DOWN SYNDROME AND OTHER ANEUPLOIDIES
PREGNANCIES AT INCREASED RISK FOR GENETIC DISORDERS
PRENATAL AND PREIMPLANTATION
Major congenital abnormalities are identified during pregnancy or shortly after birth in 2 to 3 percent of pregnancies. They account for 20 percent of infant deaths in the United States, surpassing preterm birth as the most common cause (Kochanek, 2011). Prenatal diagnosis is the science of identifying malformations, disruptions, chromosomal abnormalities, and other genetic syndromes in the fetus. It encompasses routine screening tests for aneuploidy and neural-tube defects, invasive diagnostic tests such as chorionic villus sampling and amniocentesis, additional screening and diagnostic tests offered to those at risk for specific genetic disorders, and the diagnosis of structural malformations with specialized sonography and other fetal imaging techniques discussed in Chapter 10. The goal of prenatal diagnosis is to provide accurate information regarding short- and long-term prognosis, recurrence risk, and potential therapy and to thereby improve counseling and optimize outcomes.
Structural fetal abnormalities may develop in at least three ways. The most common mechanism is malformation—an intrinsic abnormality “programmed” in development, regardless of whether a precise genetic etiology is known. Examples include spina bifida and omphalocele. A second mechanism is deformation, by which a fetus develops abnormally because of extrinsic mechanical forces imposed by the uterine environment. An example is limb contractures that develop with oligohydramnios from bilateral renal agenesis. A third type is disruption, which is a more severe change in form or function that occurs when genetically normal tissue is modified as the result of a specific insult. An example is damage from an amnionic band, which can cause a limb-reduction defect.
Multiple structural or developmental abnormalities may also present together as a syndrome, sequence, or association. A syndrome is a cluster of several anomalies or defects that have the same cause—for example, trisomy 18. A sequence describes anomalies that all developed sequentially from one initial insult. An example is the Pierre-Robin sequence in which micrognathia causes posterior displacement of the tongue—glossoptosis—which leads to a posterior rounded cleft in the palate. An association is a group of specific abnormalities that occur together frequently but do not seem to be linked etiologically. Diagnosis of the VACTERL association, for example, includes three or more of the following: vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula, renal anomalies, and limb abnormalities. Because of overlap of anomaly patterns, it is readily apparent that classification of fetal malformations is challenging, and reclassification is required periodically.
These defects include anencephaly, spina bifida, cephalocele, and other rare spinal fusion (schisis) abnormalities. Features of these anomalies are detailed in Chapter 10 (p. 201), and fetal surgery for spina bifida is discussed in Chapter 16 (p. 325). Neural-tube defects (NTDs) are the second most common class of birth defect after cardiac anomalies, and their reported frequency is approximately 0.9 per 1000 births (Cragan, 2009; Dolk, 2010). More than 40 years ago, Brock and associates (1972, 1973) observed that pregnancies complicated by NTDs had higher levels of alpha-fetoprotein (AFP) in both maternal serum and amnionic fluid. This formed the basis for the first maternal serum screening test for a fetal defect.
Selected risk factors for NTDs are listed in Table 14-1, and genetic factors represent the largest category. Isolated NTDs display multifactorial inheritance. The recurrence risk is approximately 3 to 5 percent if a couple has previously had a child with either anencephaly or spina bifida, 5 percent if either parent was born with an NTD, and as high as 10 percent if a couple has two affected children. Importantly, almost 95 percent of NTDs develop in the absence of a family history. Polymorphisms in the methylene tetrahydrofolate reductase gene, which leads to impaired homocysteine and folate metabolism, have been associated with increased risk for anencephaly and spina bifida, as well as for cardiac malformations (Aneji, 2012; Harisha, 2010; Munoz, 2007; Yin, 2012). NTDs are a component of more than 80 genetic syndromes, many including other anomalies that may be amenable to prenatal diagnosis (Milunsky, 2004).
TABLE 14-1. Risk Factors for Neural-Tube Defects
Family history—multifactorial inheritance
Syndromes with autosomal recessive inheritance—Meckel Gruber, Roberts, Joubert, Jarcho-Levin, HARDE (hydrocephalus-agyria-retinal dysplasia-encephalocele)
Aneuploidy—trisomy 13 and 18, triploidy
Hyperthermia—hot tub or sauna, fever (controversial)
Medications—valproic acid, carbamazepine, coumadin, thalidomide, efavirenz
Geographical—Ethnicity, Diet, and Other Factors
United Kingdom, India, China, Egypt, Mexico, Southern Appalachian United States
MTHFR = methylene tetrahydrofolate reductase.
Other risk factors for NTDs include hyperthermia, medications that disturb folic acid metabolism, and hyperglycemia from insulin-dependent diabetes. Although the exact mechanism by which diabetes causes these anomalies is unknown, rodent studies have found that oxidative stress from embryonic hyperglycemia is associated with apoptosis in the developing neural tube (Li, 2012; Sugimura, 2009; Yang, 2008). The risk for these defects is also increased in certain racial or ethnic groups as well as in populations from selected geographical regions. For example, recent data from population-based registries indicate an NTD prevalence of 1.0 to 1.3 per 1000 births in the United Kingdom, which compares with 0.9 per 1000 in the United States (Cragan, 2009; Dolk, 2010). In the United States, the risk may be twice as high among Mexican-born women (Velie, 2006).
Most women at increased risk for NTDs benefit from 4 mg folic acid taken daily before conception and through the first trimester. This is particularly important if a woman has one or more prior affected children or if either the pregnant woman or her partner has such a defect. Folic acid supplementation may not decrease the risk for NTDs in those with valproic acid exposure, pregestational diabetes, first-trimester fever or hot tub exposure, or defects associated with a genetic syndrome (American College of Obstetricians and Gynecologists, 2013b).
The policy of routine fortification of cereal grains with folic acid, which has been in place in the United States since 1998, provides approximately 200 additional micrograms of folic acid daily and may reduce the first occurrence of NTDs in low-risk women by approximately 20 percent (Honein, 2001). It is recommended that all women at low risk take 400 μg of folic acid orally every day before conception and through the first trimester, to reduce the NTD risk by as much as 80 percent (Chap. 8, p. 159).
Maternal Serum Alpha-Fetoprotein Screening (MSAFP)
Alpha-fetoprotein (AFP) is a glycoprotein synthesized by the fetal yolk sac and later by the fetal gastrointestinal tract and liver. It is the major serum protein in the embryo and fetus and is thus analogous to albumin. As shown in Figure 14-1, its concentration increases steadily in both fetal serum and amnionic fluid until 13 weeks’ gestation, after which, levels rapidly decline. Conversely, AFP is found in steadily increasing quantities in maternal serum after 12 weeks. 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 AFP levels.
FIGURE 14-1 Diagram of alpha-fetoprotein (AFP) concentration across gestational age in fetal plasma, amnionic fluid, and maternal serum. The scale refers to the fetal plasma level, which is approximately 150 times greater than the amnionic fluid concentration and 50,000 times greater than the maternal serum concentration.
It was shown more than 30 years ago that maternal serum AFP concentrations at 16 to 18 weeks exceeded 2.5 multiples of the median (MoM) in a large proportion of women carrying fetuses with either anencephaly or spina bifida (Wald, 1977). Since the mid-1980s, MSAFP concentration has been routinely measured as a screening test for NTDs.
Maternal serum AFP screening is generally performed from 15 through 20 weeks, within a protocol that includes quality control, counseling, and follow-up. AFP is measured in nanograms per milliliter and reported as multiples of the median (MoM) of the unaffected population. Using MoM normalizes the distribution of AFP levels and permits comparison of results from different laboratories and populations. Using a maternal serum AFP level of 2.0 or 2.5 MoM as the upper limit of normal, most laboratories report a detection rate—test sensitivity—of at least 90 percent for anencephaly and 80 percent for spina bifida at a screen-positive rate of 3 to 5 percent (Milunsky, 2004). The positive predictive value—the proportion with AFP elevation that have an affected fetus—is only 2 to 6 percent. This is explained by the overlap in AFP distributions in affected and unaffected pregnancies, as shown in Figure 14-2.
FIGURE 14-2 Maternal serum alpha-fetoprotein distribution for singleton pregnancies at 15 to 20 weeks. The screen cut-off value of 2.5 multiples of the median is expected to result in a false-positive rate of up to 5 percent (black hatched area) and false-negative rates of up to 20 percent for spina bifida (tan hatched area) and 10 percent for anencephaly (red hatched area).
Several factors influence maternal serum AFP levels and are considered when calculating the AFP MoM:
1. Maternal weight—The AFP concentration is adjusted for the maternal volume of distribution.
2. Gestational age—The maternal serum concentration increases by approximately 15 percent per week during the second trimester (Knight, 1992). In general, the MoM should be recalculated if the biparietal diameter differs from the stated gestational age by more than 1 week.
3. Race/ethnicity—African American women have at least 10-percent higher serum AFP concentrations but are at lower risk for fetal NTDs.
4. Diabetes—Serum levels may be 10 to 20 percent lower in women with insulin-treated diabetes, despite a three- to fourfold increased risk for NTDs (Greene, 1988; Huttly, 2004). There is controversy whether such adjustment remains necessary or if results should apply to all types of diabetes (Evans, 2002; Sancken, 2001; Thornburg, 2008).
5. Multifetal gestation—Higher screening threshold values are used in twin pregnancies (Cuckle, 1990). At Parkland Hospital, an AFP level is considered elevated in a twin pregnancy if greater than 3.5 MoM, but other laboratories use 4.0 or even 5.0 MoM.
According to the American College of Obstetricians and Gynecologists (2013b), all pregnant women should be offered screening for NTDs. Women who present for care early in pregnancy often have the option of several different screening tests for aneuploidy, as discussed subsequently. Those who elect second-trimester multiple marker serum screening will have a maternal serum AFP level measured as a component. Those who elect first-trimester screening or chorionic villus sampling may receive neural-tube defect screening either with serum AFP at 15 to 20 weeks or with sonography (American College of Obstetricians and Gynecologists, 2013c).
One algorithm for evaluating elevated maternal serum AFP levels is shown in Figure 14-3. The evaluation begins with a standard sonogram, if not already performed earlier in gestation, as this can reliably exclude three common causes of AFP level elevation: underestimation of gestational age, multifetal gestation, and fetal demise. 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). Once the gestational age is verified and the screening test is confirmed to be abnormal, a patient is offered diagnostic evaluation.
FIGURE 14-3 Example of an algorithm for evaluating maternal serum alpha-fetoprotein screening values (MSAFP). CVS = chorionic villus sampling; MoM = multiples of the median.
Numerous fetal and placental abnormalities have been associated with AFP elevation (Table 14-2). The likelihood of one of these abnormalities or of an adverse pregnancy outcome in the absence of a recognized abnormality increases in proportion to the AFP level. More than 40 percent of pregnancies may be abnormal if the AFP level is greater than 7 MoM (Reichler, 1994).
TABLE 14-2. Conditions Associated with Abnormal Maternal Serum Alpha-Fetoprotein Concentrations
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
Trisomies 21 or 18
Gestational trophoblastic disease
Overestimated gestational age
aAlpha-fetoprotein is adjusted for these factors when multiples of the median are calculated.
For the foregoing reasons, women with a confirmed serum AFP level elevation should be referred for additional counseling and offered a diagnostic test, either specialized sonography or amniocentesis. Some women have risk factors that warrant referral for a diagnostic test even in the setting of a normal AFP level. These include personal history of or first-degree relative with an NTD, insulin-treated diabetes, and first-trimester exposure to a medication associated with increased risk.
More than 25 years ago, Nicolaides and colleagues (1986) described frontal bone scalloping—the lemon sign, and anterior curvature of the cerebellum with effacement of the cisterna magna—the banana sign—in second-trimester fetuses with open spina bifida (Fig. 14-4). These investigators also frequently noted a small biparietal diameter and ventriculomegaly in such cases. Watson and coworkers (1991) reported that 99 percent of fetuses with open spina bifida had one or more of these findings. In addition to these cranial findings, transverse and sagittal images of the spine are increasingly used to characterize the size and location of spinal defects (Chap. 10, p. 202). Using these findings, experienced investigators have described nearly 100-percent detection of open NTDs (Norem, 2005; Sepulveda, 1995). Overall NTD risk may be reduced by at least 95 percent when no spine or cranial abnormality is observed (Morrow, 1991; Van den Hof, 1990).
FIGURE 14-4 A. Image of the fetal head at the level of the lateral ventricles in the setting of spina bifida, demonstrating inward bowing or scalloping of the frontal bones (arrows)—the lemon sign. The image also depicts ventriculomegaly. B. Image of the fetal head at the level of the posterior fossa, demonstrating anterior curvature of the cerebellum (white arrows) with effacement of the cisterna magna—the banana sign.
Most centers use targeted sonography as the primary method of evaluating maternal serum AFP level elevation. The American College of Obstetricians and Gynecologists (2013b) recommends that women be counseled regarding the risks and benefits of targeted sonography and amniocentesis, the risk associated with the degree of AFP level elevation or other risk factors, and the quality and findings of the sonographic examination before making a decision.
Although amniocentesis for amnionic fluid AFP measurement was once considered the standard for open NTD diagnosis, it has been replaced in most centers by targeted sonography. If the amnionic fluid AFP level was elevated, an assay for acetylcholinesterase was performed, and if positive, was considered diagnostic of an NTD. Acetylcholinesterase leaks directly from exposed neural tissue into the amnionic fluid. The overall sensitivity of amniocentesis is approximately 98 percent for open NTDs, with a false-positive rate of 0.4 percent (Milunsky, 2004). Other fetal abnormalities associated with elevated amnionic fluid AFP levels and positive assay for acetylcholinesterase include ventral wall defects, esophageal atresia, fetal teratoma, cloacal exstrophy, and skin abnormalities such as epidermolysis bullosa.
Unexplained Maternal Serum AFP Level Elevation
If no fetal or placental abnormality is detected after a specialized sonographic evaluation, with or without amniocentesis, then an MSAFP elevation is considered unexplained. These pregnancies are at increased risk for various subsequent adverse pregnancy outcomes. These include fetal abnormalities or genetic syndromes not detectable sonographically, fetal-growth restriction, oligohydramnios, placental abruption, preterm membrane rupture, preterm birth, and fetal death. Many of these complications are assumed to result from placental damage or dysfunction. Importantly, AFP level elevation is notconsidered to be clinically useful as a screening tool for adverse pregnancy outcomes, due to its low sensitivity and positive predictive value. 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 AFP level elevation have normal outcomes. Abnormally high or low values of other serum analytes used in aneuploidy screening protocols are reviewed on page 290.
Management of the Fetus with Spina Bifida
The optimal mode of delivery for a fetus with open spina bifida remains controversial. Some have recommended cesarean delivery before the onset of labor, positing that it may reduce the risk of mechanical trauma and spinal infection. Although some have found improved motor function in children delivered operatively, others have not identified benefit in short- or long-term outcomes (Lewis, 2004; Luthy, 1991; Merrill, 1998). The American College of Obstetricians and Gynecologists (2013b) recommends that the route of delivery for the fetus with spina bifida should be individualized.
Open fetal surgery to repair NTDs has been the subject of several clinical studies. A landmark trial designed to compare open fetal surgery for spina bifida with standard postnatal care was described by Adzick and colleagues (2011). These authors of the Management of Myelomeningocele or MOMS trial found that in selected cases, fetal surgery resulted in improved motor outcomes and reduced need for ventriculoperitoneal shunt placement at age 2 to 3 years. They also reported, however, that the surgery itself was associated with significant maternal and fetal risks. This is discussed further in Chapter 16 (p. 325).
DOWN SYNDROME AND OTHER ANEUPLOIDIES
At least 8 percent of conceptuses are aneuploid, accounting for 50 percent of first-trimester abortions and 5 to 7 percent of all stillbirths and neonatal deaths. As discussed in Chapter 13 (p. 260), the risk of fetal trisomy increases with maternal age, particularly after age 35. Specific maternal age-related aneuploidy risks for singleton and twin pregnancies are shown in Tables 14-3 and 14-4. Other significant risk factors include a prior pregnancy with autosomal trisomy or triploidy or a woman or her partner with a numerical chromosomal abnormality or structural chromosomal rearrangement, such as a balanced translocation.
TABLE 14-3. Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Singleton Pregnancies
TABLE 14-4. Maternal Age-Related Risk for Down Syndrome and Any Aneuploidy at Midtrimester and at Term in Dizygotic Twin Pregnanciesa
Types of Screening Tests
Until the mid-1980s, prenatal diagnostic testing for fetal aneuploidy was offered for “advanced maternal age.” However, age alone is a poor screening test, because approximately 70 percent of Down syndrome pregnancies are in women younger than 35 years. Nearly 30 years ago, Merkatz and associates (1984) observed that pregnancies with Down syndrome were characterized by lower maternal serum AFP levels at 15 to 20 weeks, and screening became available for younger women. During the past two decades, there have been four major advances in the area of aneuploidy screening:
1. The addition of other serum analytes to second-trimester screening has improved Down syndrome detection rates to approximately 80 percent for the quadruple marker test (Table 14-5).
2. First-trimester screening at 11 to 14 weeks’ gestation, using the fetal nuchal translucency measurement together with serum analytes, has achieved Down syndrome detection rates comparable to those for second-trimester screening in women younger than 35 years (American College of Obstetricians and Gynecologists, 2013c).
3. Combinations of first- and second-trimester screening yield Down syndrome detection rates as high as 90 to 95 percent (Malone, 2005b).
4. Maternal serum cell-free fetal DNA testing for trisomy 21, 18, and 13 has become available as a screening test for high-risk pregnancies, with a 98-percent detection rate and a false-positive rate of 0.5 percent (American College of Obstetricians and Gynecologists, 2012b; Bianchi, 2012; Palomaki, 2011, 2012).
TABLE 14-5. Selected Down Syndrome Screening Strategies and Their Detection Rate
With the exception of cell-free fetal DNA testing, each first- and/or second-trimester aneuploidy screening test is based on a composite likelihood ratio, and the maternal age-related risk is multiplied by this ratio. This principle also applies to modification of the Down syndrome risk by selected sonographic findings (p. 292). Each woman is provided with a specific risk, expressed as a ratio—1:X. However, each screening test has a predetermined value at 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 385 at term (see Table 14-3). Women with a positive screening test result should be offered a diagnostic test for fetal karyotype by either chorionic villus sampling or amniocentesis (American College of Obstetricians and Gynecologists, 2012a).
Because technology advances have resulted in improved aneuploidy detection with available screening tests, the American College of Obstetricians and Gynecologists (2013c) recommends that all women who present for prenatal care before 20 weeks be offered screening. Available screening paradigms are shown in Table 14-5. A positive screening test result indicates increased risk, but it is not diagnostic of aneuploidy. Conversely, a negative screening test indicates that the risk is not increased, but it does not guarantee a normal fetus. Although Down syndrome is the focus of most aneuploidy screening protocols, it accounts for only half of all fetal chromosomal abnormality cases. Invasive diagnostic tests such as chorionic villus sampling and amniocentesis are safe and effective. Regardless of age, all women are counseled regarding the differences between screening and diagnostic tests, and they are given the option of invasive diagnostic testing.
The most commonly used protocol involves measurement of sonographic nuchal translucency and two maternal serum analytes. This is performed between 11 and 14 weeks’ gestation.
Nuchal Translucency (NT)
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. 14-5). It is measured in the sagittal plane, when the crown-rump length measures between 38 and 84 mm. Specific criteria for NT measurement are listed in Table 10-3 (p. 196). The NT measurement is expressed as a multiple of the gestational age-specific median, similar to serum markers used for aneuploidy screening. An increased NT thickness itself is not a fetal abnormality, but rather is a marker that confers increased risk. Approximately one third of fetuses with increased nuchal translucency thickness will have a chromosome abnormality, nearly half of which are Down syndrome (Snijders, 1998).
FIGURE 14-5 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. (Image contributed by Dr. Michael Zaretsky.)
As shown in Table 14-5, as an isolated marker, NT detects 64 to 70 percent of fetuses with Down syndrome at a false-positive rate of 5 percent, and it has maximal sensitivity at 11 weeks (Malone, 2005b). The risk conferred by an increased NT thickness is independent of that of serum analytes, and combining NT with serum analyte values results in greatly improved aneuploidy detection (Spencer, 1999). Thus, NT is generally used as an isolated marker only in screening for multifetal gestations, in which serum screening is not as accurate or may not be available (American College of Obstetricians and Gynecologists, 2013c). An exception is that if the NT measurement is increased to 3 to 4 mm, then the aneuploidy risk is unlikely to be normalized using serum analyte assessment, and invasive testing should be offered (Comstock, 2006).
Increased NT thickness is also associated with other aneuploidies, genetic syndromes, and various birth defects, especially fetal cardiac anomalies (Atzei, 2005; Simpson, 2007). Because of this, if the NT measurement is 3.5 mm or greater, the patient should be offered targeted sonography, with or without fetal echocardiography, in addition to fetal karyotyping (American College of Obstetricians and Gynecologists, 2013c).
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 (NTQR) program (www.ntqr.org). Training is also available through the Fetal Medicine Foundation (www.fetalmedicineusa.com). In addition to nuchal translucency, NTQR provides an educational process leading to certification in measurement of the fetal nasal bone, which is discussed on page 294 and shown in Figure 14-5.
Two analytes used for first-trimester aneuploidy screening are human chorionic gonadotropin—either intact or free β-hCG—and pregnancy-associated plasma protein A (PAPP-A). In cases of fetal Down syndrome, the first-trimester serum free β-hCG level is higher, approximately 2.0 MoM, and the PAPP-A level is lower, approximately 0.5 MoM. With trisomy 18 and trisomy 13, levels of both analytes are lower (Cuckle, 2000; Malone, 2005b; Spencer, 1999, 2000; Tul, 1999). If gestational age is correct, the use of these serum markers—without NT measurement—results in detection rates for fetal Down syndrome up to 67 percent at a false-positive rate of 5 percent (Wapner, 2003). Aneuploidy detection is significantly greater if these first-trimester analytes are either: (1) combined with the sonographic NT measurement or (2) combined with second-trimester analytes, which is termed serum integrated screening (p. 291).
In twin pregnancies, serum free β-hCG and PAPP-A levels are approximately doubled compared with 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).
Combined First-Trimester Screening
The most commonly used screening protocol combines the NT measurement with serum hCG and PAPP-A. Using this protocol, Down syndrome detection rates in large prospective trials range from 79 to 87 percent, at a false-positive rate of 5 percent (see Table 14-5). The detection rate is approximately 5-percent higher if performed at 11 compared with 13 weeks (Malone, 2005b). The detection rate for trisomies 18 and 13 is approximately 90 percent, at a 2-percent false-positive rate (Nicolaides, 2004; Wapner, 2003).
Maternal age does affect the performance of first-trimester aneuploidy screening tests. In prospective trials, combined first-trimester screening resulted in 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, however, Down syndrome detection rates were 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 (Dugoff, 2004). Similarly, low levels of free β-hCG have been associated with fetal demise (Goetzl, 2004). The sensitivity and positive-predictive values of these markers are considered too low to be clinically useful as screening tests. As with other serum analyte level abnormalities, no management strategies have been demonstrated to improve pregnancy outcomes when these marker levels are abnormally low (Dugoff, 2010).
Pregnancies with fetal Down syndrome are characterized by lower maternal serum AFP levels—approximately 0.7 MoM, higher hCG levels—approximately 2.0 MoM, and lower unconjugated estriol levels—approximately 0.8 MoM (Merkatz, 1984; Wald, 1988). This triple test can detect 61 to 70 percent of Down syndrome cases as shown in Table 14-5 (Alldred, 2012). Levels of all three markers are decreased in the setting of trisomy 18, with a detection rate similar to that for Down syndrome at a false-positive rate of only 0.5 percent (Benn, 1999).
Levels of a fourth marker—dimeric inhibin alpha—are elevated in Down syndrome, with an average value of 1.8 MoM (Spencer, 1996). The addition of dimeric inhibin to the other three markers is the quadruple or quad test, which has a trisomy 21 detection rate of approximately 80 percent at a false-positive rate of 5 percent (see Table 14-5). As with first-trimester screening, aneuploidy detection rates will be slightly 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).
The quad test is the most commonly used second-trimester serum screening test for aneuploidy. 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. As subsequently discussed, combining the quad test with first-trimester screening yields even greater aneuploidy detection rates.
Unexplained Abnormalities of Second-Trimester Analytes
There is a significant association between second-trimester elevation of either hCG or dimeric inhibin alpha levels and adverse pregnancy outcomes. The outcomes reported are similar to those associated with AFP level elevation and include fetal-growth restriction, preeclampsia, preterm birth, fetal demise, and stillbirth. Moreover, the likelihood of adverse outcome is increased when multiple marker levels are elevated (Dugoff, 2005). However, the sensitivity and positive predictive values of these markers are considered too low to be useful for screening or management (Dugoff, 2010).
Low Maternal Serum Estriol Levels. A maternal serum estriol level < 0.25 MoM has been associated with two uncommon but important conditions. The first, Smith-Lemli-Opitz syndrome, is an autosomal recessive condition characterized by mutations in the 7-dehydrocholesterol reductase gene. It may be associated with central nervous system, heart, kidney, and extremity abnormalities, 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, p. 266). 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, fluorescence in situ hybridization to assess the steroid sulfatase locus on the X-chromosome may be considered (Dugoff, 2010).
Combined First- and Second-Trimester Screening
Combined screening strategies enhance aneuploidy detection. For this reason, the American College of Obstetricians and Gynecologists (2013c) recommends that a strategy incorporating both first- and second-trimester screening should be offered to women who seek prenatal care in the first trimester. Three types of screening strategies are available:
1. 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 15 to 20 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 at a false-positive rate of 5 percent (see Table 14-5). If NT measurement is not available, serum integrated screening includes all six serum markers to calculate risk. This screening, however, is less effective.
2. Sequential screening discloses the results of first-trimester screening to women at highest risk, who are then offered invasive testing with chorionic villus sampling or amniocentesis. 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. The threshold is set at approximately 1 percent, because in a screened population, the 1 percent at highest risk includes approximately 70 percent of Down syndrome pregnancies (Cuckle, 2005). This method of screening may achieve up to a 95-percent detection rate (see Table 14-5).
• With contingent sequential screening, women are divided into high-, moderate-, and low-risk groups. Those at highest risk, for example, the top 1 percent, are offered invasive testing. Women at moderate risk, who comprise 15 to 20 percent of the population, undergo second-trimester screening. The remaining 80 to 85 percent, who are at or below a 1:1000 risk, receive negative screening test results and have no further testing (Cuckle, 2005). Thus, most of those screened are provided with results almost immediately while still maintaining a high detection rate. This rate ranges from 88 to 94 percent (see Table 14-5). This option is also more cost-effective because a second-trimester test is obviated in up to 85 percent of patients.
Integrated and sequential screening strategies require coordination between the provider and laboratory to ensure that the second sample is obtained during the appropriate gestational age window, sent to the same laboratory, and linked to the first-trimester results.
Cell-Free Fetal DNA Screening
Using massively parallel sequencing or chromosome selective sequencing to isolate cell-free fetal DNA from maternal plasma, fetal Down syndrome and other autosomal trisomies may be detected as early as 10 weeks’ gestation (Chap. 13, p. 279). Recent trials of these techniques in high-risk pregnancies have yielded detection rates for trisomies 21, 18, and 13 of approximately 98 percent at a false-positive rate of 0.5 percent or less (American College of Obstetricians and Gynecologists, 2012b; Bianchi, 2012; Palomaki, 2011, 2012; Sparks, 2012). This novel technology has recently become clinically available as a screening test, but it is not considered a replacement diagnostic test. Pretest counseling is recommended. If an abnormal result is identified, genetic counseling should be performed, and invasive prenatal diagnostic testing should be offered to confirm the results. The American College of Obstetricians and Gynecologists (2012b) currently recommends that the test may be offered to the following groups:
• Women 35 years or older at delivery
• Those with sonographic findings indicating increased risk for fetal aneuploidy
• Those with a prior pregnancy complicated by trisomy 21, 18, or 13
• Patient or partner carries a balanced robertsonian translocation indicating increased risk for fetal trisomy 21 or 13
• Those with an abnormal first-, second-, or combined first- and second-trimester screening test result for aneuploidy.
The College does not recommend offering the test to women with low-risk pregnancies or multifetal gestations (American College of Obstetricians and Gynecologists, 2012b).
Major abnormalities and minor sonographic markers contribute significantly to aneuploidy detection. As shown in Table 14-6, with few exceptions, the aneuploidy risk associated with any major abnormality is high enough to warrant offering an invasive test for fetal karyotype and/or chromosomal microarray analysis (Chap. 13, p. 275). Importantly, a fetus with one abnormality may have others that are less likely to be detected sonographically or even undetectable sonographically but that greatly affect the prognosis nonetheless. 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. However, only 25 to 30 percent of second-trimester fetuses with Down syndrome will have a major malformation that can be identified sonographically (Vintzileos, 1995).
TABLE 14-6. Aneuploidy Risk Associated with Selected Major Fetal Anomalies
Second-Trimester Sonographic Markers—“Soft Signs”
For more than two 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. Examples of these sonographic findings are listed in Table 14-7. Six of these markers have been the focus of genetic sonogram studies, in which likelihood ratios have been derived that allow a numerical risk to be calculated (Table 14-8). They are generally used only from 15 to 20 or 22 weeks’ gestation. The aneuploidy risk increases steeply with the number of markers identified.
TABLE 14-7. Second-Trimester Sonographic Markers or “Soft Signs” Associated with Down Syndrome Fetuses
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 14-8. Likelihood Ratios and False-Positive Rates for Isolated Second-Trimester Markers Used in Down Syndrome Screening Protocols
Unfortunately, at least 10 percent of unaffected pregnancies will have one of these soft signs, significantly limiting their utility for general population screening (Bromley, 2002; Nyberg, 2003). The incorporation of minor markers into second-trimester screening protocols has been studied primarily in high-risk populations. In this setting, detection rates of 50 to 75 percent for Down syndrome have been reported (American College of Obstetricians and Gynecologists, 2013c). With the exception of increased nuchal skinfold thickness, the identification of an isolated second-trimester marker in an otherwise low-risk pregnancy is not generally considered sufficient to warrant “high-risk” status. A metaanalysis concluded that if minor markers were used as a basis to decide whether to offer amniocentesis, more fetal losses would result than cases of Down syndrome identified (Smith-Bindman, 2001). The American College of Obstetricians and Gynecologists (2013c) recommends that risk adjustment based on second-trimester sonographic markers be limited to specialized centers.
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 (Fig. 14-6A). 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). Unlike the other markers listed in Table 14-8, nuchal skinfold thickening should prompt targeted sonography and consideration of amniocentesis even as an isolated finding in an otherwise low-risk patient.
FIGURE 14-6 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.”
An echogenic intracardiac focus (EIF) is a focal papillary muscle calcification that is neither a structural nor functional cardiac abnormality. It is usually left-sided (Fig. 14-6B). An EIF 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, an EIF approximately doubles the risk for fetal Down syndrome (see Table 14-8). Particularly if bilateral, they are also common with trisomy 13 (Nyberg, 2001).
As discussed in Chapter 10 (p. 214), mild renal pelvis dilatation is usually transient or physiological and does not represent an underlying abnormality (Nguyen, 2010). 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 (Fig. 14-6C). A measurement 4 mm or greater is found in about 2 percent of fetuses and approximately doubles the risk for Down syndrome (see Table 14-8). 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 34 weeks (Chap. 10, p. 215).
Echogenic fetal bowel appears as bright as bone and is seen in approximately 0.5 percent of pregnancies (Fig. 14-6D). Although typically associated with normal outcomes, it increases the risk for Down syndrome approximately sixfold (see Table 14-8). Echogenic bowel may represent small amounts of swallowed blood and may be seen in the setting of AFP level elevation (p. 287). It 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, although the femur length to abdominal circumference (FL/AC) ratio is generally within the normal range in the second trimester. The femur is considered “short” for Down syndrome screening if it measures ≤ 90 percent of that expected. The expected femur length is that which correlates with the measured biparietal diameter (Benacerraf, 1987). Although this finding may be identified in approximately 4 percent of fetuses, its sensitivity may vary with ethnicity. 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 expected, based on a given biparietal diameter, has also been associated with an increased risk for Down syndrome (see Table 14-8).
First-Trimester Sonographic Findings
Unlike second-trimester soft signs, which may be readily visible during a standard sonogram, first-trimester findings associated with aneuploidy require specialized training. The fetal NT is unique in that it has become a component of aneuploidy screening offered to all women. Other first-trimester findings associated with an increased risk for fetal Down syndrome include an absent fetal nasal bone, wider frontomaxillary facial angle—indicating a flat facial profile, tricuspid regurgitation, and abnormal ductus venosus flow (Borenstein, 2008; Cicero, 2001; Faiola, 2005; Huggon, 2003; Matias, 1998; Sonek, 2007). Each has also been associated with an increased risk for trisomies 18 and 13 and other aneuploidies. However, these signs have not become widely adopted for routine use in the United States.
Fetal Nasal Bone. In approximately two thirds of fetuses with Down syndrome, the nasal bone is not visible at the 11- to 14-week examination (Cicero, 2004; Rosen, 2007; Sonek, 2006). Currently, this is the only first-trimester marker, other than NT, for which the Nuchal Translucency Quality Review Program has established a training program. Criteria for adequate assessment include that the fetus occupies most of the image; that there be a 45-degree angle of insonation with the fetal profile; that the profile be well defined in the midsagittal plane, with the tip of the nose and the third and fourth ventricles visible; and that the nasal bone brightness be greater than or equal to that of the overlying skin (Nuchal Translucency Quality Review Program, 2013). An example is shown in Figure 14-5. Initial optimism for this marker was somewhat dampened when the FASTER (First- and Second-Trimester Evaluation of Risk) trial concluded that difficulty in performing the assessment would limit its usefulness for aneuploidy screening (Malone, 2004, 2005a).
PREGNANCIES AT INCREASED RISK FOR GENETIC DISORDERS
Couples with a personal or family history of a heritable genetic disorder should be offered genetic counseling. They should be given an estimated risk of having an affected infant and provided information concerning benefits and limitations of available prenatal testing options. As discussed in Chapter 13 (p. 260), the publicly funded GeneTests website contains detailed information regarding hundreds of specific genetic conditions and laboratory testing information for more than 3000 genetic disorders (http://www.ncbi.nlm.nih.gov/sites/GeneTests). Prenatal diagnosis may be available if the disease-causing mutation or mutations are known. That said, many genetic disorders are characterized by a high degree of penetrance but variable expressivity, such that prediction of phenotype—even when family members are affected—is not currently possible. Common examples include neurofibromatosis, tuberous sclerosis, and Marfan syndrome. There are also conditions for which risk may be refined by detection of associated sonographic abnormalities or by gender determination if X-linked.
Ethnicity-based carrier screening is performed for certain autosomal recessive disorders that are found with increased frequency in specific racial or ethnic groups (Table 14-9). When an otherwise rare gene is found with increased frequency within a certain population and can be traced back to a single family member or small group of ancestors, it is called the founder effect. This phenomenon may occur when generations of individuals procreate only within their own groups because of religious or ethnic prohibitions or geographical isolation. Carrier screening should be offered to those at increased risk for selected autosomal recessive conditions, either prior to conception or early in pregnancy.
TABLE 14-9. Autosomal Recessive Diseases Found with Increased Frequency in Certain Ethnic Groups
This disorder is caused by a mutation in the cystic fibrosis conductance transmembrane regulator (CFTR) gene, which is located on the long arm of chromosome 7 and encodes a chloride-channel protein. Although the most common CFTR gene mutation associated with classic cystic fibrosis (CF) is the ΔF508 mutation, more than 1900 mutations have been identified (Cystic Fibrosis Mutation Database, 2012). Cystic fibrosis may be caused by either homozygosity or compound heterozygosity for mutations in the CFTR gene. In other words, one mutation must be present in each copy of the gene, but they need not be the same mutation. As expected, this results in a tremendous range of clinical disease severity. Median survival is approximately 37 years, but approximately 15 percent have a milder disease form and can survive for decades longer (American College of Obstetricians and Gynecologists, 2011). Care for the pregnant woman with CF is discussed in Chapter 51 (p. 1022).
The current screening panel contains 23 panethnic CF gene mutations, selected because they are present in at least 0.1 percent of patients with classic CF (American College of Obstetricians and Gynecologists, 2011). The CF carrier frequency is approximately 1 in 25 in non-Hispanic white Americans and those of Ashkenazi Jewish descent, who are from Eastern Europe. Thus, the incidence of CF in a child born to a non-Hispanic white couple is approximately ¼ × × , or 1:2500. As shown in Table 14-10, both CF incidence and the sensitivity of the screening test are lower for other ethnicities.
TABLE 14-10. Cystic Fibrosis Detection and Carrier Rates before and after Testing
Current recommendations for CF carrier screening from the American College of Obstetricians and Gynecologists (2011) are as follows:
• Information regarding CF carrier screening should be made available to all couples presenting for preconceptional counseling or prenatal care.
• When both partners are from a high-risk ethnicity, carrier screening should be offered before conception or early in pregnancy.
• Acknowledging that it is becoming increasingly difficult to assign individuals a single ethnicity, it is reasonable to offer CF carrier screening to patients of all ethnicities.
• For individuals with a family or personal history of CF, screening with an expanded mutation panel or even complete CFTR gene sequencing may be necessary if not already obtained.
Although a negative screening test result does not preclude the possibility of carrying a less-common mutation, it reduces the risk substantively from the background rate (see Table 14-10). If both parents are carriers, the fetus can be tested using chorionic villus sampling or amniocentesis to determine whether he or she has inherited one or both of the parental mutations. Counseling following identification of two disease-causing mutations is challenging, because phenotype prediction is reasonably accurate only for pancreatic disease, and only then for well-characterized mutations. Prognosis depends most on degree of pulmonary disease, which varies considerably even among individuals with the most common genotype associated with classic disease, that is, those homozygous for the ΔF508 mutation. This likely reflects the effect of genetic modifiers on protein function, which may further vary depending on CFTR mutation and on exposure and susceptibility to environmental factors (Cutting, 2005; Drumm, 2005).
This group includes sickle-cell anemia, sickle-cell hemoglobin C disease, and sickle-cell β-thalassemia. Sickle-cell anemia is the most common inherited life-shortening disease of childhood onset in the United States. Normal adult hemoglobin consists of two α-chains and two β-chains. As discussed in Chapter 56 (p. 1107), hemoglobin S results from a single point mutation in the gene that encodes the β-chain. A heterozygous individual has one copy each of hemoglobin A and S, that is, hemoglobin AS or sickle-cell trait. A homozygous individual inherits one copy of hemoglobin S from each parent to express hemoglobin SS or sickle-cell anemia. Hemoglobin C is inherited in a similar manner and SC disease results from one copy each of hemoglobin S and C.
African and African American patients are at increased risk to carry hemoglobin S and other hemoglobinopathies and should be offered preconceptional or prenatal screening. One in 12 African Americans has sickle-cell trait, one in 40 carries hemoglobin C, and one in 40 carries the trait for β-thalassemia. Hemoglobin S is also more common among individuals of Mediterranean, Middle Eastern, and Asian Indian descent (Davies, 2000). The American College of Obstetricians and Gynecologists (2013a) recommends that patients of African descent be offered hemoglobin electrophoresis. If a couple is at risk to have a child with a sickle hemoglobinopathy, genetic counseling should be offered. Prenatal diagnosis can be performed with either chorionic villus sampling or amniocentesis.
These syndromes are the most common single-gene disorders worldwide, and up to 200 million people carry a gene for one of these hemoglobinopathies. Some individuals with thalassemia have microcytic anemia secondary to decreased synthesis of either α- or β-hemoglobin chains. In general, deletions of α-globin chains cause α-thalassemia, whereas mutations in β-globin chains cause β-thalassemia. Less commonly, an α-globin chain mutation also causes α-thalassemia. Care for the pregnant woman with thalassemia is discussed in Chapter 56 (p. 1112).
The number of α-globin genes that are deleted may range from one to all four. If two α-globin genes are deleted, both may be deleted from the same chromosome—cis configuration (αα/–), or one may be deleted from each chromosome—trans configuration (α-/α-). Alpha-thalassemia trait is common among individuals of African, Mediterranean, Middle Eastern, West Indian, and Southeast Asian descent and results in mild anemia. The cisconfiguration is more prevalent among Southeast Asians, whereas those of African descent are more likely to inherit the trans configuration. The clinical significance of this difference is that when both parents carry cis deletions, offspring are at risk for the absence of α-hemoglobin, called Hb Barts disease, which typically leads to hydrops and fetal loss (Chap. 15, p. 315).
Detection of α-thalassemia or α-thalassemia trait is based on molecular genetic testing and is not detectable using hemoglobin electrophoresis. Because of this, routine carrier screening is not offered. If there is microcytic anemia in the absence of iron deficiency, and the hemoglobin electrophoresis is normal, then testing for α-thalassemia should be considered, particularly among individuals of Southeast Asian descent (American College of Obstetricians and Gynecologists, 2013a).
Mutations in β-globin genes may cause reduced or absent production of β-globin chains. If the mutation affects one gene, it results in β-thalassemia minor. If both copies are affected, the result is either β-thalassemia major—termed Cooley anemia—or β-thalassemia intermedia. Because of reduced production of hemoglobin A among carriers, electrophoresis demonstrates elevation of hemoglobins that do not contain β-chains, including hemoglobins F and A2.
Beta-thalassemia minor is more common among individuals of African, Mediterranean, and Southeast Asian descent. The American College of Obstetricians and Gynecologists (2013a) recommends that they be offered carrier screening with hemoglobin electrophoresis, particularly if found to have microcytic anemia in the absence of iron deficiency. Hemoglobin A2 levels exceeding 3.5 percent confirm the diagnosis. Other ethnicities at increased risk include those of Middle Eastern, West Indian, and Hispanic descent.
This autosomal recessive lysosomal-storage disease is characterized by absence of the hexosaminidase A enzyme. This leads to a buildup of GM2 gangliosides in the central nervous system, progressive neurodegeneration, and death in early childhood. Affected individuals have almost complete absence of the enzyme, whereas carriers are asymptomatic but have less than 55-percent hexosaminidase A activity. The carrier frequency of Tay-Sachs disease in Jewish individuals of Eastern European (Ashkenazi) descent is approximately 1 in 30, but it is much lower, only about 1 in 300, in the general population. Other groups at increased risk for Tay-Sachs disease include those of French-Canadian and Cajun descent. An international Tay-Sachs carrier-screening campaign was initiated in the 1970s and met with unprecedented success in the Ashkenazi Jewish population. The incidence of Tay-Sachs disease subsequently declined more than 90 percent (Kaback, 1993). Most cases of Tay-Sachs now occur in non-Jewish individuals.
The American College of Obstetricians and Gynecologists (2010) has the following screening recommendations for Tay-Sachs disease:
• Screening should be offered before pregnancy if both members of a couple are of Ashkenazi Jewish, French-Canadian, or Cajun descent, or if there is a family history of Tay-Sachs disease.
• When only one member of the couple is of one of the above ethnicities, the high-risk partner may be screened first, and if found to be a carrier, the other partner also should be offered screening. If the couple is already pregnant, then both partners may be screened simultaneously.
• Targeted mutation analysis of the HEXA gene has a sensitivity of 94 percent in Ashkenazi Jewish individuals but is not recommended for screening in low-risk groups because the detection rate may be below 50 percent.
• Biochemical analysis by determining the hexosaminidase A serum level has a sensitivity of 98 percent and is the test that should be performed in individuals from low-risk ethnicities. Leukocyte testing must be used if the woman is already pregnant or taking oral contraceptives.
• Ambiguous or positive screening test results should be confirmed by biochemical and DNA analysis for the most common mutation. This will detect patients who carry genes associated with mild disease or pseudodeficiency states. Referral to a genetics specialist may be helpful in such cases.
• If both partners are found to be carriers of Tay-Sachs disease, genetic counseling and prenatal diagnosis should be offered. Hexosaminidase activity may be measured from chorionic villus sampling or amniocentesis specimens.
Other Recessive Diseases in Ashkenazi Jewish Individuals
The carrier rate among individuals of Eastern European (Ashkenazi) Jewish descent is approximately 1 in 30 for Tay-Sachs disease, 1 in 40 for Canavan disease, and 1 in 32 for familial dysautonomia. Fortunately, the detection rate of screening tests for each is at least 98 percent in this population. Because of their relatively high prevalence and consistently severe and predictable phenotype, the American College of Obstetricians and Gynecologists (2009a) recommends that carrier screening for these three conditions be offered to Ashkenazi Jewish individuals, either before conception or during early pregnancy. In addition, other conditions for which carrier screening should be made available include mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia group C, Bloom syndrome, and Gaucher disease. Features of these conditions are shown in Table 14-11. Gaucher disease differs from the other conditions listed in that it is has a wide range in phenotype—from childhood illness to absence of symptoms throughout life. Also, there is effective treatment available in the form of enzyme therapy (Zuckerman, 2007).
TABLE 14-11. Autosomal Recessive Genetic Diseases More Common in Individuals of Eastern European Jewish Descent
The American College of Obstetricians and Gynecologists (2009a) has the following recommendations for carrier screening:
• When only one partner is of Ashkenazi Jewish descent, that individual should be screened first, and if found to be a carrier, the other partner is offered screening. With the exception of cystic fibrosis and Tay-Sachs disease, the carrier frequency and detection rate for each of the conditions listed in Table 14-11 is unknown.
• Individuals with a positive family history of one of these disorders should be offered carrier screening for it and may benefit from genetic counseling.
• When both partners are carriers of one of these disorders, they should be referred for genetic counseling and offered prenatal diagnosis.
• When a carrier is identified, they should be encouraged to inform relatives at risk for carrying the same mutation.
PRENATAL AND PREIMPLANTATION DIAGNOSTIC TESTING
Invasive procedures used in prenatal diagnosis—amniocentesis, chorionic villus sampling, and fetal blood sampling—enable a vast array of sophisticated genetic diagnoses to be made before birth. Preimplantation genetic diagnosis permits similar diagnoses to be made in oocytes or embryos before implantation.
Improvements in aneuploidy screening tests during the past decade as described in the preceding section have resulted in a significant decrease in the number of prenatal diagnostic procedures. In a study of more than 160,000 pregnant women 35 years and older, patient acceptance of amniocentesis procedures decreased from 56 to 36 percent between 2001 and 2008, while that of chorionic villus sampling decreased from 36 to 24 percent (Nakata, 2010). Fetal blood sampling procedures have also decreased, but for different reasons. Namely, amniocentesis with fluorescence in situ hybridization (FISH) has decreased the need for rapid karyotyping from fetal blood (Chap. 13, p. 276); the number of DNA-based tests performed on amnionic fluid has greatly expanded; and fetal middle cerebral artery Doppler studies have improved the accuracy of fetal anemia detection (Chap. 10, p. 221).
Transabdominal withdrawal of amnionic fluid remains the most common procedure used to diagnose fetal aneuploidy and other genetic conditions. It is generally performed between 15 and 20 weeks’ gestation but may be performed later as well. The indication is usually to assess fetal karyotype, although use of FISH and array-based comparative genomic hybridization studies have increased considerably as discussed in Chapter 13 (p. 275). Because the amniocytes must be cultured before fetal karyotype can be assessed, the time needed for karyotyping is 7 to 10 days. Outside the context of prenatal genetic analysis, amnionic fluid occasionally may be removed in large amounts therapeutically to relieve symptomatic hydramnios (Chap. 11, p. 236). The same technique described next is also used for this indication.
Amniocentesis is performed using aseptic technique, under direct sonographic guidance, using a 20- to 22-gauge spinal needle (Fig. 14-7). A standard spinal needle is approximately 9 cm long, and depending on patient habitus, a longer needle may be required. The needle is directed into a clear pocket of amnionic fluid, while avoiding the fetus and umbilical cord and ideally without traversing the placenta. Efforts are made to puncture the chorioamnion rather than to “tent” it away from the underlying uterine wall. Discomfort from the procedure is considered minor, and local anesthetic is not typically used (Mujezinovic, 2011).
FIGURE 14-7 Amniocentesis.
The volume of fluid generally needed for commonly performed analyses is shown in Table 14-12. Because the initial 1 to 2 mL of fluid aspirate may be contaminated with maternal cells, it is generally discarded. Approximately 20 mL of fluid is then collected for fetal chromosomal analysis before removing the needle. Sonography is used to observe the uterine puncture site for bleeding, and fetal cardiac motion is documented at the end of the procedure. If the patient is Rh D-negative and unsensitized, anti-D immune globulin is administered following the procedure (Chap. 15, p. 311).
TABLE 14-12. Selected Tests Performed on Amnionic Fluid and Typical Volume of Fluid Required
The color and clarity of the fluid are documented. Amnionic fluid should be clear and colorless or pale yellow. Blood-tinged fluid is more frequent if there is transplacental passage of the needle, however, it generally clears with continued aspiration. The placenta is attached to the anterior uterine wall in approximately half of pregnancies, and if this is the case, it will be traversed by the needle about 60 percent of the time (Bombard, 1995). Fortunately, this has not been associated with pregnancy loss (Marthin, 1997). Dark brown or greenish fluid may represent a past episode of intraamnionic bleeding.
Amniocentesis in Multifetal Pregnancy. For twin gestations, a small quantity of dilute indigo carmine dye is often injected before removing the needle from the first sac. This can be accomplished using 2 mL of a solution in which 1 mL of indigo carmine has been diluted in 10 mL of sterile saline. When the second sac is entered, the return of clear amnionic fluid verifies needle positioning within the second sac. Methylene blue dye is contraindicated because it has been associated with jejunal atresia and neonatal methemoglobinemia (Cowett, 1976; van der Pol, 1992). Because isolated cases of jejunal atresia have also been reported following use of indigo carmine dye, it has been suggested that sonographic visualization should be clear enough to avert the need for dye (Brandenburg, 1997).
The procedure-related loss rate following midtrimester amniocentesis is considered to be 1 per 300 to 500 (American College of Obstetricians and Gynecologists, 2012a). The loss rate may be doubled in women with class 3 obesity–those with a body mass index (BMI) ≥ 40 kg/m2 (Harper, 2012). In twin pregnancies, Cahill and coworkers (2009) reported an increased loss rate attributable to amniocentesis of 1.8 percent. Some losses are unrelated to the procedure and instead are due to abnormal placental implantation or abruption, uterine abnormalities, fetal anomalies, or infection. Wenstrom and colleagues (1990) analyzed 66 fetal deaths following nearly 12,000 second-trimester amniocenteses and found that 12 percent were caused by preexisting intrauterine infection.
Other complications of amniocentesis include amnionic fluid leakage in 1 to 2 percent and chorioamnionitis in less than 0.1 percent (American College of Obstetricians and Gynecologists, 2012a). Following leakage of amnionic fluid, which generally occurs within 48 hours of the procedure, fetal survival exceeds 90 percent (Borgida, 2000). Needle injuries to the fetus are rare. Amnionic fluid culture is successful in more than 99 percent of cases, although cells are less likely to grow if the fetus is abnormal (Persutte, 1995).
If performed between 11 and 14 weeks, amniocentesis is termed “early.” The technique is the same as for traditional amniocentesis, although sac puncture may be more challenging due to lack of membrane fusion to the uterine wall. Also, less fluid is typically withdrawn—approximately 1 mL for each gestational week (Shulman, 1994; Sundberg, 1997).
Early amniocentesis is associated with significantly higher rates of procedure-related complications than other fetal procedures. In the Canadian Early and Mid-Trimester Amniocentesis Trial (1998) that involved more than 4000 women undergoing early amniocentesis, rates of amnionic fluid leakage, fetal loss, and talipes equinovarus (clubfoot) were all significantly higher following early amniocentesis than with traditional amniocentesis. Compared with chorionic villus sampling, early amniocentesis was similarly found to be associated with a fourfold increased rate of talipes equinovarus (Philip, 2004). Another problem with early amniocentesis is that the cell culture failure rate is higher, thus necessitating a second procedure. For all these reasons, the American College of Obstetricians and Gynecologists (2012a) recommends against the use of early amniocentesis.
Chorionic Villus Sampling (CVS)
Biopsy of chorionic villi is generally performed between 10 and 13 weeks’ gestation. Although most procedures are performed to assess fetal karyotype, numerous specialized genetic tests can also be performed by chorionic villus sampling (CVS). Very few analyses specifically require either amnionic fluid or placental tissue. The primary advantage of villus biopsy is that results are available earlier in pregnancy, allowing safer pregnancy termination, if desired. A full karyotype is available in 7 to 10 days, and some laboratories provide preliminary results within 48 hours.
Chorionic villi may be obtained transcervically or transabdominally, using aseptic technique. Both approaches are considered equally safe and effective (American College of Obstetricians and Gynecologists, 2012a; Jackson, 1992). Transcervical villus sampling is performed using a specifically designed catheter made from flexible polyethylene that contains a blunt-tipped, malleable stylet. Transabdominal sampling is performed using an 18- or 20-gauge spinal needle. With either technique, transabdominal sonography is used to guide the catheter or needle into the early placenta—chorion frondosum, followed by aspiration of villi into a syringe containing tissue culture media (Fig. 14-8). Fetal cardiac motion is documented following the procedure.
FIGURE 14-8 Transcervical chorionic villus sampling (CVS).
Relative contraindications include vaginal bleeding or spotting, active genital tract infection, extreme uterine ante- or retroflexion, or body habitus precluding adequate visualization. If the patient is Rh D-negative and unsensitized, anti-D immune globulin is administered following the procedure as discussed in Chapter 15 (p. 311).
The overall loss rate following CVS is higher than that following midtrimester amniocentesis because of background spontaneous losses, that is, those that would have occurred between the first and second trimester in the absence of a fetal procedure. The procedure-related fetal loss rate is comparable to that with amniocentesis (American College of Obstetricians and Gynecologists, 2012a). Caughey and colleagues (2006) found that the overall loss rate following CVS was approximately 2 percent compared with less than 1 percent following amniocentesis. However, the adjusted procedure-related loss rate was about 1 per 400 for either procedure. The indication for CVS will also affect the loss rate. For example, fetuses with increased nuchal translucency thickness have a higher likelihood of demise. Finally, there is a “learning curve” effect associated with safe performance of CVS (Silver, 1990; Wijnberger, 2003).
An early problem with CVS was its association with limb-reduction defects and oromandibular limb hypogenesis (Burton, 1992; Firth, 1991, 1994; Hsieh, 1995). These were subsequently found to be associated with procedures performed at 7 weeks’ gestation (Holmes, 1993). When performed at ≥ 10 weeks’ gestation, as is commonly done today, the incidence of limb defects does not exceed the background rate of 1 per 1000 (Evans, 2005; Kuliev, 1996).
Vaginal spotting is not uncommon following transcervical sampling, but it is self-limited and not associated with pregnancy loss. The incidence of infection is less than 0.5 percent (American College of Obstetricians and Gynecologists, 2012a).
A limitation of CVS is that chromosomal mosaicism is identified in up to 2 percent of specimens. In most cases, the mosaicism reflects confined placental mosaicism rather than a true second cell line within the fetus. Amniocentesis should be offered, and if the result is normal, the mosaicism is presumed to be confined to the placenta. Confined placental mosaicism has been associated with fetal-growth impairment and stillbirth.
Fetal Blood Sampling
This procedure is also called cordocentesis or percutaneous umbilical blood sampling (PUBS). It was initially described for fetal transfusion of red blood cells in the setting of anemia from alloimmunization, as discussed in Chapter 15 (p. 310), and fetal anemia assessment remains the most common indication. Fetal blood sampling is also performed for assessment and treatment of platelet alloimmunization and for fetal karyotype determination, particularly in cases of mosaicism identified following amniocentesis or CVS. Fetal blood karyotyping can be accomplished within 24 to 48 hours. Thus, it is significantly quicker than the 7- to 10-day turnaround time with amniocentesis or CVS. Although fetal blood can be analyzed for virtually any test performed on neonatal blood, improvements in tests available with amniocentesis and CVS have eliminated the need for fetal venipuncture in most cases (Society for Maternal-Fetal Medicine, 2013).
Under direct sonographic guidance, using aseptic technique, the operator introduces a 22- or 23-gauge spinal needle into the umbilical vein, and blood is slowly withdrawn into a heparinized syringe. Adequate visualization of the needle is essential. As with amniocentesis, a longer needle may be required depending on patient habitus. Fetal blood sampling is often performed near the placental cord insertion site, where it may be easier to enter the cord if the placenta is anterior (Fig. 14-9). Alternatively, a free loop of cord may be punctured. Because fetal blood sampling requires more time than other fetal procedures, a local anesthetic may be administered. Prophylactic antibiotics are used at some centers, although there are no trials to support this policy. Arterial puncture is avoided, because it may result in vasospasm and fetal bradycardia. After the needle is removed, fetal cardiac motion is documented, and the site is observed for bleeding.
FIGURE 14-9 Fetal blood sampling. Access to the umbilical vein varies depending on placental location and cord position. With an anterior placenta, the needle may traverse the placenta. Inset: With posterior placentation, the needle passes through amnionic fluid before penetrating the umbilical vein. Alternatively, a free loop of cord may be accessed.
The procedure-related fetal loss rate following fetal blood sampling is approximately 1.4 percent (Ghidini, 1993; Maxwell, 1991; Tongsong 2001). The actual loss rate varies according to the procedure indication and the fetal status. Other complications may include cord vessel bleeding in 20 to 30 percent of cases, fetal-maternal bleeding in approximately 40 percent of cases in which the placenta is traversed, and fetal bradycardia in 5 to 10 percent (Boupaijit, 2012; Ghidini, 1993; Society for Maternal-Fetal Medicine, 2013). Most complications are transitory, with complete recovery, but some result in fetal loss.
In a series of more than 2000 procedures comparing fetal blood sampling near the placental cord insertion site with puncture of a free loop, there were no differences in rates of procedure success, pregnancy loss, visible bleeding from the cord, or fetal bradycardia. Time to complete the procedure was significantly shorter if the cord was sampled at the placental insertion site rather than at a free loop—5 versus 7 minutes. However, sampling at the insertion site had a higher rate of maternal blood contamination (Tangshewinsirikul, 2011).
Preimplantation Genetic Testing
For couples undergoing in vitro fertilization (IVF), genetic testing performed on oocytes or embryos before implantation may provide valuable information regarding the chromosomal complement and single-gene disorders. There are two separate categories of testing—preimplantation genetic diagnosis and preimplantation genetic screening—each with different indications. Comprehensive genetic counseling is required before consideration of these procedures. There are three techniques that are used for both categories of preimplantation genetic testing:
1. Polar body analysis is a technique used to infer whether a developing oocyte is affected by a maternally inherited genetic disorder. The first and second polar bodies are normally extruded from the developing oocyte following meiosis I and II, and their sampling should not affect fetal development (Fig. 5-8, p. 89). In one recent series, this technique was used to diagnose 146 mendelian disorders, with reported accuracy exceeding 99 percent. The main disadvantages of polar body analysis are that the paternal genetic contribution is not evaluated and that an additional procedure may be required in complex cases (Kuliev, 2011).
2. Blastomere biopsy is done at the 6- to 8-cell (cleavage) stage when an embryo is 3 days old, and it is the technique most commonly used for preimplantation testing. One cell is typically removed through a hole made in the zona pellucida, as shown in Figure 14-10. The technique is associated with a 10-percent reduction in the pregnancy rate (Mastenbroek, 2007, 2011; Simpson, 2012). As discussed subsequently, a particular limitation of using this technique for aneuploidy assessment is that mosaicism of the blastomeres may not reflect the chromosomal complement of the developing embryo (American Society for Reproductive Medicine, 2008).
3. Trophectoderm biopsy involves removal of 5 to 7 cells from a 5- to 6-day blastocyst (Fig. 14-11). An advantage is that because the trophectoderm cells give rise to the trophoblast—the placenta—no cells are removed from the developing embryo. Disadvantageously, because the procedure is performed later in development, if genetic analysis cannot be performed rapidly, then cryopreservation and embryo transfer during a later IVF cycle may be required.
FIGURE 14-10 Blastomere biopsy. A. A blastomere is selected. B. This cell is then drawn into the pipette. (From Doody, 2012, with permission.)
FIGURE 14-11 Photomicrograph of trophectoderm biopsy used in preimplantation genetic testing. The trophectoderm is distinct from the embryonic inner cell mass and gives rise to trophoblastic cells, which initiate placental development. (From Doody, 2012, with permission.)
Preimplantation Genetic Diagnosis (PGD)
A genetic abnormality—rather than infertility—may be the reason why a couple has elected IVF. When either or both members of a couple are known carriers of a specific genetic disease or a balanced chromosomal rearrangement, preimplantation genetic diagnosis (PGD) may be performed to determine if an oocyte or embryo has the defect (American Society for Reproductive Medicine, 2008). Only embryos without the abnormality would be implanted.
This procedure has a vast number of applications. It is used to diagnose single-gene disorders such as cystic fibrosis, β-thalassemia, and hemophilia; to determine gender in X-linked diseases; to identify mutations such as BRCA-1 that do not cause disease but confer significantly increased risk; and to match human leukocyte antigens for umbilical cord stem cell transplantation for a sibling (de Wert, 2007; Flake, 2003; Fragouli, 2007; Grewal, 2004; Jiao, 2003; Rund, 2005; Xu, 2004).
To determine whether the known carrier has transmitted a specific genetic mutation, polymerase chain reaction (PCR) is used to amplify the genome region containing the segment of interest (Chap. 13, p. 277). If PGD is performed to identify a translocation or other structural chromosomal rearrangement carried by either parent, FISH is typically used. Because typically only one or two cells are available for analysis and because a rapid completion time is essential, this procedure is technically challenging. Risks include failure to amplify the genetic region of interest, selection of a cell that does not contain a nucleus, and maternal cell contamination. Infrequently, affected embryos thought to be normal are implanted, and unaffected embryos are misdiagnosed as abnormal and discarded. Because of this, the American Society for Reproductive Medicine (2008) encourages further prenatal diagnostic testing—either CVS or amniocentesis—to confirm PGD results.
Preimplantation Genetic Screening (PGS)
This term is used for aneuploidy screening that is performed on oocytes or embryos before IVF transfer. Such screening is used with couples who are not known to have or carry a genetic abnormality. Although preimplantation screening has obvious theoretical advantages, it has faced significant challenges in practice.
Most commonly, FISH is used to identify the copy number of selected chromosomes, and it is performed on a single blastomere (American Society for Reproductive Medicine, 2008). Because the number of chromosome pairs per cell nucleus that can be evaluated with FISH is limited, efforts have also focused on the use of chromosomal microarray analysis (Chap. 13, p. 277). Mosaicism is common in cleavage-stage embryo blastomeres, and it may not be clinically significant because it often does not reflect the actual embryonic chromosomal complement (American College of Obstetricians and Gynecologists, 2009b). In addition, among women 35 years or older, pregnancy rates following preimplantation screening are significantly lower than those observed following IVF without it (Mastenbroek, 2007, 2011). For these reasons, the American College of Obstetricians and Gynecologists (2009b) recommends against use of preimplantation screening with FISH for advanced maternal age screening. It also recommends against it for women with recurrent pregnancy loss or recurrent implantation failure outside the setting of a research trial.
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