Because the prevalence of aneuploidy is so low, the negative-predictive value of all aneuploidy screening tests generally exceeds 99 percent (Gil, 2015; Norton, 2015).
TABLE 14-5Positive-Predictive Value of Cell-Free DNA Screening for Autosomal Trisomies and Selected Sex Chromosome Abnormalities, According to Maternal Age ||Download (.pdf) TABLE 14-5 Positive-Predictive Value of Cell-Free DNA Screening for Autosomal Trisomies and Selected Sex Chromosome Abnormalities, According to Maternal Age
|Maternal Age ||Trisomy 21 ||Trisomy 18 ||Trisomy 13 ||45,X ||47,XXY |
|20 ||48% ||14% || 6% ||41% ||29% |
|25 ||51% ||15% || 7% ||41% ||29% |
|30 ||61% ||21% ||10% ||41% ||29% |
|35 ||79% ||39% ||21% ||41% ||30% |
|40 ||93% ||69% ||50% ||41% ||52% |
|45 ||98% ||90% ||NA ||41% ||77% |
Traditional Aneuploidy Screening Tests
These screening tests have multiple markers or analytes and are also called conventional or traditional to differentiate them from cell-free DNA-based screening. There are three categories: first-trimester screens, second-trimester screens, and combinations of first- and second-trimester screens. If the test has a first-trimester component, it almost always includes a measurement of the sonographic nuchal translucency, which is discussed in the next section.
Each maternal serum analyte is measured as a concentration—for example, nanograms per milliliter of AFP. The concentration is converted to a multiple of the median (MoM) by adjusting for maternal age, maternal weight, and gestational age. The nuchal translucency measurement increases with crown-rump length (CRL), and thus its value is adjusted for CRL and also reported as an MoM. The AFP analyte is further adjusted for maternal race and ethnicity and for presence of diabetes, which all affect the calculation of neural-tube defect risk rather than of aneuploidy risk (Greene, 1988; Huttly, 2004). Reporting these results as an MoM of the unaffected population normalizes the distribution of analyte levels and permits comparison of results from different laboratories and populations.
The analyte-based aneuploidy screening result is based on a composite likelihood ratio, and the maternal age-related risk is multiplied by this ratio. This principle similarly applies to modification of fetal Down syndrome risk by selected sonographic markers, which are discussed later in Sonographic Screening. Each woman is provided with a specific risk for trisomy 21 and for trisomy 18—or in the first trimester, for trisomy 18 or 13 in some cases. The result is expressed as a ratio that represents the positive-predictive value.
Importantly, each screening test also has a predetermined value at which or above which it is deemed “positive” or abnormal. For second-trimester tests, this threshold has traditionally been set at the risk for fetal Down syndrome in a woman aged 35 years—approximately 1 in 270 in the second trimester (see Table 14-1). The threshold selected for a positive screen reflects the laboratory requirement but is somewhat problematic, as it may bear no relationship to patient preference. However, a positive screening result may affect whether the patient is deemed “high risk,” receives formal genetic counseling, and is offered diagnostic testing with chorionic villus sampling or amniocentesis. Thus, it behooves the provider to discuss the patient’s preferences prior to screening.
First-Trimester Aneuploidy Screening
Also called combined first-trimester screening, this test combines two maternal serum analytes, human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A), with the sonographic measurement of the nuchal translucency (NT). It is performed between 11 and 14 weeks’ gestation. In cases of fetal Down syndrome, the first-trimester serum free β-hCG level is higher and the PAPP-A level is lower. With trisomy 18 and trisomy 13, levels of both analytes are lower (Cuckle, 2000; Malone, 2005b).
This is the maximum thickness of the subcutaneous translucent area between the skin and soft tissue overlying the fetal spine at the back of the neck (Fig. 14-2). An increased NT thickness is not a fetal abnormality but rather is a marker that confers increased risk. It is measured in the sagittal plane and is valid when the CRL value lies between 38 to 45 mm and 84 mm, with the lower limit varying according to the laboratory. Specific criteria for NT measurement are listed in Table 10-4. Whenever possible, it is helpful to differentiate increased NT from cystic hygroma, which is a venolymphatic malformation that appears as a septated hypoechoic space behind the neck, extending along the length of the back (Fig. 10-22). Cystic hygroma confers a fivefold increased aneuploidy risk when identified in the first trimester (Malone, 2005a).
Sagittal image of a normal, 12-week fetus demonstrating correct caliper placement (+) for nuchal translucency measurement. The fetal nasal bone and overlying skin are indicated. The nasal tip and the 3rd and 4th ventricles (asterisk), which are other landmarks that should be visible in the nasal bone image, are also shown. (Used with permission from Dr. Michael Zaretsky.)
In addition to aneuploidy, an increased NT thickness is associated with other genetic syndromes and various birth defects, especially fetal cardiac anomalies (Simpson, 2007). And, if the NT measurement reaches 3 mm or more, the aneuploidy risk is unlikely to normalize using serum analyte assessment (Comstock, 2006). Because of this, if the NT measurement is at least 3 mm or exceeds the 99th percentile, the patient should receive counseling and be offered targeted sonography with fetal echocardiography. In addition, she should be offered cell-free DNA screening and prenatal diagnosis (American College of Obstetricians and Gynecologists, 2016c).
The NT must be imaged and measured with a high degree of precision for aneuploidy detection to be accurate. This has led to standardized training, certification, and ongoing quality review programs. In the United States, training, credentialing, and monitoring are available through the Nuchal Translucency Quality Review program of the Perinatal Quality Foundation and through the Fetal Medicine Foundation.
Efficacy of First-Trimester Screening
Before first-trimester screening became widely adopted, four large prospective trials were conducted, together including more than 100,000 pregnancies (Reddy, 2006). When the false-positive rate was set at 5 percent, the overall rate for trisomy 21 detection was 84 percent, comparable to quadruple-marker screening (see Table 14-4). The detection rate is approximately 5 percent higher if performed at 11 weeks’ compared with 13 weeks’ gestation, and slightly lower—80 to 82 percent—when cases with cystic hygroma are analyzed separately (Malone, 2005a). In a recent multicenter trial, first-trimester screening detected approximately 80 percent of fetuses with trisomy 21, 80 percent with trisomy 18, and 50 percent with trisomy 13 (Norton, 2015).
As an isolated marker, NT detects approximately two thirds of fetuses with Down syndrome, with a false-positive rate of 5 percent (Malone, 2005b). However, NT is generally used as an isolated marker only in screening for multifetal gestations, in which serum screening is less accurate or may not be available. The NT distribution is similar in twins and singletons (Cleary-Goldman, 2005). In twin pregnancies, serum free β‑hCG and PAPP-A levels are approximately double the singleton values (Vink, 2012). Even with specific curves, a normal dichorionic cotwin will tend to normalize screening results, and thus, the aneuploidy detection rate is at least 15-percent lower (Bush, 2005).
Maternal age affects the performance of first-trimester aneuploidy screening. Prospective trials have demonstrated Down syndrome detection rates of 67 to 75 percent in women younger than 35 years at delivery, which are 10 percent lower than the overall detection rates in these studies (Malone, 2005b; Wapner, 2003). Among women older than 35 at delivery, Down syndrome detection reached 90 to 95 percent, albeit at a higher false-positive rate of 15 to 22 percent.
Unexplained Abnormalities of First-Trimester Analytes
There is a significant association between serum PAPP-A levels below the 5th percentile and preterm birth, growth restriction, preeclampsia, and fetal demise (Cignini, 2016; Dugoff, 2004; Jelliffe-Pawlowski, 2015). Similarly, low levels of free β-hCG have been associated with fetal demise (Goetzl, 2004). The sensitivity and positive-predictive values of isolated markers are generally too low to make them clinically useful as screening tests.
There has been renewed interest in low-dose aspirin for prevention of early preeclampsia in women identified as at risk based on mean arterial pressure, uterine artery Doppler values, and PAPP-A levels. However, these observations are preliminary (Park, 2015).
Second-Trimester Aneuploidy Screening
Currently, the only second-trimester multiple marker test widely used in the United States is the quadruple marker or “quad” screening test. It is performed from 15 to 21 weeks’ gestation, and inclusive gestational age ranges vary according to individual laboratories. Pregnancies with fetal Down syndrome are characterized by lower maternal serum AFP, higher hCG, lower unconjugated estriol, and higher dimeric inhibin levels. When the quad screen was initially described, the Down syndrome detection rate approximated 70 percent. But, by the early 2000s, the reported detection rate in two large prospective trials had improved to 81 to 83 percent, with a 5-percent screen-positive rate (Malone, 2005b; Wald, 1996, 2003). The improved detection rate is attributable, at least in part, to accurate gestational age assessment with sonography. In a review of more than 500,000 pregnancies receiving quadruple-marker screening through the statewide California Prenatal Screening Program, trisomy 21 detection was 78 percent with sonographic gestational age assessment but only 67 percent when the screen was calculated based on last menstrual period alone (Kazerouni, 2011). As with first-trimester screening, aneuploidy detection rates are lower in younger women and higher in women older than 35 years at delivery. If second-trimester serum screening is used in twin pregnancies, aneuploidy detection rates are significantly lower (Vink, 2012). With trisomy 18, the levels of the first three analytes are all decreased, and inhibin is not part of the calculation. Trisomy 18 detection is similar to that for Down syndrome, with a false-positive rate of only 0.5 percent (Benn, 1999).
Although the quadruple-marker screening test is used to screen for Down syndrome and trisomy 18, pregnancies with other chromosomal abnormalities may be identified as well. The California Prenatal Screening Program found that the quadruple-marker screen result was abnormal in 96 percent of those with triploidy, in 75 percent with Turner syndrome (45,X), in 44 percent with trisomy 13, and in more than 40 percent of those with other major chromosomal abnormalities (Kazerouni, 2011). Although a specific risk for these aneuploidies cannot be provided based on the test result, the information may be relevant for women considering amniocentesis.
Quadruple-marker screening offers no benefit over first-trimester screening from the standpoint of trisomy 21 or trisomy 18 detection. As a stand-alone test, it is generally used if women do not begin care until the second trimester or if first-trimester screening is not available. In 2011, women who initiated prenatal care beyond the first trimester made up nearly 25 percent of pregnancies in the United States. As subsequently discussed, combining first- and second-trimester screening yields an even greater aneuploidy detection rate.
Maternal Serum AFP Elevation: Neural-Tube Defect Screening
All pregnant women are offered screening for fetal open neural-tube defects in the second trimester, either with MSAFP screening or with sonography (American College of Obstetricians and Gynecologists, 2016c). Measurement of the MSAFP concentration between 15 and 20 weeks’ gestation has been offered as part of routine prenatal care for more than 30 years. Because AFP is the major protein in fetal serum, analogous to albumin in a child or adult, the normal concentration gradient between fetal plasma and maternal serum is on the order of 50,000:1. Defects in fetal integument, such as neural-tube and ventral-wall defects, permit AFP to leak into the amnionic fluid, resulting in dramatically increased maternal serum levels. The AFP value rises by about 15 percent per week during the screening window (Knight, 1992). The MoM is generally recalculated if the first-trimester CRL or second-trimester biparietal diameter differs from the stated gestational age by more than 1 week.
Using an MSAFP level of 2.5 MoM as the upper limit of normal, the neural-tube defect detection rate is at least 90 percent for anencephaly and 80 percent for spina bifida, with a screen-positive rate of 3 to 5 percent (American College of Obstetricians and Gynecologists, 2016a; Milunsky, 2004). Higher screening threshold values are used in twin pregnancies (Cuckle, 1990).
Virtually all cases of anencephaly and many cases of spina bifida may be detected or suspected during a standard second-trimester sonographic examination (Dashe, 2006). Most centers now use targeted sonography as the primary method to evaluate elevated MSAFP levels and as the prenatal diagnostic test of choice for neural-tube defects (Chap. 10, Neural-Tube Defects). If targeted sonography is not available and myelomeningocele cannot be excluded, amniocentesis may be considered for measurement of amnionic fluid AFP and acetylcholinesterase levels. That said, we recommend additional imaging prior to establishing the diagnosis, with the understanding that other abnormalities or conditions can result in elevation of these amnionic fluid analytes (Table 14-6). Sonographic findings characteristic of fetal neural-tube defects are reviewed in Chapter 10 (Neural-Tube Defects). Fetal surgery for myelomeningocele is discussed in Chapter 16 (Open Fetal Surgery).
TABLE 14-6Conditions Associated with an Elevated MSAFP Concentration ||Download (.pdf) TABLE 14-6 Conditions Associated with an Elevated MSAFP Concentration
|Underestimated gestational age |
Esophageal or intestinal obstruction
Renal anomalies—polycystic kidneys, renal agenesis, congenital nephrosis, urinary tract obstruction
Congenital skin abnormality
Chorioangioma of placenta
Placenta intervillous thrombosis
Maternal hepatoma or teratoma
Unexplained Abnormalities of Second-Trimester Analytes
The positive-predictive value of an elevated MSAFP value is only 2 percent. Approximately 98 percent of pregnancies with an MSAFP level exceeding 2.5 MoM have an etiology other than a neural-tube defect. Thus, counseling is indicated not only to inform the patient about the benefits and limitations of targeted sonography for the diagnosis of neural-tube defects but also to review the numerous other conditions. Some of these include fetal anomalies, placental abnormalities, and adverse outcomes associated with MSAFP level elevation (see Table 14-6). The likelihood of one of these abnormalities or of an adverse pregnancy outcome in the absence of a recognized abnormality rises in proportion to the AFP level. Adverse outcomes include fetal-growth restriction, preeclampsia, preterm birth, fetal demise, and stillbirth. More than 40 percent of pregnancies may be abnormal if the MSAFP level is greater than 7 MoM (Reichler, 1994).
Second-trimester elevation of either hCG or dimeric inhibin alpha levels also shows significant association with adverse pregnancy outcomes. The outcomes reported are similar to those associated with MSAFP level elevation. Moreover, the likelihood of adverse outcome is augmented when levels of several markers are elevated (Dugoff, 2005).
Many of these complications are assumed to result from placental damage or dysfunction. However, the sensitivity and positive-predictive values of these markers are considered too low to be useful for screening or management. No specific program of maternal or fetal surveillance has been found to favorably affect pregnancy outcomes (Dugoff, 2010). At Parkland Hospital, prenatal care for these women is not altered unless a specific complication arises. Despite the extensive list of possible adverse outcomes, it is reassuring that most women with unexplained elevation of these analytes have normal outcomes.
Low Maternal Serum Estriol Level
A maternal serum estriol level less than 0.25 MoM has been associated with two uncommon but important conditions. The first, Smith-Lemli-Opitz syndrome, is an autosomal recessive condition resulting from mutations in the 7-dehydrocholesterol reductase gene. It is characterized by abnormalities of the central nervous system, heart, kidney, and extremities; with ambiguous genitalia; and with fetal-growth restriction. For this reason, the Society for Maternal-Fetal Medicine has recommended that sonographic evaluation be performed if an unconjugated estriol level is <0.25 MoM (Dugoff, 2010). If abnormalities are identified, an elevated amnionic fluid 7-dehydrocholesterol level can confirm the diagnosis.
The second condition is steroid sulfatase deficiency, also known as X-linked ichthyosis. It is typically an isolated condition, but it may also occur in the setting of a contiguous gene deletion syndrome (Chap. 13, Abnormalities of Chromosome Structure). In such cases, it may be associated with Kallmann syndrome, chondrodysplasia punctata, and/or mental retardation (Langlois, 2009). If the estriol level is <0.25 MoM and the fetus appears to be male, chromosomal microarray analysis or fluorescence in situ hybridization to assess the steroid sulfatase locus on the X-chromosome may be considered.
Integrated and Sequential Screening
As shown in Table 14-4, if first-trimester screening is combined with second-trimester screening, aneuploidy detection is significantly improved. Combined screening test options require coordination between the provider and laboratory. Specifically, if a second sample is required, it is obtained during the appropriate gestational age window, sent to the same laboratory, and linked to the first-trimester results. The first- and second-trimester components cannot be performed independently because if either component yields positive results, then providing accurate risk assessment would be problematic.
Three types of screening strategies are available:
Integrated screening combines results of first- and second-trimester tests. This includes a combined measurement of fetal NT and serum analyte levels at 11 to 14 weeks’ gestation plus quadruple markers at approximately 15 to 21 weeks. An aneuploidy risk is then calculated from these seven parameters. As expected, integrated screening has the highest Down syndrome detection rate—94 to 96 percent, with a false-positive rate of 5 percent (see Table 14-4). If NT measurement is not available, then serum integrated screening includes all six serum markers to calculate risk. This screening, however, is less effective, and Down syndrome detection rates are 85 to 88 percent (Malone, 2005b).
Sequential screening involves performing first-trimester screening and informing the patient of the results. This is coupled with the understanding that if the calculated risk value lies above a specified threshold, she will receive counseling and will be offered diagnostic testing. There are two testing strategies in this category:
With stepwise sequential screening, women with first-trimester screen results that confer risk for Down syndrome above a particular threshold are offered invasive testing, and the remaining women receive second-trimester screening. Using data from the First- and Second-Trimester Evaluation of Risk (FaSTER) trial, when the first-trimester threshold is set at approximately 1:30, and the overall threshold is set at 1:270, stepwise sequential screening resulted in a 92-percent detection rate of Down syndrome pregnancies, with a false-positive rate of 5 percent (see Table 14-4) (Cuckle, 2008).
With contingent sequential screening, women are divided into high-, moderate-, and low-risk groups. Those at highest risk for Down syndrome—for example, risk >1:30, are counseled and offered invasive testing. Women at moderate risk, between 1:30 and 1:1500, undergo second-trimester screening, whereas those at lowest risk of <1:1500 receive negative screening test results and have no further testing (Cuckle, 2008). Using this strategy, more than 75 percent of those screened are provided with reassuring results almost immediately, while still maintaining a high detection rate of about 91 percent, with a 5-percent false-positive rate (see Table 14-4). This option is also more cost effective because a second-trimester test is obviated in most patients.
In a population-based review of 450,000 pregnancies from the California Prenatal Screening Program, integrated screening detected 94 percent of trisomy 21 fetuses and 93 percent with trisomy 18 (Baer, 2015). Additionally, the screening result was abnormal in 93 percent of cases of trisomy 13, in 91 percent with triploidy, and in 80 percent with Turner syndrome. Women considering options of integrated screening and cell-free DNA screening may find this information helpful.
This was introduced in 2011 and has completely changed the prenatal screening paradigm. The test works by identifying DNA fragments that are derived primarily from apoptotic trophoblasts, which are placental cells undergoing programmed cell death. Thus, the term cell-free fetal DNA is somewhat of a misnomer. Screening is not gestational-age dependent and can be performed at any time after 9 to 10 weeks’ gestation. Results are available in 7 to 10 days (American College of Obstetricians and Gynecologists, 2017c). Three types of assays are currently available: whole-genome sequencing, which is also called massively parallel or shotgun sequencing; chromosome selective or targeted sequencing; and analysis of single nucleotide polymorphisms.
The screening performance of cell-free DNA is excellent. In a metaanalysis of 37 studies of largely high-risk pregnancies, the pooled sensitivity to detect Down syndrome was 99 percent, and for trisomies 18 and 13, 96 percent and 91 percent, respectively. For each of these autosomal trisomies, the specificity was 99.9 percent. Thus, most unaffected pregnancies received a normal screening result. Cell-free DNA also detects 90 percent with Turner syndrome (45,X) and 93 percent with sex chromosome aneuploidies other than 45,X (Gil, 2015). The false-positive rate is cumulative for each aneuploidy for which screening is performed, but it is usually only 0.5 to 1 percent. As a result, cell-free DNA screening is recommended as a screening option in those at increased risk for fetal autosomal trisomy (American College of Obstetricians and Gynecologists, 2017c; Society for Maternal-Fetal Medicine, 2015). This includes the following categories:
Women who will be 35 years or older at delivery
A positive first- or second-trimester analyte-based screening test
Sonogram with a minor aneuploidy marker
Prior pregnancy with autosomal trisomy
Known carriage (patient or partner) of a balanced robertsonian translocation involving chromosome 21 or 13.
Cell-Free DNA for Secondary Screening
If cell-free DNA screening is performed as a secondary screen following a positive first- or second-trimester analyte-based test result, a normal result is not quite as reassuring. The residual risk for chromosomal abnormality is estimated to be 2 percent (Norton, 2014). Compared with amniocentesis, use of cell-free DNA screening after an initial abnormal analyte-based test result is estimated to yield a 20-percent reduction in aneuploidy diagnoses. This takes into consideration false-negative diagnoses and aneuploidies not detectable with cell-free DNA screening (Davis, 2014; Norton, 2014). In addition, definitive diagnosis may be delayed, potentially affecting management. Concurrent or parallel screening is not recommended, and if an aneuploidy screening test of any type yields a negative result, additional screening is not indicated (American College of Obstetricians and Gynecologists, 2016b, 2017c).
The association between increased NT values and fetal structural and genetic abnormalities has raised questions about the role of a NT measurements following cell-free DNA screening. The College (2016b) has stated that NT measurement is not necessary at the time of cell-free DNA screening but that sonography may help to confirm fetal number and viability and to assign gestational age. The Society for Maternal-Fetal Medicine (2015) states that after a negative cell-free DNA screening test result, the additional clinical utility of NT measurement to detect other chromosomal or structural abnormalities is unknown but appears to be limited.
Cell-Free DNA Screening in Low-Risk Pregnancies
Most studies of cell-free DNA have been conducted in high-risk pregnancies. Pragmatically, chromosomal abnormalities are individually so rare that even large studies of low-risk pregnancies contain few affected cases. Available data suggest that the high sensitivity and specificity for Down syndrome detection are preserved in low-risk pregnancies (Norton, 2015; Pergament, 2014; Zhang, 2015). Importantly, the positive-predictive value of cell-free DNA screening still depends greatly on maternal age and the specific aneuploidy in question (see Table 14-5). For a woman in her early 20s, the positive-predictive value is approximately 50 percent for fetal trisomy 21, 15 percent for trisomy 18, and <10 percent for trisomy 13. Hence, decisions for irreversible medical intervention should not be based on the results of this or other screening test alone.
Cell-Free DNA Screening Limitations
Important caveats are considered with selection of cell-free DNA aneuploidy screening. Because the cell-free DNA that is analyzed is maternal and placental, results may not reflect the fetal DNA complement but rather may indicate confined placental mosaicism, early demise of an aneuploid co- twin, maternal mosaicism, or even occult maternal malignancy (Bianchi, 2015; Curnow, 2015; Grati, 2014; Wang, 2014). In addition, if a twin pregnancy is identified sonographically, cell-free DNA screening is not currently recommended because of limited evidence regarding efficacy.
Another limitation is that cell-free DNA testing does not yield a result in approximately 4 to 8 percent of screened pregnancies, due to assay failure, high assay variance, or low fetal fraction (Norton, 2012; Pergament, 2014; Quezada, 2015). Most cell-free DNA is maternal. The fetal fraction is the proportion derived from the placenta and generally is about 10 percent of the total. A low fetal fraction is usually defined as <4 percent of the total and confers significantly higher risk for fetal aneuploidy (Ashoor, 2013; Norton, 2015; Pergament, 2014). Women with a low fetal fraction or “no-call” results have fetal aneuploidy rates as high as 4 percent, a percentage comparable to the average predictive value conferred by a positive first-trimester screening test result (see Table 14-4). The fetal fraction is not related to maternal age or analyte-based screening test results. However, it is lower earlier in pregnancy and appears to be reduced in women of greater weight (Ashoor, 2013).
Because of the increased risk for fetal aneuploidy in cases not generating a cell-free DNA screening result (no-call result), genetic counseling is indicated, and amniocentesis should be offered. If the patient elects repeat screening, the risk for screen failure may exceed 40 percent (Dar, 2014; Quezada, 2015). Targeted sonography is recommended but is not a substitute for amniocentesis, because it is unclear what the residual risk would be with normal sonogram findings. (American College of Obstetricians and Gynecologists, 2016b, 2017c). Pretest counseling should include the possibility of a low fetal fraction or no-call result and its clinical significance.
Comparison with Analyte-Based Screening
Cell-free DNA screening has obvious advantages, but it is not simply a “better” test—because no screening test is superior for all test characteristics (American College of Obstetricians and Gynecologists, 2016c). Compared with analyte-based tests, benefits of cell-free DNA screening in women 35 years and older include the lower likelihood of a false-positive result, its higher positive-predictive value, and the fact that isolated minor aneuploidy markers are generally not a concern (Sonographic Screening).
But, analyte-based tests are frequently positive with a large range of chromosomal abnormalities, whereas cell-free DNA screens are specific for individual aneuploidies (Baer, 2015; Kazerouni, 2011). Women younger than 35 are at lower risk for the specific autosomal trisomies for which cell-free DNA screening is typically performed. Thus, if the goal is to select a screening test that will identify the highest proportion of fetuses with any chromosomal abnormality, the yield may be comparable or even slightly higher with integrated or sequential screening than with current cell-free DNA screening (Baer, 2015; Norton, 2014).
Sonography can augment aneuploidy screening by providing accurate gestational age assessment, by detecting multifetal gestations, and by identifying major structural abnormalities and minor sonographic markers. As shown in Table 14-7, with rare exceptions, the aneuploidy risk associated with any major abnormality is high enough to warrant offering prenatal diagnosis. Generally, chromosomal microarray analysis is recommended as the first-line test. Importantly, a fetus with one abnormality may have others that are less likely to be detected sonographically but that greatly affect the prognosis. Aneuploidy screening—including cell-free DNA—is not recommended if a major abnormality has been identified. The fetal risk cannot be normalized with a normal screening result, not merely because screening results can be falsely negative, but also because major anomalies confer risk for genetic syndromes not identified through screening tests.
TABLE 14-7Aneuploidy Risk Associated with Selected Major Fetal Anomalies ||Download (.pdf) TABLE 14-7 Aneuploidy Risk Associated with Selected Major Fetal Anomalies
|Abnormality ||Birth Prevalence ||Aneuploidy Risk (%) ||Common Aneuploidiesa |
|Cystic hygroma ||1/5000 ||50–70 ||45,X; 21; 18; 13; triploidy |
|Nonimmune hydrops ||1/1500–4000 ||10–20 ||21, 18, 13, 45X, triploidy |
|Ventriculomegaly ||1/1000–2000 ||5–25 ||13, 18, 21, triploidy |
|Holoprosencephaly ||1/10,000–15,000 ||30–40 ||13, 18, 22, triploidy |
|Dandy-Walker malformation ||1/12,000 ||40 ||18, 13, 21, triploidy |
|Cleft lip/palate ||1/1000 ||5–15 ||18, 13 |
|Cardiac defects ||5–8/1000 ||10–30 ||21; 18; 13; 45,X; 22q11.2 microdeletion |
|Diaphragmatic hernia ||1/3000–4000 ||5–15 ||18, 13, 21 |
|Esophageal atresia ||1/4000 ||10 ||18, 21 |
|Duodenal atresia ||1/10,000 ||30 ||21 |
|Gastroschisis ||1/2000–4000 ||No increase || |
|Omphalocele ||1/4000 ||30–50 ||18, 13, 21, triploidy |
|Clubfoot ||1/1000 ||5–30 ||18, 13 |
If a major abnormality is identified, targeted sonography is indicated. Sonography is not an alternative to prenatal diagnosis, but the aneuploidy risk is further increased if additional findings are identified. An earlier study reported that only 25 to 30 percent of second-trimester fetuses with Down syndrome had a major malformation that could be identified sonographically (Vintzileos, 1995). When both major anomalies and minor aneuploidy markers are considered, it is estimated that 50 to 60 percent of Down syndrome pregnancies can be detected sonographically (American College of Obstetricians and Gynecologists, 2016c). Fortunately, most fetuses with aneuploidy that is likely to be lethal in utero—such as trisomy 18 and 13 and triploidy—usually have sonographic abnormalities that can be seen by the second trimester.
Second-Trimester Markers—“Soft Signs”
For three decades, investigators have recognized that the sonographic detection of aneuploidy, particularly Down syndrome, may be improved by minor markers that are collectively referred to as “soft signs.” Minor markers are normal variants rather than fetal abnormalities, and in the absence of aneuploidy or an associated abnormality, they do not significantly affect prognosis. They are present in at least 10 percent of unaffected pregnancies (Bromley, 2002; Nyberg, 2003). Examples of these sonographic findings are listed in Table 14-8 and depicted in Figure 14-3. Findings are generally useful from 15 to 20 or 22 weeks’ gestation. Six of these markers have been the focus of sonographic studies, in which likelihood ratios have been derived that allow a numerical aneuploidy risk to be calculated (Table 14-9). The risk rises steeply with the number of markers identified. Alternatively, absence of a minor marker has been used to reduce the calculated risk (Agathokleous, 2013). This should be done systematically, following a protocol that specifies the markers included in a model, the definition for what constitutes a finding, and positive- and negative-likelihood ratios (Reddy, 2014).
TABLE 14-8Second-Trimester Sonographic Markers or “Soft Signs” Associated with Fetal Trisomy 21a ||Download (.pdf) TABLE 14-8 Second-Trimester Sonographic Markers or “Soft Signs” Associated with Fetal Trisomy 21a
|Aberrant right subclavian artery |
Brachycephaly or shortened frontal lobe
Clinodactyly (hypoplasia of the 5th digit middle phalanx)
Echogenic intracardiac focus
Nasal bone absence or hypoplasia
Nuchal fold thickening
Renal pelvis dilation (mild)
“Sandal gap” between first and second toes
Shortened ear length
Single transverse palmar crease
Single umbilical artery
Widened iliac angle
TABLE 14-9Likelihood Ratios and False-Positive Rates for Isolated Second-Trimester Markers Used in Down Syndrome Screening Protocols ||Download (.pdf) TABLE 14-9 Likelihood Ratios and False-Positive Rates for Isolated Second-Trimester Markers Used in Down Syndrome Screening Protocols
|Sonographic Marker ||Likelihood Ratio ||Prevalence in Unaffected Fetuses (%) |
|Nuchal skinfold thickening ||11–17 ||0.5 |
|Renal pelvis dilation ||1.5–1.9 ||2.0–2.2 |
|Echogenic intracardiac focus ||1.4–2.8 ||3.8–3.9a |
|Echogenic bowel ||6.1–6.7 ||0.5–0.7 |
|Short femur ||1.2–2.7 ||3.7–3.9 |
|Short humerus ||5.1–7.5 ||0.4 |
|Any one marker ||1.9–2.0 ||10.0–11.3 |
|Two markers ||6.2–9.7 ||1.6–2.0 |
|Three or more ||80–115 ||0.1–0.3 |
Minor sonographic markers that are associated with increased risk for fetal Down syndrome. A. Nuchal skinfold thickening (bracket). B. Echogenic intracardiac focus (arrow). C. Mild renal pelvis dilatation (pyelectasis) (arrows). D. Echogenic bowel (arrow). E. Clinodactyly—hypoplasia of the 5th finger middle phalanx creates an inward curvature (arrow). F. “Sandal-gap” (arrow).
The nuchal skinfold is measured in the transcerebellar view of the fetal head, from the outer edge of the skull to the outer border of the skin (see Fig. 14-3A). A measurement ≥6 mm is typically considered abnormal (Benacerraf, 1985). This finding is present in approximately 1 per 200 pregnancies and confers a more than tenfold risk for Down syndrome (Bromley, 2002; Nyberg, 2001; Smith-Bindman, 2001).
An echogenic intracardiac focus is a focal papillary muscle calcification that is neither a structural nor functional cardiac abnormality. It is usually left-sided (see Fig. 14-3B). Such a focus is present in approximately 4 percent of fetuses, but it may be found in up to 30 percent of Asian individuals (Shipp, 2000). As an isolated finding, this approximately doubles the risk for fetal Down syndrome (see Table 14-9). Bilateral echogenic foci are associated with trisomy 13 (Nyberg, 2001).
Mild renal pelvis dilatation is usually transient or physiological and does not represent an underlying abnormality (Chap. 10, Renal Pelvis Dilatation). The renal pelves are measured in a transverse image of the kidneys, anterior-to-posterior, with calipers placed at the inner borders of the fluid collection (see Fig. 14-3C). A measurement ≥4 mm is found in about 2 percent of fetuses and approximately doubles the risk for Down syndrome. The degree of pelvic dilatation beyond 4 mm correlates with the likelihood of an underlying renal abnormality, and additional evaluation is generally performed at approximately 32 weeks.
Echogenic fetal bowel is defined as bowel that appears as bright as fetal bone (see Fig. 14-3D). It is identified in approximately 0.5 percent of pregnancies and most commonly represents small amounts of swallowed blood, frequently in the setting of maternal serum AFP level elevation. Although typically associated with normal outcomes, it raises the risk for Down syndrome approximately sixfold. Echogenic bowel has also been associated with fetal cytomegalovirus infection and cystic fibrosis—representing inspissated meconium in the latter.
The femur and humerus are slightly shorter in Down syndrome fetuses. The femur is considered “short” for Down syndrome screening if it measures below the 2.5th percentile or is shortened to ≤90 percent of that expected based on the measured biparietal diameter (American College of Obstetricians and Gynecologists, 2016c; Benacerraf, 1987). As an isolated finding in an otherwise low-risk pregnancy, it is generally not considered to pose great enough risk to warrant counseling modification. Similarly, a humerus shortened to ≤89 percent of that expected, based on a given biparietal diameter, has also been associated with an elevated risk for Down syndrome.
If an isolated minor marker is identified in a woman who has not yet received aneuploidy screening, screening should be offered, and a minor marker is considered an indication to offer cell-free DNA screening (American College of Obstetricians and Gynecologists, 2016c). If cell-free DNA screening has already been performed, the association between isolated minor markers and aneuploidy risk is no longer considered relevant (Reddy, 2014). And, if the cell-free DNA screening result is negative, the fetal aneuploidy risk is not modified by the marker. Conversely, if a cell-free DNA screening result is positive, the absence of minor markers is not considered reassuring.
First-Trimester Sonographic Findings
Unlike second-trimester soft signs, which may be readily visible during standard sonography, first-trimester findings associated with aneuploidy require specialized training. The fetal NT measurement has gained widespread use for aneuploidy screening. Other first-trimester sonographic findings are not routinely used in the United States but may be available in specialized centers. The Perinatal Quality Foundation’s Nuchal Translucency Quality Review Program offers an education program in first-trimester nasal bone assessment (see Fig. 14-2). The Fetal Medicine Foundation also provides online instruction and certification in first-trimester assessment of nasal bone, ductus venosus flow, and tricuspid flow.
Other benefits of first-trimester sonography in women who elect aneuploidy screening include accurate assessment of gestational age and early detection of multifetal gestation or fetal demise. As discussed in Chapter 10 (Second- and Third-Trimester Sonography), first-trimester sonography may identify selected major anomalies associated with aneuploidy, such as cystic hygroma.