What Is the History of Serum Screening?
Prenatal screening for aneuploidy has evolved dramatically over a short period of time. The purpose of prenatal screening for aneuploidy is to identify women who are at an increased risk for the most common aneuploidies. Down syndrome is the most common aneuploidy seen in live births. Chromosomal abnormalities occur in approximately 1 in 150 live births with Down syndrome being the most common with a prevalence of 1 in 800.
Screening tests for open neural tube defects started in the 1970s after the discovery that elevated levels of maternal serum alpha-fetoprotein (MSAFP) in the second trimester were associated with open spina bifida and anencephaly. The combination of reduced serum levels of second trimester AFP and maternal age was used to screen for Down syndrome beginning in 1984. Test performance improved with the introduction of other second trimester analytes namely unconjugated estriol (uE3), human chorionic gonadotropin (hCG), free beta hCG, and dimeric inhibin A, . Decreased maternal serum levels of uE3 and increased levels of hCG and inhibin A are associated with Down syndrome. Second trimester screening using maternal age and the quadruple serum markers alpha fetoprotein, total hCG, uE3, and inhibin A has been validated as an effective tool for Down syndrome.
First trimester maternal serum analytes were introduced in the mid-1980s, including pregnancy-associated plasma protein A (PAPP-A) and free beta hCG. Decreased levels of PAPP-A and increased levels of free beta hCG are associated with Down syndrome. The nuchal translucency (NT) is the most effective first trimester ultrasound marker for fetal aneuploidy and was first used in 1990. First trimester screening, using NT, combined with maternal age and serum analytes (the combined test) was found to be equivalent in performance to the second trimester quadruple test.
The integrated test was introduced in 1993 and includes first trimester NT and PAPP-A and the second trimester quad screen markers with the final interpretation provided only after analysis of the second trimester analyte levels. Integrated screening can also be performed using first and second trimester analytes without an NT. This may prove to be beneficial in cases in which women do not have access to first trimester NT assessment or in cases in which the NT is not obtainable.
Both the stepwise sequential and the contingent sequential screen are a type of integrated test. The results are available in the first trimester for both tests. In the stepwise sequential screen, the first trimester and the quadruple screen are performed with the results available after the first trimester screen. If a patient returns with a high risk for aneuploidy, this allows for earlier options. In the contingent screen, all women undergo the first trimester screen. They then get stratified into high-, medium-, and low-risk groups. The low-risk group does not require any further testing. The intermediate group is offered quadruple screening and the high-risk group is offered diagnostic testing.
Cell-free DNA (cfDNA) screening became commercially available in 2011 after Palomaki et al. conducted a blinded nested case–control study designed within a cohort of more than 4600 pregnancies at high risk for Down syndrome. The Down syndrome detection rate was 98.6% with a 0.8% false-positive rate. cfDNA screening is reviewed in Chapter 9 .
Who Should Be Offered Serum Screening for Aneuploidy?
All women should be offered the option of screening or diagnostic testing for aneuploidy regardless of age. There are identifiable risk factors that increase a woman’s risk of having a child affected with aneuploidy including advancing age ( Table 8.1 ). Other risk factors for aneuploidy are as follows: a history of a prior fetus with aneuploidy, a fetal anomaly or structural malformation, or a parental translocation. The decision to perform screening and/or diagnostic testing depends on the woman’s goals and desires for her pregnancy. Some women choose screening for knowledge, whereas others want to obtain information to make decisions regarding pregnancy continuation versus termination. Patients considering aneuploidy screening should have pretest counseling regarding the benefits, risks, and limitations of the test.
|Age at Term||Risk of Trisomy 21||Risk of Any Chromosome Abnormality|
What Are the Different Serum Screening Tests? What Are the Benefits and Limitations of Each One?
Screening tests for aneuploidy include serum screening, ultrasound, and cfDNA. Fetal aneuploidy risk can be evaluated on the basis of maternal age, maternal serum results and ultrasound markers. No one screening test is superior for all testing characteristics, and not all tests are available in all centers. Factors to be considered in the choice of screening tests are the availably of NT assessment, gestational age at the time of presentation, cost, screening test sensitivity, and limitations. An overview of serum screening tests is described below and summarized in Table 8.2 . Detection rates and false-positive rates for common screening tests for aneuploidy are displayed. Each test has advantages and disadvantages that should be discussed with the patient before screening.
|Test||Gestational Age||Detection Rate T21, %||Detection Rate of All Aneuploidies, %||Screen-Positive Rate, % b|
|First trimester screen a (PAPP-A, hCG and NT)||10 0/7 to 13/67||80||69||5|
|Sequential screen a |
1st trimester PAPP – A, hCG, NT and 2nd trimester MSAFP, hCG, uE3, and inhibin A)
|10 0/7 to 13/67, then 15 0/7 to 22 6/7||93||82||5|
|Cell-free DNA||10 0/7 to term||99||72||1–9|
|Chorionic villus sampling||10 0/7–13/67||>99||>99||1|
|Amniocentesis||15 0/7 to term||>99||>99||0.2|
Screening based on the NT alone is insufficient for aneuploidy risk evaluation because of a lower detection rate of approximately 70%, although NT alone may be used to screen women with high-order multiple gestations (triplets or quadruplets), as there are currently no effective serum screening options for these pregnancies. The first trimester screen includes NT measurement by ultrasound along with serum testing for free beta hCG or total hCG, and PAPP-A levels. First trimester screening can be performed between 10 0/7 and 13 6/7 weeks’ gestation. The NT is dependent on the crown-rump length (CRL) of the fetus being within the appropriate range (36–84 mm) at the time of ultrasound. The NT and serum marker data are combined with gestational age and information regarding maternal factors such as maternal age, prior history of aneuploidy, weight, race and number of fetuses, to calculate a risk estimate for the risk of aneuploidy. The first trimester screen detects approximately 85% of cases of trisomy 21 at a 5% false-positive rate.
Trisomy 18 is characterized by increased NT and decreased free beta hCG and PAPP-A. Screening using the combination of all three markers can detect 86%–89% of trisomy 18 at a 0.5–1.0% false-positive rate. The benefits of first trimester screening include the early gestational age at which results are provided. cfDNA or diagnostic testing can then be offered and pursued at an earlier gestational age. A limitation of first trimester screening is the accuracy required for the NT scan.
The quadruple screen can be performed between 15 0/7 and 22 6/7 weeks of gestation, but for optimal screening of neural tube defects the ideal timing is between 16 and 18 weeks. The test is comprised of hCG, AFP, dimeric inhibin A, and uE3. Similar to the first trimester screen, these results, along with maternal factors including maternal age, weight, race, the presence of diabetes, and number of fetuses, are used to calculate an estimate for the specific risk of aneuploidy or neural tube defect. First trimester and quad screening have similar detection rates for Down syndrome with comparable false-positive rates. There is an 80% detection rate with a 5% false-positive rate for Down syndrome.
The benefits of the quadruple screen are that it screens for open neural tube defects in addition to aneuploidy. A skilled sonographer is not needed as there is no ultrasound component to the exam. The limitation is the late gestational age at which it is performed.
Sequential screening combines the first trimester screen and the quadruple screen for increased aneuploidy detection rates over either test in isolation. In the sequential screen, results from the first trimester screen are shared with the patient. This offers women the opportunity for early diagnostic testing if the early risk for aneuploidy is increased. Women without an increased risk of aneuploidy after the early screen then undergo quadruple screening in the second trimester, which is incorporated with their first trimester screen results for a final estimate of the risk of aneuploidy.
An integrated screen offers both first and second trimester testing but without disclosing the results of the first trimester screen. The detection rate for Down syndrome is approximately 96% with a false-positive rate of 5%. The benefit of integrated screening is the high detection rate. The limitation is the unavailability of a result until the second trimester.
Stepwise Sequential and Contingent Screening
The stepwise sequential screen is a combination of the first trimester portion of the integrated screen with an NT scan and serum analytes. Patients at very high risk for aneuploidy with greater or equal to 1 in 50 or at the highest 0.5% of having an affected fetus are offered counseling and diagnostic testing. Women who do not have an increased risk (less than 1 in 50) proceed to the second trimester portion of the test. These women at lower risk for not receiving the first trimester results do not receive their results until the second trimester. The limitations of this approach are the withholding of first trimester results to some women until the second trimester and the potential for nonadherence of the second blood draw.
Both the stepwise sequential screen and the contingent sequential make the first trimester screening results available to patients. In the stepwise sequential screen, both the first trimester screen and the quad screen are performed. The results are available after their first trimester screen. This allows for earlier counseling for patients at high risk for aneuploidy. In the contingent screen, all women undergo the first trimester screen and then get stratified into high-, medium-, and low-risk groups. The high-risk group is then offered cfDNA screening or diagnostic testing. The low-risk group requires no further testing. The intermediate-risk group is offered quad screening. The detection rate for the contingent screen is between 80%–94% with a false-positive rate of 5%.
What Is the Role of First Trimester Ultrasound Markers in the Detection of Aneuploidy?
First trimester ultrasound can be used to assess the risk for fetal aneuploidy. Markers of interest are summarized below.
Increased Nuchal Translucency and Cystic Hygroma
NT is the subcutaneous fluid-filled space between the back of the fetal neck and overlying skin. The fetal NT should be measured between 10 and 14 weeks’ gestation, when the CRL is between 36 and 84 mm, by a clinician with expertise in this technique. A cystic hygroma is an enlarged hypoechoic space at the back of the fetal neck, extending along the length of the fetal back, and in which septations are clearly visible.
Increased NT is associated with an increased risk for chromosomal abnormalities, including trisomy 21, 13, 18, and monosomy X. The detection rate for Down syndrome using NT ranges between 63% and 77% with a 5% false-positive rate. The risk for fetal aneuploidy increases with NT measurement. One study examined 11,315 pregnancies, which included 19% (2168) with an abnormal karyotype. In that study, an NT measurement less than 3 mm was associated with a 7% risk for a chromosome abnormality, whereas the risk increased to 75% when the NT measured 8.5 mm or greater. Furthermore, the NT measurement varied depending on the fetal karyotype with the majority of fetuses affected with trisomy 21 having an NT measurement less than 4.5 mm, fetuses affected with trisomy 13 or 18 between 4.5 and 8.4 mm, and fetuses affected with Turner syndrome above 8.5 mm. A cystic hygroma is associated with approximately 50% risk for chromosome abnormalities, particularly trisomy 21, trisomy 18, and Turner syndrome. Some studies have shown that septated cystic hygromas have a higher risk of aneuploidy than simple cystic hygromas, but other studies have not found this association.
NT measurement is currently used as a component of first trimester screening, integrated screening, and sequential screening. Comstock and colleagues evaluated data from 36, 120 subjects enrolled in the FASTER Trial to determine the utility of serum analytes in the setting of an increased NT. In this study, 32 patients had an NT measurement greater than 4 mm, and 128 patients had an NT measurement between 3 and 4 mm. There was minimal benefit of measuring serum analytes in cases with an NT measurement between 3 and 4 mm, and no benefit to measuring serum analytes when the NT measurement is above 4 mm. The authors concluded that chorionic villus sampling (CVS) should be offered to patients with an NT of 3 mm or greater.
NT measurement may not be useful in identifying aneuploidy for woman already undergoing cfDNA screening. The DNA First study examined 2691 women in Rhode Island who had cfDNA testing as the primary mode of aneuploidy screening between September 2014 and July 2015. Ten women had an NT above 3 mm or a cystic hygroma as well as cfDNA testing, and the cfDNA testing was able to correctly identify both euploid and aneuploid fetuses. In 2017, the Society for Maternal Fetal Medicine issued a statement recommending that NT measurement is not useful for women already undergoing cfDNA testing.
In contrast, first trimester ultrasound can still be useful for women who are deciding between aneuploidy screening and diagnostic testing via CVS. In one retrospective study of more than 2400 women of advanced maternal age, 2337 were eligible for cfDNA testing, and 237 of those women had an ultrasound abnormality at the time of testing that could have changed the testing strategy such as an anomaly, incorrect dating, multiple gestation, or nonviable pregnancy. Similarly, a retrospective cohort study examined 1739 women at increased risk for aneuploidy based on age or medical history who had negative cfDNA testing. Sixty women had an unexpected fetal finding: 33 had an NT measurement above 3 mm (4 of these cases had a cystic hygroma, and 3 had a structural abnormality); 13 had unrecognized twins; and 10 had a fetal demise. A normal karyotype was confirmed in 98.7% of these cases. Another retrospective cohort study examined 1906 women undergoing first trimester ultrasound between 2013 and 2014. Negative cfDNA testing results were available for half of the women (956), and 37% of these women had a clinically significant first trimester ultrasound finding (42 fetal, 286 gynecological, and 317 placental). Fetal findings included NT abnormalities (8, 19%), fetal abnormality with NT abnormality (8, 19%), and fetal abnormality without NT abnormality (26, 61%). Gynecological abnormalities included ovarian (148, 52%) and uterine findings (138, 48%). Placental abnormalities included location (258, 81%), placentation (1, 0.3%), bleeding (56, 18%), and cord insertion (2, 0.6%).
Fetuses with increased NT and cystic hygroma that have normal karyotypes are at increased risk for structural abnormalities, particularly cardiac defects. The risk of cardiac defects increases with increasing NT measurement, with approximately one-third risk for cardiac defects when a cystic hygroma is detected. A metaanalysis that included 20 studies involving 205,232 fetuses, including 537 diagnosed with a congenital heart defect, reported a pooled positive likelihood ratio of 30.5 (95% CI, 24.3–38.6) for NT > 99th percentile. Other fetal anomalies that have been seen in association with increased NT have involved the neurologic, gastrointestinal, urogenital, and skeletal systems. Therefore, a detailed fetal survey and fetal echocardiogram are recommended for all fetuses with an increased NT or cystic hygroma.
Several genetic syndromes have been associated with an increased NT and cystic hygroma, with Noonan syndrome being the most frequent syndrome diagnosed. Lee et al. performed a retrospective review of 134 fetuses tested for mutations in PTPN11, which is the most common gene associated with Noonan syndrome and 12/134 fetuses had a PTPN11 mutation. Cystic hygroma had a higher likelihood of a positive result (16%) than increased NT (2%). Another study performed by Croonen et al. found a mutation in genes associated with Noonan syndrome in 13/75 fetuses with a normal karyotype and abnormal ultrasound (PTPN11, KRAS, RAF1) and 10/60 fetuses with an abnormal ultrasound only (PTPN11, RAF1, BRAF, MAP2K1). The authors recommended prenatal testing for Noonan syndrome (PTPN11, RAF1, KRAS) in fetuses with an increased NT and at least one additional ultrasound finding such as polyhydramnios, hydrops, renal abnormalities, distended jugular lymphatic sac, hydrothorax, cardiac abnormalities, cystic hygroma, and ascites. They also recommended considering mutation analysis of BRAF and MAP2K1. A recent study calculated a 10% risk for Noonan syndrome in fetuses with an NT over 3 mm based on findings of a Noonan syndrome–related mutation in 4/39 fetuses with an NT over 3 mm and a normal karyotype. Testing for other genetic syndromes should be determined based on additional ultrasound findings at the time of the fetal survey ultrasound.
Chromosome microarray should be considered for all fetuses with an increased NT or cystic hygroma. A metaanalysis showed that an NT measurement above 3.5 mm was associated with a 5% pooled rate of copy number variants on chromosome microarray. The most common findings were 22q11.2 deletion, 22q11.2 duplication, 10q26.12q26.3 deletion, and 12q21q22 deletion. Variants of uncertain significance occurred 1% of the time. Fetuses with additional abnormalities were more likely to have a copy number variant. Similarly, Yang et al. identified a submicroscopic chromosomal abnormality in 20 of 220 fetuses with an increased NT and normal karyotype (9.1%). Fetuses with additional ultrasound abnormalities on a second-trimester ultrasound were more likely to have a submicroscopic abnormality (26.9%) than those with otherwise normal ultrasounds (6.7%). Maya et al. assessed NT cut-off levels as an indication for CMA by examining 462 fetuses with a normal NT (less than 3 mm), 170 with NT between 3–3.4 mm, and 138 with an NT greater than 3.5 mm. Pathogenic copy number variants were detected more often when NT was larger (1.7% NT less than 3 mm, 6.5% NT 3–3.4 mm 13.8% NT greater than 3.5 mm).
Some studies have examined long-term neurodevelopmental outcomes in fetuses with increased NT. There does not appear to be an increased risk for neurodevelopmental problems in early childhood for children with normal karyotypes, no structural abnormalities, and a history of an increased NT measurement or cystic hygroma. Iculano et al. reported that the risk for adverse outcome in school age children with a history of an increased NT is comparable with the general population.
Multiple studies have identified an association between absent fetal nasal bone in the first trimester and trisomy 21. Nasal bone sonography has been shown to improve the detection rate and lower the false-positive rate for trisomy 21 on first trimester screening. However, nasal bone measurement is not uniformly included in all first trimester screening protocols because there are multiple factors besides fetal aneuploidy that can impact the nasal bone and measurement requires an experienced sonographer. Cicero et al. noted that the incidence of absent nasal bone was related to the ethnic origin of the mother (less common in Caucasians than African Americans or Asians), fetal CRL (less common as CRL increases), and NT thickness (more common with increasing NT thickness). Another concern about nasal bone sonography is its lack of sensitivity. Malone et al. studied 6324 patients who had an NT ultrasound and nasal bone sonography. Of the 4801 patients who had an acceptable nasal bone measurement, 0.5% had an absent nasal bone (22). The majority of fetuses with trisomy 21 had a nasal bone (9/11), whereas 50% of the fetuses with trisomy 18 had a nasal bone (1/2). Overall, the authors found the absence of a nasal bone to have a sensitivity of 7.7% for fetal aneuploidy, positive predictive value of 4.5%, and false-positive rate of 0.3%. Chanprapaph et al. also found nasal bone sonography to be specific, but lacking sensitivity.
Ductus Venosus Blood Flow
There are differences in ductus venosus blood flow between aneuploid and euploid fetuses. Reversed flow at the time of atrial contraction has been associated with aneuploidy and fetal cardiac defects, while forward triphasic pulsatile ductus venosus flow is normal. Abnormal ductus venosus blood flow has been observed in 59%–93% of aneuploid fetuses and 3%–21% of euploid fetuses. Measurement of ductus venosus blood flow and NT increases the sensitivity of first trimester ultrasound in the detection of Down syndrome to 94%. Measurement of ductus venosus flow can be helpful in identifying increased risks for adverse fetal outcomes. One study showed adverse outcomes in 11/42 (26.2%) fetuses with a normal NT and reversed or absent ductus venosus blood flow. Adverse outcomes included heart defects, fetal growth restriction, and multiple fetal anomalies.
Tricuspid regurgitation (TR) diagnosed by pulse wave Doppler studies between 11 and 13 weeks 6 days gestation has been shown to be a marker for fetal aneuploidy, particularly trisomy 21. A metaanalysis of 15 studies showed that TR detected at 11–14 weeks of gestation has a likelihood ratio of 25 for the fetus to have Down syndrome (95% CI 14.9–41.9). Additionally, many studies have suggested that TR is a marker for heart defects in euploid fetuses. A metaanalysis showed that the strength of association between TR and heart defects persisted when there was another factor associated with heart defects, such as an increased NT, but no association between TR and CHD when screening a low-risk population for heart defects. Overall, the sensitivity of TR was 35.2%, and the specificity was 98.6%.