CHAPTER 14: Prenatal Diagnosis

In the initial edition of Williams Obstetrics, very few fetal disorders could be identified before delivery. Now, more than 100 years later, prenatal diagnosis has become a separate field of its own. Strictly speaking, prenatal diagnosis is the science of identifying congenital abnormalities, aneuploidies, and other genetic syndromes in the fetus. It encompasses the diagnosis of structural malformations with specialized sonography; routine screening tests for aneuploidy and neural-tube defects; diagnostic tests such as karyotyping and chromosomal microarray analysis performed on chorionic villi and amniocentesis specimens; and additional screening and diagnostic tests offered to those with pregnancies at risk for specific genetic disorders. The goal of prenatal diagnosis is to provide accurate information regarding short- and long-term prognosis, recurrence risk, and potential therapy, thereby improving patient counseling and optimizing outcomes.

Management of an affected pregnancy, including whether a woman would elect pregnancy termination, may be incorporated into the discussion of screening and testing options. However, nondirective counseling is central to prenatal diagnosis. This practice provides the patient with unbiased knowledge regarding a diagnosis and preserves her autonomy (Flessel, 2011). Fetal imaging of congenital anomalies is discussed in Chapter 10, and pregnancy termination is discussed in Chapter 18.

More than 40 years ago, Brock (1972, 1973) observed that pregnancies complicated by neural-tube defects had higher levels of alpha-fetoprotein (AFP) in maternal serum and amnionic fluid. This formed the basis for the first maternal serum screening test for a fetal condition. The beginning of widespread serum screening came in 1977, after a collaborative trial from the United Kingdom established the association between elevated maternal serum AFP levels (MSAFP) and fetal open neural-tube defects (Wald, 1977). When screening was performed at 16 to 18 weeks’ gestation, detection approached 90 percent for pregnancies with fetal anencephaly and 80 percent for those with myelomeningocele (spina bifida). These sensitivities are comparable to current testing (American College of Obstetricians and Gynecologists, 2016a).

The terms level I and level II sonography were coined in this context. In the California MSAFP Screening Program of the 1980s and early 1990s, women received serum screening prior to sonography, and those with an elevated MSAFP level would undergo level I sonography to identify an incorrect gestational age, multifetal gestation, or fetal demise (Filly, 1993). A third of pregnancies with an elevated MSAFP level had one of these three etiologies. Although birth defects were occasionally detected during level I sonography, this was not the expectation. If level I sonography did not identify an etiology for the MSAFP level elevation, amniocentesis would be offered. Then, only if the amnionic fluid AFP concentration were elevated would the woman undergo level II sonography. This more detailed and comprehensive survey of fetal anatomy was performed to detect and characterize the fetal abnormality.

If the amnionic fluid AFP level was elevated, an assay for amnionic fluid acetylcholinesterase was concurrently performed. This capitalized on the tendency of acetylcholinesterase to leak directly from exposed neural tissue into the amnionic fluid. The presence of both analytes in the amnionic fluid was considered diagnostic for neural-tube defects (American College of Obstetricians and Gynecologists, 2016a).

The overall sensitivity of amniocentesis to diagnose open neural-tube defects approximates 98 percent, with a false-positive rate of 0.4 percent (Milunsky, 2004). Importantly, other fetal abnormalities are associated with elevated amnionic fluid AFP levels and positive assay results for acetylcholinesterase. These include ventral wall defects, esophageal atresia, fetal teratoma, cloacal exstrophy, and skin abnormalities such as epidermolysis bullosa. Thus, by current standards, these amnionic fluid analytes would be considered ancillary screening tests, understanding that a positive result would prompt additional fetal imaging.

With current imaging technology, most neural-tube defects are detected with sonography, and targeted sonography is the diagnostic test of choice (Dashe, 2006). Pregnant women now have the option of neural-tube defect screening with either MSAFP or sonography (American College of Obstetricians and Gynecologists, 2016c). Although level II is used as a synonym for targeted sonography, the former might best be removed from our lexicon. Namely, today’s targeted sonography includes a much more comprehensive evaluation of fetal anatomy (Chap. 10, Fetal Anomaly Detection).

As MSAFP screening was being adopted, the designation “advanced maternal age” (AMA) became popular. A 1979 National Institutes of Health Consensus Development Conference recommended advising pregnant women who were 35 years and older about the possibility of amniocentesis for fetal karyotyping. The threshold was based on the greater risk for selected fetal chromosomal abnormalities with increasing maternal age and on the assumption that at that time, the loss rate attributable to amniocentesis was equivalent to the fetal Down syndrome risk at maternal age 35. Notably, this is no longer the case, as discussed later (Chorionic Villus Sampling).

Serum aneuploidy screening soon became available for women who would be younger than 35 at delivery. In 1984, Merkatz and colleagues reported that MSAFP levels were lower in pregnancies with trisomies 21 and 18 at 15 to 21 weeks’ gestation. Maternal age was incorporated into the calculation, such that a specific risk could be assigned (DiMaio, 1987; New England Regional Genetics Group, 1989). The MSAFP screen detected approximately 25 percent of cases of fetal trisomy 21 when the threshold ratio for a positive result was set at 1:270. This ratio reflects the approximate second-trimester risk for Down syndrome at maternal age 35. This trisomy 21 risk threshold and the associated 5-percent false-positive rate became standards that remain in use in some laboratories today.

For more than a decade after its introduction, serum aneuploidy screening was intended for women younger than 35, because it simply did not have sufficient sensitivity to be offered to women who had higher a priori risk. This is also no longer the case. And, because the prevalence of fetal aneuploidy rises sharply with maternal age, the positive-predictive value of all aneuploidy screening tests—whether analyte-based or cell-free DNA tests—is higher in women aged 35 years or older. Women 35 and older now make up more than 15 percent of deliveries in the United States (Fig. 14-1). At Parkland Hospital, this age group accounts for half of births with Down syndrome (Hussamy, 2017).

Aneuploidy is the presence of one or more extra chromosomes, usually resulting in trisomy, or loss of a chromosome—monosomy. Data from population-based registries that include births, fetal deaths, and pregnancy terminations indicate an overall prevalence of 4 such abnormalities per 1000 births (Wellesley, 2012). Aneuploidy accounts for more than 50 percent of first-trimester abortions, about 20 percent of second-trimester losses, and 6 to 8 percent of stillbirths and early-childhood deaths (Reddy, 2012; Stevenson, 2004; Wou, 2016). Of recognized pregnancies with chromosomal abnormalities, trisomy 21 composes approximately half of cases; trisomy 18 accounts for 15 percent; trisomy 13, for 5 percent; and the sex chromosomal abnormalities—45,X, 47,XXX, 47,XXY, and 47,XYY—for approximately 12 percent (Wellesley, 2012).

The risk for fetal trisomy increases with maternal age, particularly after age 35 (Fig. 13-2). When counseling, a provider includes specific maternal-age-related aneuploidy risks (Tables 14-1 and 14-2). Other important fetal aneuploidy risk factors include a numerical chromosomal abnormality or structural chromosomal rearrangement in the woman or her partner—such as balanced translocation—or a prior pregnancy with autosomal trisomy or triploidy.

Broadly speaking, there are two types of aneuploidy screening tests, those that are traditional or analyte-based and those that are cell-free DNA-based. All pregnant women should be offered aneuploidy screening or diagnostic testing early in pregnancy (American College of Obstetricians and Gynecologists, 2016c). Considerations prior to screening are as follows:

1. Has the patient elected screening? At least 20 percent of women elect not to receive aneuploidy screening, even when financial barriers are removed. Fewer than 40 percent of women with a positive screening result elect prenatal diagnosis (Dar, 2014; Kuppermann, 2014).

2. Would the patient prefer prenatal diagnosis? Diagnostic testing is safe and effective, and chromosomal microarray analysis provides information about genetic conditions that screening tests and karyotyping alone currently do not (American College of Obstetricians and Gynecologists, 2016b). This is discussed further in Prenatal Diagnostic Procedures and Preimplantation Testing and in Chapter 13 (Chromosomal Microarray Analysis).

3. Is this a multifetal gestation? All traditional (analyte-based) aneuploidy screening tests are significantly less effective in multifetal gestations, and cell-free DNA screening is not currently recommended with multifetal gestations (Cell-Free DNA for Secondary Screening).

4. What method will be used for neural-tube defect screening? Whenever a patient elects an aneuploidy screening test that does not include second-trimester serum analytes, screening for neural-tube defects should be performed separately, either with MSAFP assessment or with sonography (American College of Obstetricians and Gynecologists, 2016c).

5. Does the fetus have a major anomaly? If so, diagnostic testing is recommended rather than screening.

The American College of Obstetricians and Gynecologists (2016c) has affirmed that screening for aneuploidy should be an informed patient choice, with an underlying foundation of shared decision making that fits her clinical circumstances, values, interests, and goals. Elements of counseling prior to aneuploidy screening are listed in Table 14-3.

Statistical Considerations

Aneuploidy screening can be challenging because the test characteristics of each option may vary with maternal age and with whether the test is analyte-based or cell-free DNA-based.

The test sensitivity is the detection rate—that is, the proportion of aneuploid fetuses identified by the screening test. Its converse, the false-negative rate, is the percentage of cases that the test is expected to miss. A first-trimester screening test with a sensitivity of 80 percent is expected to miss 1 in 5 cases. The sensitivity of screening tests for Down syndrome has increased steadily during the past 30 years, from just 25 percent with serum AFP alone to more than 90 percent with integrated or sequential screening.

Another key characteristic is the false-positive rate, the percentage of unaffected pregnancies that will “falsely” screen positive. This approximates 5 percent for first-trimester screening, quadruple-marker screening, or integrated screening options (Baer, 2015; Kazerouni, 2011; Malone, 2005b; Norton, 2015). The converse of false-positive rate is specificity— analyte-based screens will be reassuring in approximately 95 percent of unaffected pregnancies. Although test sensitivity has improved, the false-positive rate has been held constant for many different aneuploidy screening tests (Table 14-4). Both statistics are relevant for counseling. An additional consideration is that with all analyte-based screening tests, women 35 and older have higher rates of positive results (Kazerouni, 2011; Malone, 2005b).

Importantly, neither sensitivity nor false-positive rate conveys individual risk. The statistic that patients and providers usually consider to be the test result is the positive-predictive value, which is the proportion of those with a positive screening result who actually have an aneuploid fetus. It may be expressed as a 1:X ratio or as a percentage. The positive-predictive value is directly affected by disease prevalence, so it is much higher for women aged 35 years and older than for younger women (Table 14-5). Positive-predictive values can also be reported for cohorts of pregnancies. For example, the positive-predictive value reported in a research trial is the proportion of women with positive screening results who have affected fetuses (see Table 14-4). Negative-predictive value is the proportion of those with a negative screening test result who have unaffected (euploid) fetuses. Because the prevalence of aneuploidy is so low, the negative-predictive value of all aneuploidy screening tests generally exceeds 99 percent (Gil, 2015; Norton, 2015).

Traditional Aneuploidy Screening Tests

These screening tests have multiple markers or analytes and are also called conventional or traditional to differentiate them from cell-free DNA-based screening. There are three categories: first-trimester screens, second-trimester screens, and combinations of first- and second-trimester screens. If the test has a first-trimester component, it almost always includes a measurement of the sonographic nuchal translucency, which is discussed in the next section.

Each maternal serum analyte is measured as a concentration—for example, nanograms per milliliter of AFP. The concentration is converted to a multiple of the median (MoM) by adjusting for maternal age, maternal weight, and gestational age. The nuchal translucency measurement increases with crown-rump length (CRL), and thus its value is adjusted for CRL and also reported as an MoM. The AFP analyte is further adjusted for maternal race and ethnicity and for presence of diabetes, which all affect the calculation of neural-tube defect risk rather than of aneuploidy risk (Greene, 1988; Huttly, 2004). Reporting these results as an MoM of the unaffected population normalizes the distribution of analyte levels and permits comparison of results from different laboratories and populations.

The analyte-based aneuploidy screening result is based on a composite likelihood ratio, and the maternal age-related risk is multiplied by this ratio. This principle similarly applies to modification of fetal Down syndrome risk by selected sonographic markers, which are discussed later in Sonographic Screening. Each woman is provided with a specific risk for trisomy 21 and for trisomy 18—or in the first trimester, for trisomy 18 or 13 in some cases. The result is expressed as a ratio that represents the positive-predictive value.

Importantly, each screening test also has a predetermined value at which or above which it is deemed “positive” or abnormal. For second-trimester tests, this threshold has traditionally been set at the risk for fetal Down syndrome in a woman aged 35 years—approximately 1 in 270 in the second trimester (see Table 14-1). The threshold selected for a positive screen reflects the laboratory requirement but is somewhat problematic, as it may bear no relationship to patient preference. However, a positive screening result may affect whether the patient is deemed “high risk,” receives formal genetic counseling, and is offered diagnostic testing with chorionic villus sampling or amniocentesis. Thus, it behooves the provider to discuss the patient’s preferences prior to screening.

First-Trimester Aneuploidy Screening

Also called combined first-trimester screening, this test combines two maternal serum analytes, human chorionic gonadotropin (hCG) and pregnancy-associated plasma protein A (PAPP-A), with the sonographic measurement of the nuchal translucency (NT). It is performed between 11 and 14 weeks’ gestation. In cases of fetal Down syndrome, the first-trimester serum free β-hCG level is higher and the PAPP-A level is lower. With trisomy 18 and trisomy 13, levels of both analytes are lower (Cuckle, 2000; Malone, 2005b).

Nuchal Translucency

This is the maximum thickness of the subcutaneous translucent area between the skin and soft tissue overlying the fetal spine at the back of the neck (Fig. 14-2). An increased NT thickness is not a fetal abnormality but rather is a marker that confers increased risk. It is measured in the sagittal plane and is valid when the CRL value lies between 38 to 45 mm and 84 mm, with the lower limit varying according to the laboratory. Specific criteria for NT measurement are listed in Table 10-4. Whenever possible, it is helpful to differentiate increased NT from cystic hygroma, which is a venolymphatic malformation that appears as a septated hypoechoic space behind the neck, extending along the length of the back (Fig. 10-22). Cystic hygroma confers a fivefold increased aneuploidy risk when identified in the first trimester (Malone, 2005a).

In addition to aneuploidy, an increased NT thickness is associated with other genetic syndromes and various birth defects, especially fetal cardiac anomalies (Simpson, 2007). And, if the NT measurement reaches 3 mm or more, the aneuploidy risk is unlikely to normalize using serum analyte assessment (Comstock, 2006). Because of this, if the NT measurement is at least 3 mm or exceeds the 99th percentile, the patient should receive counseling and be offered targeted sonography with fetal echocardiography. In addition, she should be offered cell-free DNA screening and prenatal diagnosis (American College of Obstetricians and Gynecologists, 2016c).

The NT must be imaged and measured with a high degree of precision for aneuploidy detection to be accurate. This has led to standardized training, certification, and ongoing quality review programs. In the United States, training, credentialing, and monitoring are available through the Nuchal Translucency Quality Review program of the Perinatal Quality Foundation and through the Fetal Medicine Foundation.

Efficacy of First-Trimester Screening

Before first-trimester screening became widely adopted, four large prospective trials were conducted, together including more than 100,000 pregnancies (Reddy, 2006). When the false-positive rate was set at 5 percent, the overall rate for trisomy 21 detection was 84 percent, comparable to quadruple-marker screening (see Table 14-4). The detection rate is approximately 5 percent higher if performed at 11 weeks’ compared with 13 weeks’ gestation, and slightly lower—80 to 82 percent—when cases with cystic hygroma are analyzed separately (Malone, 2005a). In a recent multicenter trial, first-trimester screening detected approximately 80 percent of fetuses with trisomy 21, 80 percent with trisomy 18, and 50 percent with trisomy 13 (Norton, 2015).

As an isolated marker, NT detects approximately two thirds of fetuses with Down syndrome, with a false-positive rate of 5 percent (Malone, 2005b). However, NT is generally used as an isolated marker only in screening for multifetal gestations, in which serum screening is less accurate or may not be available. The NT distribution is similar in twins and singletons (Cleary-Goldman, 2005). In twin pregnancies, serum free β‑hCG and PAPP-A levels are approximately double the singleton values (Vink, 2012). Even with specific curves, a normal dichorionic cotwin will tend to normalize screening results, and thus, the aneuploidy detection rate is at least 15-percent lower (Bush, 2005).

Maternal age affects the performance of first-trimester aneuploidy screening. Prospective trials have demonstrated Down syndrome detection rates of 67 to 75 percent in women younger than 35 years at delivery, which are 10 percent lower than the overall detection rates in these studies (Malone, 2005b; Wapner, 2003). Among women older than 35 at delivery, Down syndrome detection reached 90 to 95 percent, albeit at a higher false-positive rate of 15 to 22 percent.

Unexplained Abnormalities of First-Trimester Analytes

There is a significant association between serum PAPP-A levels below the 5th percentile and preterm birth, growth restriction, preeclampsia, and fetal demise (Cignini, 2016; Dugoff, 2004; Jelliffe-Pawlowski, 2015). Similarly, low levels of free β-hCG have been associated with fetal demise (Goetzl, 2004). The sensitivity and positive-predictive values of isolated markers are generally too low to make them clinically useful as screening tests.

There has been renewed interest in low-dose aspirin for prevention of early preeclampsia in women identified as at risk based on mean arterial pressure, uterine artery Doppler values, and PAPP-A levels. However, these observations are preliminary (Park, 2015).

Second-Trimester Aneuploidy Screening

Currently, the only second-trimester multiple marker test widely used in the United States is the quadruple marker or “quad” screening test. It is performed from 15 to 21 weeks’ gestation, and inclusive gestational age ranges vary according to individual laboratories. Pregnancies with fetal Down syndrome are characterized by lower maternal serum AFP, higher hCG, lower unconjugated estriol, and higher dimeric inhibin levels. When the quad screen was initially described, the Down syndrome detection rate approximated 70 percent. But, by the early 2000s, the reported detection rate in two large prospective trials had improved to 81 to 83 percent, with a 5-percent screen-positive rate (Malone, 2005b; Wald, 1996, 2003). The improved detection rate is attributable, at least in part, to accurate gestational age assessment with sonography. In a review of more than 500,000 pregnancies receiving quadruple-marker screening through the statewide California Prenatal Screening Program, trisomy 21 detection was 78 percent with sonographic gestational age assessment but only 67 percent when the screen was calculated based on last menstrual period alone (Kazerouni, 2011). As with first-trimester screening, aneuploidy detection rates are lower in younger women and higher in women older than 35 years at delivery. If second-trimester serum screening is used in twin pregnancies, aneuploidy detection rates are significantly lower (Vink, 2012). With trisomy 18, the levels of the first three analytes are all decreased, and inhibin is not part of the calculation. Trisomy 18 detection is similar to that for Down syndrome, with a false-positive rate of only 0.5 percent (Benn, 1999).

Although the quadruple-marker screening test is used to screen for Down syndrome and trisomy 18, pregnancies with other chromosomal abnormalities may be identified as well. The California Prenatal Screening Program found that the quadruple-marker screen result was abnormal in 96 percent of those with triploidy, in 75 percent with Turner syndrome (45,X), in 44 percent with trisomy 13, and in more than 40 percent of those with other major chromosomal abnormalities (Kazerouni, 2011). Although a specific risk for these aneuploidies cannot be provided based on the test result, the information may be relevant for women considering amniocentesis.

Quadruple-marker screening offers no benefit over first-trimester screening from the standpoint of trisomy 21 or trisomy 18 detection. As a stand-alone test, it is generally used if women do not begin care until the second trimester or if first-trimester screening is not available. In 2011, women who initiated prenatal care beyond the first trimester made up nearly 25 percent of pregnancies in the United States. As subsequently discussed, combining first- and second-trimester screening yields an even greater aneuploidy detection rate.

Maternal Serum AFP Elevation: Neural-Tube Defect Screening

All pregnant women are offered screening for fetal open neural-tube defects in the second trimester, either with MSAFP screening or with sonography (American College of Obstetricians and Gynecologists, 2016c). Measurement of the MSAFP concentration between 15 and 20 weeks’ gestation has been offered as part of routine prenatal care for more than 30 years. Because AFP is the major protein in fetal serum, analogous to albumin in a child or adult, the normal concentration gradient between fetal plasma and maternal serum is on the order of 50,000:1. Defects in fetal integument, such as neural-tube and ventral-wall defects, permit AFP to leak into the amnionic fluid, resulting in dramatically increased maternal serum levels. The AFP value rises by about 15 percent per week during the screening window (Knight, 1992). The MoM is generally recalculated if the first-trimester CRL or second-trimester biparietal diameter differs from the stated gestational age by more than 1 week.

Using an MSAFP level of 2.5 MoM as the upper limit of normal, the neural-tube defect detection rate is at least 90 percent for anencephaly and 80 percent for spina bifida, with a screen-positive rate of 3 to 5 percent (American College of Obstetricians and Gynecologists, 2016a; Milunsky, 2004). Higher screening threshold values are used in twin pregnancies (Cuckle, 1990).

Virtually all cases of anencephaly and many cases of spina bifida may be detected or suspected during a standard second-trimester sonographic examination (Dashe, 2006). Most centers now use targeted sonography as the primary method to evaluate elevated MSAFP levels and as the prenatal diagnostic test of choice for neural-tube defects (Chap. 10, Neural-Tube Defects). If targeted sonography is not available and myelomeningocele cannot be excluded, amniocentesis may be considered for measurement of amnionic fluid AFP and acetylcholinesterase levels. That said, we recommend additional imaging prior to establishing the diagnosis, with the understanding that other abnormalities or conditions can result in elevation of these amnionic fluid analytes (Table 14-6). Sonographic findings characteristic of fetal neural-tube defects are reviewed in Chapter 10 (Neural-Tube Defects). Fetal surgery for myelomeningocele is discussed in Chapter 16 (Open Fetal Surgery).

Unexplained Abnormalities of Second-Trimester Analytes

The positive-predictive value of an elevated MSAFP value is only 2 percent. Approximately 98 percent of pregnancies with an MSAFP level exceeding 2.5 MoM have an etiology other than a neural-tube defect. Thus, counseling is indicated not only to inform the patient about the benefits and limitations of targeted sonography for the diagnosis of neural-tube defects but also to review the numerous other conditions. Some of these include fetal anomalies, placental abnormalities, and adverse outcomes associated with MSAFP level elevation (see Table 14-6). The likelihood of one of these abnormalities or of an adverse pregnancy outcome in the absence of a recognized abnormality rises in proportion to the AFP level. Adverse outcomes include fetal-growth restriction, preeclampsia, preterm birth, fetal demise, and stillbirth. More than 40 percent of pregnancies may be abnormal if the MSAFP level is greater than 7 MoM (Reichler, 1994).

Second-trimester elevation of either hCG or dimeric inhibin alpha levels also shows significant association with adverse pregnancy outcomes. The outcomes reported are similar to those associated with MSAFP level elevation. Moreover, the likelihood of adverse outcome is augmented when levels of several markers are elevated (Dugoff, 2005).

Many of these complications are assumed to result from placental damage or dysfunction. However, the sensitivity and positive-predictive values of these markers are considered too low to be useful for screening or management. No specific program of maternal or fetal surveillance has been found to favorably affect pregnancy outcomes (Dugoff, 2010). At Parkland Hospital, prenatal care for these women is not altered unless a specific complication arises. Despite the extensive list of possible adverse outcomes, it is reassuring that most women with unexplained elevation of these analytes have normal outcomes.

Low Maternal Serum Estriol Level

A maternal serum estriol level less than 0.25 MoM has been associated with two uncommon but important conditions. The first, Smith-Lemli-Opitz syndrome, is an autosomal recessive condition resulting from mutations in the 7-dehydrocholesterol reductase gene. It is characterized by abnormalities of the central nervous system, heart, kidney, and extremities; with ambiguous genitalia; and with fetal-growth restriction. For this reason, the Society for Maternal-Fetal Medicine has recommended that sonographic evaluation be performed if an unconjugated estriol level is <0.25 MoM (Dugoff, 2010). If abnormalities are identified, an elevated amnionic fluid 7-dehydrocholesterol level can confirm the diagnosis.

The second condition is steroid sulfatase deficiency, also known as X-linked ichthyosis. It is typically an isolated condition, but it may also occur in the setting of a contiguous gene deletion syndrome (Chap. 13, Abnormalities of Chromosome Structure). In such cases, it may be associated with Kallmann syndrome, chondrodysplasia punctata, and/or mental retardation (Langlois, 2009). If the estriol level is <0.25 MoM and the fetus appears to be male, chromosomal microarray analysis or fluorescence in situ hybridization to assess the steroid sulfatase locus on the X-chromosome may be considered.

Integrated and Sequential Screening

As shown in Table 14-4, if first-trimester screening is combined with second-trimester screening, aneuploidy detection is significantly improved. Combined screening test options require coordination between the provider and laboratory. Specifically, if a second sample is required, it is obtained during the appropriate gestational age window, sent to the same laboratory, and linked to the first-trimester results. The first- and second-trimester components cannot be performed independently because if either component yields positive results, then providing accurate risk assessment would be problematic.

Three types of screening strategies are available:

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 approximately 15 to 21 weeks. An aneuploidy risk is then calculated from these seven parameters. As expected, integrated screening has the highest Down syndrome detection rate—94 to 96 percent, with a false-positive rate of 5 percent (see Table 14-4). If NT measurement is not available, then serum integrated screening includes all six serum markers to calculate risk. This screening, however, is less effective, and Down syndrome detection rates are 85 to 88 percent (Malone, 2005b).

2. Sequential screening involves performing first-trimester screening and informing the patient of the results. This is coupled with the understanding that if the calculated risk value lies above a specified threshold, she will receive counseling and will be offered diagnostic testing. There are two testing strategies in this category:

   • With stepwise sequential screening, women with first-trimester screen results that confer risk for Down syndrome above a particular threshold are offered invasive testing, and the remaining women receive second-trimester screening. Using data from the First- and Second-Trimester Evaluation of Risk (FaSTER) trial, when the first-trimester threshold is set at approximately 1:30, and the overall threshold is set at 1:270, stepwise sequential screening resulted in a 92-percent detection rate of Down syndrome pregnancies, with a false-positive rate of 5 percent (see Table 14-4) (Cuckle, 2008).

   • With contingent sequential screening, women are divided into high-, moderate-, and low-risk groups. Those at highest risk for Down syndrome—for example, risk >1:30, are counseled and offered invasive testing. Women at moderate risk, between 1:30 and 1:1500, undergo second-trimester screening, whereas those at lowest risk of <1:1500 receive negative screening test results and have no further testing (Cuckle, 2008). Using this strategy, more than 75 percent of those screened are provided with reassuring results almost immediately, while still maintaining a high detection rate of about 91 percent, with a 5-percent false-positive rate (see Table 14-4). This option is also more cost effective because a second-trimester test is obviated in most patients.

In a population-based review of 450,000 pregnancies from the California Prenatal Screening Program, integrated screening detected 94 percent of trisomy 21 fetuses and 93 percent with trisomy 18 (Baer, 2015). Additionally, the screening result was abnormal in 93 percent of cases of trisomy 13, in 91 percent with triploidy, and in 80 percent with Turner syndrome. Women considering options of integrated screening and cell-free DNA screening may find this information helpful.

Cell-Free DNA Screening

This was introduced in 2011 and has completely changed the prenatal screening paradigm. The test works by identifying DNA fragments that are derived primarily from apoptotic trophoblasts, which are placental cells undergoing programmed cell death. Thus, the term cell-free fetal DNA is somewhat of a misnomer. Screening is not gestational-age dependent and can be performed at any time after 9 to 10 weeks’ gestation. Results are available in 7 to 10 days (American College of Obstetricians and Gynecologists, 2017c). Three types of assays are currently available: whole-genome sequencing, which is also called massively parallel or shotgun sequencing; chromosome selective or targeted sequencing; and analysis of single nucleotide polymorphisms.

The screening performance of cell-free DNA is excellent. In a metaanalysis of 37 studies of largely high-risk pregnancies, the pooled sensitivity to detect Down syndrome was 99 percent, and for trisomies 18 and 13, 96 percent and 91 percent, respectively. For each of these autosomal trisomies, the specificity was 99.9 percent. Thus, most unaffected pregnancies received a normal screening result. Cell-free DNA also detects 90 percent with Turner syndrome (45,X) and 93 percent with sex chromosome aneuploidies other than 45,X (Gil, 2015). The false-positive rate is cumulative for each aneuploidy for which screening is performed, but it is usually only 0.5 to 1 percent. As a result, cell-free DNA screening is recommended as a screening option in those at increased risk for fetal autosomal trisomy (American College of Obstetricians and Gynecologists, 2017c; Society for Maternal-Fetal Medicine, 2015). This includes the following categories:

1. Women who will be 35 years or older at delivery

2. A positive first- or second-trimester analyte-based screening test

3. Sonogram with a minor aneuploidy marker

4. Prior pregnancy with autosomal trisomy

5. Known carriage (patient or partner) of a balanced robertsonian translocation involving chromosome 21 or 13.

Cell-Free DNA for Secondary Screening

If cell-free DNA screening is performed as a secondary screen following a positive first- or second-trimester analyte-based test result, a normal result is not quite as reassuring. The residual risk for chromosomal abnormality is estimated to be 2 percent (Norton, 2014). Compared with amniocentesis, use of cell-free DNA screening after an initial abnormal analyte-based test result is estimated to yield a 20-percent reduction in aneuploidy diagnoses. This takes into consideration false-negative diagnoses and aneuploidies not detectable with cell-free DNA screening (Davis, 2014; Norton, 2014). In addition, definitive diagnosis may be delayed, potentially affecting management. Concurrent or parallel screening is not recommended, and if an aneuploidy screening test of any type yields a negative result, additional screening is not indicated (American College of Obstetricians and Gynecologists, 2016b, 2017c).

The association between increased NT values and fetal structural and genetic abnormalities has raised questions about the role of a NT measurements following cell-free DNA screening. The College (2016b) has stated that NT measurement is not necessary at the time of cell-free DNA screening but that sonography may help to confirm fetal number and viability and to assign gestational age. The Society for Maternal-Fetal Medicine (2015) states that after a negative cell-free DNA screening test result, the additional clinical utility of NT measurement to detect other chromosomal or structural abnormalities is unknown but appears to be limited.

Cell-Free DNA Screening in Low-Risk Pregnancies

Most studies of cell-free DNA have been conducted in high-risk pregnancies. Pragmatically, chromosomal abnormalities are individually so rare that even large studies of low-risk pregnancies contain few affected cases. Available data suggest that the high sensitivity and specificity for Down syndrome detection are preserved in low-risk pregnancies (Norton, 2015; Pergament, 2014; Zhang, 2015). Importantly, the positive-predictive value of cell-free DNA screening still depends greatly on maternal age and the specific aneuploidy in question (see Table 14-5). For a woman in her early 20s, the positive-predictive value is approximately 50 percent for fetal trisomy 21, 15 percent for trisomy 18, and <10 percent for trisomy 13. Hence, decisions for irreversible medical intervention should not be based on the results of this or other screening test alone.

Cell-Free DNA Screening Limitations

Important caveats are considered with selection of cell-free DNA aneuploidy screening. Because the cell-free DNA that is analyzed is maternal and placental, results may not reflect the fetal DNA complement but rather may indicate confined placental mosaicism, early demise of an aneuploid co- twin, maternal mosaicism, or even occult maternal malignancy (Bianchi, 2015; Curnow, 2015; Grati, 2014; Wang, 2014). In addition, if a twin pregnancy is identified sonographically, cell-free DNA screening is not currently recommended because of limited evidence regarding efficacy.

Another limitation is that cell-free DNA testing does not yield a result in approximately 4 to 8 percent of screened pregnancies, due to assay failure, high assay variance, or low fetal fraction (Norton, 2012; Pergament, 2014; Quezada, 2015). Most cell-free DNA is maternal. The fetal fraction is the proportion derived from the placenta and generally is about 10 percent of the total. A low fetal fraction is usually defined as <4 percent of the total and confers significantly higher risk for fetal aneuploidy (Ashoor, 2013; Norton, 2015; Pergament, 2014). Women with a low fetal fraction or “no-call” results have fetal aneuploidy rates as high as 4 percent, a percentage comparable to the average predictive value conferred by a positive first-trimester screening test result (see Table 14-4). The fetal fraction is not related to maternal age or analyte-based screening test results. However, it is lower earlier in pregnancy and appears to be reduced in women of greater weight (Ashoor, 2013).

Because of the increased risk for fetal aneuploidy in cases not generating a cell-free DNA screening result (no-call result), genetic counseling is indicated, and amniocentesis should be offered. If the patient elects repeat screening, the risk for screen failure may exceed 40 percent (Dar, 2014; Quezada, 2015). Targeted sonography is recommended but is not a substitute for amniocentesis, because it is unclear what the residual risk would be with normal sonogram findings. (American College of Obstetricians and Gynecologists, 2016b, 2017c). Pretest counseling should include the possibility of a low fetal fraction or no-call result and its clinical significance.

Comparison with Analyte-Based Screening

Cell-free DNA screening has obvious advantages, but it is not simply a “better” test—because no screening test is superior for all test characteristics (American College of Obstetricians and Gynecologists, 2016c). Compared with analyte-based tests, benefits of cell-free DNA screening in women 35 years and older include the lower likelihood of a false-positive result, its higher positive-predictive value, and the fact that isolated minor aneuploidy markers are generally not a concern (Sonographic Screening).

But, analyte-based tests are frequently positive with a large range of chromosomal abnormalities, whereas cell-free DNA screens are specific for individual aneuploidies (Baer, 2015; Kazerouni, 2011). Women younger than 35 are at lower risk for the specific autosomal trisomies for which cell-free DNA screening is typically performed. Thus, if the goal is to select a screening test that will identify the highest proportion of fetuses with any chromosomal abnormality, the yield may be comparable or even slightly higher with integrated or sequential screening than with current cell-free DNA screening (Baer, 2015; Norton, 2014).

Sonographic Screening

Sonography can augment aneuploidy screening by providing accurate gestational age assessment, by detecting multifetal gestations, and by identifying major structural abnormalities and minor sonographic markers. As shown in Table 14-7, with rare exceptions, the aneuploidy risk associated with any major abnormality is high enough to warrant offering prenatal diagnosis. Generally, chromosomal microarray analysis is recommended as the first-line test. Importantly, a fetus with one abnormality may have others that are less likely to be detected sonographically but that greatly affect the prognosis. Aneuploidy screening—including cell-free DNA—is not recommended if a major abnormality has been identified. The fetal risk cannot be normalized with a normal screening result, not merely because screening results can be falsely negative, but also because major anomalies confer risk for genetic syndromes not identified through screening tests.

If a major abnormality is identified, targeted sonography is indicated. Sonography is not an alternative to prenatal diagnosis, but the aneuploidy risk is further increased if additional findings are identified. An earlier study reported that only 25 to 30 percent of second-trimester fetuses with Down syndrome had a major malformation that could be identified sonographically (Vintzileos, 1995). When both major anomalies and minor aneuploidy markers are considered, it is estimated that 50 to 60 percent of Down syndrome pregnancies can be detected sonographically (American College of Obstetricians and Gynecologists, 2016c). Fortunately, most fetuses with aneuploidy that is likely to be lethal in utero—such as trisomy 18 and 13 and triploidy—usually have sonographic abnormalities that can be seen by the second trimester.

Second-Trimester Markers—“Soft Signs”

For three decades, investigators have recognized that the sonographic detection of aneuploidy, particularly Down syndrome, may be improved by minor markers that are collectively referred to as “soft signs.” Minor markers are normal variants rather than fetal abnormalities, and in the absence of aneuploidy or an associated abnormality, they do not significantly affect prognosis. They are present in at least 10 percent of unaffected pregnancies (Bromley, 2002; Nyberg, 2003). Examples of these sonographic findings are listed in Table 14-8 and depicted in Figure 14-3. Findings are generally useful from 15 to 20 or 22 weeks’ gestation. Six of these markers have been the focus of sonographic studies, in which likelihood ratios have been derived that allow a numerical aneuploidy risk to be calculated (Table 14-9). The risk rises steeply with the number of markers identified. Alternatively, absence of a minor marker has been used to reduce the calculated risk (Agathokleous, 2013). This should be done systematically, following a protocol that specifies the markers included in a model, the definition for what constitutes a finding, and positive- and negative-likelihood ratios (Reddy, 2014).

The nuchal skinfold is measured in the transcerebellar view of the fetal head, from the outer edge of the skull to the outer border of the skin (see Fig. 14-3A). A measurement ≥6 mm is typically considered abnormal (Benacerraf, 1985). This finding is present in approximately 1 per 200 pregnancies and confers a more than tenfold risk for Down syndrome (Bromley, 2002; Nyberg, 2001; Smith-Bindman, 2001).

An echogenic intracardiac focus is a focal papillary muscle calcification that is neither a structural nor functional cardiac abnormality. It is usually left-sided (see Fig. 14-3B). Such a focus is present in approximately 4 percent of fetuses, but it may be found in up to 30 percent of Asian individuals (Shipp, 2000). As an isolated finding, this approximately doubles the risk for fetal Down syndrome (see Table 14-9). Bilateral echogenic foci are associated with trisomy 13 (Nyberg, 2001).

Mild renal pelvis dilatation is usually transient or physiological and does not represent an underlying abnormality (Chap. 10, Renal Pelvis Dilatation). The renal pelves are measured in a transverse image of the kidneys, anterior-to-posterior, with calipers placed at the inner borders of the fluid collection (see Fig. 14-3C). A measurement ≥4 mm is found in about 2 percent of fetuses and approximately doubles the risk for Down syndrome. The degree of pelvic dilatation beyond 4 mm correlates with the likelihood of an underlying renal abnormality, and additional evaluation is generally performed at approximately 32 weeks.

Echogenic fetal bowel is defined as bowel that appears as bright as fetal bone (see Fig. 14-3D). It is identified in approximately 0.5 percent of pregnancies and most commonly represents small amounts of swallowed blood, frequently in the setting of maternal serum AFP level elevation. Although typically associated with normal outcomes, it raises the risk for Down syndrome approximately sixfold. Echogenic bowel has also been associated with fetal cytomegalovirus infection and cystic fibrosis—representing inspissated meconium in the latter.

The femur and humerus are slightly shorter in Down syndrome fetuses. The femur is considered “short” for Down syndrome screening if it measures below the 2.5th percentile or is shortened to ≤90 percent of that expected based on the measured biparietal diameter (American College of Obstetricians and Gynecologists, 2016c; Benacerraf, 1987). As an isolated finding in an otherwise low-risk pregnancy, it is generally not considered to pose great enough risk to warrant counseling modification. Similarly, a humerus shortened to ≤89 percent of that expected, based on a given biparietal diameter, has also been associated with an elevated risk for Down syndrome.

If an isolated minor marker is identified in a woman who has not yet received aneuploidy screening, screening should be offered, and a minor marker is considered an indication to offer cell-free DNA screening (American College of Obstetricians and Gynecologists, 2016c). If cell-free DNA screening has already been performed, the association between isolated minor markers and aneuploidy risk is no longer considered relevant (Reddy, 2014). And, if the cell-free DNA screening result is negative, the fetal aneuploidy risk is not modified by the marker. Conversely, if a cell-free DNA screening result is positive, the absence of minor markers is not considered reassuring.

First-Trimester Sonographic Findings

Unlike second-trimester soft signs, which may be readily visible during standard sonography, first-trimester findings associated with aneuploidy require specialized training. The fetal NT measurement has gained widespread use for aneuploidy screening. Other first-trimester sonographic findings are not routinely used in the United States but may be available in specialized centers. The Perinatal Quality Foundation’s Nuchal Translucency Quality Review Program offers an education program in first-trimester nasal bone assessment (see Fig. 14-2). The Fetal Medicine Foundation also provides online instruction and certification in first-trimester assessment of nasal bone, ductus venosus flow, and tricuspid flow.

Other benefits of first-trimester sonography in women who elect aneuploidy screening include accurate assessment of gestational age and early detection of multifetal gestation or fetal demise. As discussed in Chapter 10 (Second- and Third-Trimester Sonography), first-trimester sonography may identify selected major anomalies associated with aneuploidy, such as cystic hygroma.

Three types of carrier screening may be offered: ethnicity-based screening, panethnic screening (performed regardless of ethnicity), and expanded carrier screening—which is a type of panethnic screening performed for a larger number of conditions, potentially 100 or more. The goal of screening is to provide individuals with meaningful information to guide pregnancy planning according to their values (American College of Obstetricians and Gynecologists, 2017a). Each type of screening has benefits, risks, and limitations. For example, so many disorders are included in expanded carrier screening panels that more than 50 percent of those screened may be identified to be carriers for at least 1 condition. This can cause anxiety for families and may pose challenges if genetic counseling resources are limited. Recognizing that each type of screening is an acceptable strategy, it is recommended that obstetrical providers develop a standard approach to offer one of these three types of carrier screening to pregnant women and couples considering pregnancy (American College of Obstetricians and Gynecologists, 2017a). All carrier screening is optional and should be an informed choice.

Couples with a personal or family history of a heritable genetic disorder should be offered genetic counseling. They are provided an estimated risk of having an affected newborn and given information concerning benefits and limitations of available prenatal testing options. Prenatal diagnosis may be available if the disease-causing mutation or mutations are known. The publicly funded Genetic Testing Registry website contains detailed information regarding more than 10,000 genetic conditions and 48,000 genetic tests (www.ncbi.nlm.nih.gov/gtr/). That said, many genetic disorders are characterized by a high degree of penetrance but variable expressivity. Thus, prediction of phenotype may not be possible, even when family members are affected. 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 offered for certain autosomal recessive disorders that are found in greater frequency in specific racial or ethnic groups (Table 14-10). The founder effect occurs when an otherwise rare gene is found with greater frequency in a certain population and can be traced back to a single family member or small group of ancestors. This phenomenon may develop when generations of individuals procreate only within their own groups because of religious or ethnic prohibitions or geographical isolation. Because it is becoming increasingly difficult to assign a single ethnicity, a panethnic screening panel is another option.

The American College of Obstetricians and Gynecologists (2017a) has developed the following criteria for expanded carrier screening panels:

1. Conditions included in the panel should have a carrier frequency of at least 1:100, which correlates with a population frequency, at minimum, of 1:40,000.

2. Conditions should have a well-defined phenotype, detrimental effect on quality of life, cognitive or physical impairment, early onset, or require surgical or medical intervention.

3. Conditions primarily associated with disease of adult onset are not recommended for inclusion.

4. If an individual is at increased risk for a specific condition, such as Tay-Sachs disease or β-thalassemia, the provider should consider that the test included in the panel may not be the most sensitive one for that condition.

Cystic Fibrosis

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 2000 mutations have been identified (Cystic Fibrosis Mutation Database, 2016). CF may develop from 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 milder disease and can survive for decades longer. Care for the pregnant woman with CF is discussed in Chapter 51 (Pathophysiology).

The American College of Obstetricians and Gynecologists (2017a,b) recommends that all patients who are considering pregnancy or who are already pregnant should be offered carrier screening for CF, regardless of ethnicity.

The current recommended 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, 2017b). The CF carrier frequency approximates 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 approximates ¼ × ^¹/_₂₅ × ^¹/_₂₅, or 1:2500. As shown in Table 14-11, both CF incidence and the sensitivity of the screening test are lower for other ethnicities.

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. If both parents are carriers, chorionic villus sampling or amniocentesis can help determine whether the fetus 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 then only for well-characterized mutations. Prognosis is most heavily affected by the 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 the CFTR mutation and on exposure and susceptibility to environmental factors (Cutting, 2005; Drumm, 2005).

Spinal Muscular Atrophy

This autosomal recessive disorder results in spinal cord motor neuron degeneration that leads to skeletal muscle atrophy and generalized weakness. There is currently no effective treatment. The prevalence of spinal muscular atrophy (SMA) is 1 in 6,000 to 10,000 live births. Types I, II, III, and IV are caused by mutations in the survival motor neuron (SMN1) gene, which is located on the long arm of chromosome 5 (5q13.2) and encodes the SMN protein. Types I and II account for 80 percent of cases and are both lethal (American College of Obstetricians and Gynecologists, 2017b). SMA type I, known as Werdnig-Hoffmann, is the most severe. Disease onset is within the first 6 months, and affected children die of respiratory failure by age 2 years. Type II generally has onset before age 2 years, and the age at death can range from 2 years to the third decade of life. Type III also presents before age 2 years, with disease severity that is milder and more variable. Type IV does not present until adulthood.

The American College of Obstetricians and Gynecologists (2017b) recommends that carrier screening for SMA be offered to all women who are considering pregnancy or are currently pregnant. The SMA carrier frequency approximates 1:35 in those of non-Hispanic white (caucasian) ethnicity, 1:41 in Ashkenazi Jews, 1:53 in Asians, 1:66 in African Americans, and 1:117 in those of Hispanic white ethnicity (Hendrickson, 2009). Carrier detection rates range from 90 to 95 percent for each race/ethnicity except African Americans, in whom it just exceeds 70 percent. Approximately 2 percent of individuals with SMN1 mutations are not identified with carrier screening. In addition, although there is usually one copy of the SMN1 gene on each chromosome, approximately 3 to 4 percent of individuals have two copies of this gene on one chromosome and no copies on the other. These individuals are carriers for the disease. African Americans are more likely to have this genetic variation, which explains the lower sensitivity of screening in this group. The American College of Obstetricians and Gynecologists (2017b) recommends that prior to screening for SMA, providers counsel about its potential spectrum of severity, carrier frequency, and its detection rate. Posttest counseling should include the residual risk after a negative screening result, which differs according to the patient’s ethnicity and also according to the number of SMN1 copies detected. Most unaffected individuals have two copies, but a small percentage has three copies and is at even lower risk. If the patient or her partner has a family history of SMA, or if carrier screening is positive, genetic counseling is recommended.

Sickle Hemoglobinopathies

These include sickle-cell anemia, sickle-cell hemoglobin C disease, and sickle-cell β-thalassemia. Their pathophysiology and inheritance are discussed in detail in Chapter 56 (Polycythemias).

African and African-American patients are at increased risk to carry hemoglobin S and other hemoglobinopathies and should be offered preconceptional or prenatal screening. Of African Americans, 1 in 12 has sickle-cell trait, 1 in 40 carries hemoglobin C, and 1 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 (2015) 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.

Thalassemias

These syndromes are the most common single-gene disorders worldwide, and up to 200 million people carry a gene for one of these hemoglobinopathies (Chap. 56, Thalassemia Syndromes). 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.

Alpha-Thalassemia

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 cis configuration is more prevalent among Southeast Asians, whereas those of African descent are more likely to inherit the trans configuration. Clinically, when both parents carry cis deletions, offspring are at risk for an absence of α-hemoglobin, called Hb Barts disease. This typically leads to hydrops and fetal loss as discussed in Chapter 15 (Hydrops Fetalis).

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 can be considered, particularly among individuals of Southeast Asian descent (American College of Obstetricians and Gynecologists, 2015).

Beta-Thalassemia

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 elevated levels of hemoglobins that do not contain β-chains. These include hemoglobins F and A₂.

β-Thalassemia minor is more common among individuals of African, Mediterranean, and Southeast Asian descent. The American College of Obstetricians and Gynecologists (2015) 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.

Tay-Sachs Disease

This autosomal recessive lysosomal-storage disease is characterized by absence of the hexosaminidase A enzyme. This leads to accumulation 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 approximates 1 in 30, but it is much lower, only about 1 in 300, in the general population. Other groups at greater 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 disease now occur in non-Jewish individuals.

The American College of Obstetricians and Gynecologists (2017b) has the following screening recommendations for Tay-Sachs disease:

1. 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.

2. 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 there is a family history of Tay-Sachs disease, an expanded carrier screening panel may not be the best approach unless the familial mutation is included in the panel.

3. Molecular testing (DNA-based mutation analysis) is highly effective in Ashkenazi Jewish individuals and other high-risk groups, but the detection rate in low-risk groups is more limited.

4. Biochemical analysis of 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.

5. 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 approximates 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 (2017b) recommends that carrier screening for these three conditions be offered to Ashkenazi Jewish individuals, either before conception or during early pregnancy. This is in addition to carrier screening for cystic fibrosis and spinal muscular atrophy, which are offered to all women who are considering pregnancy or who are currently pregnant. Further, there are several other autosomal recessive conditions for which the College recommends that screening be considered (American College of Obstetricians and Gynecologists, 2017b). As of 2017, these include Bloom syndrome, familial hyperinsulinism, Fanconi anemia, Gaucher disease, glycogen storage disease type I (von Gierke disease), Joubert syndrome, maple syrup urine disease, mucolipidosis type IV, Niemann-Pick disease, and Usher syndrome. 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, effective treatment is available in the form of enzyme therapy.

Diagnostic procedures used in prenatal diagnosis include amniocentesis, chorionic villus sampling (CVS), and rarely fetal blood sampling. These enable characterization of an increasingly large number of genetic abnormalities before birth. Karyotype analysis has a diagnostic accuracy of more than 99 percent for aneuploidy and chromosomal abnormalities larger than 5 to 10 megabases. In the setting of a fetal structural abnormality, chromosomal microarray analysis (CMA) is recommended as the first-line genetic test performed, as it may detect clinically significant chromosomal abnormalities in approximately 6 percent of fetuses with normal standard karyotype (Callaway, 2013; de Wit, 2014). An exception would be if the structural abnormality strongly suggests a particular karyotype—such as endocardial cushion defect with trisomy 21 or holoprosencephaly with trisomy 13. In such cases, karyotyping with or without fluorescence in-situ hybridization (FISH) may be offered as the initial test (American College of Obstetricians and Gynecologists, 2016b). Among those without evidence of a fetal structural abnormality and with a normal karyotype, CMA has detected additional chromosomal abnormalities (pathogenic copy number variants) in approximately 1 percent. It is therefore made available whenever a prenatal diagnostic procedure is performed (American College of Obstetricians and Gynecologists, 2016b; Callaway, 2013). Types of CMA platforms and their benefits and limitations are reviewed in Chapter 13 (Chromosomal Microarray Analysis).

Ironically, improvements in aneuploidy screening tests, in particular the widespread use of cell-free DNA screening, have resulted in a dramatic drop in the number of prenatal diagnostic procedures. Larion and colleagues (2014) reported a 70-percent decline in CVS procedures and a nearly 50-percent drop in amniocentesis procedures following introduction of cell-free DNA screening in 2012. This further amplified the decrease in amniocentesis procedures that began following adoption of first-trimester screening (Warsof, 2015). In addition, because so many disorders can be diagnosed from amnionic fluid specimens, fetal blood sampling is now rarely, if ever, indicated for genetic diagnoses.

Amniocentesis

This is the most common prenatal diagnostic procedure. Transabdominal withdrawal of amnionic fluid is generally done between 15 and 20 weeks but may be performed at any point later in gestation. Indications include diagnosis of fetal genetic disorders, congenital infections, and alloimmunization, as well as assessment of fetal lung maturity. The most common types of prenatal diagnostic tests are CMA to assess copy-number gains or losses, karyotype analysis to test for aneuploidy, and FISH to identify gain or loss of specific chromosomes or chromosome regions (Chap. 13, Genetic Tests). Because amniocytes must be cultured before fetal karyotype can be assessed, the time needed for karyotyping is 7 to 10 days. In contrast, FISH studies are usually completed within 24 to 48 hours. CMA can often be performed directly on uncultured amniocytes with a turnaround time of only 3 to 5 days, and if amniocyte culture is required, turnaround time is 10 to 14 days (American College of Obstetricians and Gynecologists, 2016b).

Technique

Amniocentesis is performed using aseptic technique, with a 20- or 22-gauge spinal needle and ultrasound guidance (Fig. 14-4). A standard spinal needle is 9 cm long, and depending on patient habitus, a longer needle may be required. Measurement of the sonographic distance from skin to amnionic fluid pocket may aid needle selection. Sonography is used to identify a pocket of amnionic fluid that is close to the midline, being cognizant of uterine size and shape. The needle is inserted perpendicular to the skin and guided into the deepest portion of the fluid pocket, avoiding fetal parts and umbilical cord. Efforts are made to puncture the chorioamnion rather than to push or “tent” it away from the uterine wall. The amnion usually fuses with the adjacent chorion by 16 weeks’ gestation, and the procedure is generally deferred until after chorioamnion fusion has occurred. Discomfort from the procedure is considered minor, and local anesthetic has not been found to be beneficial (Mujezinovic, 2011).

Following the procedure, 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 implants along the anterior uterine wall in approximately half of pregnancies. In these cases, the placenta will be traversed by the needle approximately 60 percent of the time (Bombard, 1995). Needle passage through the placenta is avoided when possible, although fortunately this has not been associated with greater pregnancy loss rates (Marthin, 1997). Dark brown or greenish fluid may represent a past episode of intraamnionic bleeding.

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 to 30 mL of fluid is then collected for either fetal CMA or karyotyping before removing the needle. Sonography is used to observe the uterine puncture site for bleeding, and fetal cardiac motion is documented at the procedure’s end. If the patient is Rh D-negative and unsensitized, anti-D immune globulin is administered following the procedure (Chap. 15, Prevention of Anti-D Alloimmunization).

Multifetal Pregnancy

When performing the procedure in a diamnionic twin gestation, careful attention is paid to the location of each sac and the dividing membrane. Until recently, a small quantity of dilute indigo carmine dye was often injected before removing the needle from the first sac, with return of clear amnionic fluid anticipated following needle placement into the second sac. Because of widespread shortages of indigo carmine dye, most experienced providers offer amniocentesis in multifetal gestations when indicated, without dye injection. Methylene blue dye is contraindicated because it has been associated with jejunal atresia and neonatal methemoglobinemia (Cowett, 1976; van der Pol, 1992).

Complications

The procedure-related loss rate following midtrimester amniocentesis has decreased with improvements in imaging technology. Based on single-center studies and metaanalysis data, the amniocentesis procedure-related loss rate approximates 0.1 to 0.3 percent when performed by an experienced provider—about 1 per 500 procedures (Akolekar, 2015; American College of Obstetricians and Gynecologists, 2016b; Odibo, 2008). The loss rate may be doubled in women with class 3 obesity, which is a body mass index (BMI) >40 kg/m² (Harper, 2012). In twin pregnancies, Cahill and coworkers (2009) reported a loss rate of 1.8 percent attributable to amniocentesis.

The amniocentesis indication can influence loss rates, which can be greater with some fetal abnormalities, aneuploidies, and conditions such as hydrops. And, some losses are due to abnormal placental implantation or abruption, uterine abnormalities, or infection. Wenstrom and colleagues (1990) analyzed 66 fetal deaths following nearly 12,000 procedures and found that 12 percent were associated with preexisting intrauterine infection.

Other complications of amniocentesis include amnionic fluid leakage or transient vaginal spotting in 1 to 2 percent. 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).

Early Amniocentesis

This describes amniocentesis performed between 11 and 14 weeks’ gestation. The technique is the same as for traditional amniocentesis, but sac puncture may be more challenging due to lack of membrane fusion to the uterine wall. 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. These include development of talipes equinovarus (clubfoot), amnionic fluid leakage, and fetal loss (Canadian Early and Mid-Trimester Amniocentesis Trial, 1998; Philip, 2004). Given these risks, the American College of Obstetricians and Gynecologists (2016b) recommends that early amniocentesis not be performed.

Chorionic Villus Sampling

Biopsy of chorionic villi is typically performed between 10 and 13 weeks’ gestation. As with amniocentesis, the specimen is generally sent for karyotyping or CMA. The primary advantage of villus biopsy is that results are available earlier in pregnancy, permitting more time for decision-making and safer pregnancy termination, if desired. Very few analyses specifically require either amnionic fluid or placental tissue.

Technique

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, 2016b). Transcervical CVS 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. 14-5).

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.

Complications

The overall loss rate following CVS is higher than that following midtrimester amniocentesis. This is because of background spontaneous losses, that is, losses 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 with that of amniocentesis. 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 approximated 1 per 400 for either procedure. The indication for CVS will also affect the loss rate. For example, fetuses with increased NT thickness have a greater likelihood of demise. Finally, there is a learning curve 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, shown in Figure 14-6 (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, the incidence of limb defects does not exceed the background population rate of about 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, 2016c).

A limitation of CVS is that chromosomal mosaicism is identified in up to 2 percent of specimens (Malvestiti, 2015). In most cases, the mosaicism reflects confined placental mosaicism rather than a true second cell line within the fetus. This is discussed in Chapter 13 (Chromosomal Mosaicism). Amniocentesis should be offered, and if the result is normal, the mosaicism is usually presumed to be confined to the placenta. Confined placental mosaicism has been associated with growth-restricted newborns (Baffero, 2012).

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, and fetal anemia assessment remains the most common indication (Chap. 16, Surgical Therapy). 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).

Technique

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 (Fig. 14-7). 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 lies anteriorly. 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 no trials 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.

Complications

The procedure-related fetal loss rate following fetal blood sampling approximates 1.4 percent (Ghidini, 1993; 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; Society for Maternal-Fetal Medicine, 2013). Most complications are transitory, with complete recovery, but some result in fetal loss.

In one series of more than 2000 procedures comparing fetal blood sampling near the placental cord insertion site with puncture of a free loop, rates of procedure success, pregnancy loss, visible bleeding from the cord, and fetal bradycardia did not differ. Time to complete the procedure was significantly shorter if the cord was sampled at the placental insertion site than in 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 (PGD) and preimplantation genetic screening (PGS)—each with different indications. Comprehensive genetic counseling is required before consideration of these procedures. There are three techniques that are used for both categories:

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. However, two separate micromanipulation procedures are required, and genetic abnormalities of paternal origin are not detected. This technique has been used to diagnose 146 mendelian disorders, and the reported accuracy exceeds 99 percent (Kuliev, 2011).

2. Blastomere biopsy is done at the 6- to 8-cell (cleavage) stage when an embryo is 3 days old. This allows both maternal and paternal genomes to be evaluated. One cell is typically removed through a hole made in the zona pellucida (Fig. 14-8). A limitation of using this technique for aneuploidy assessment is that because of mitotic nondisjunction, mosaicism of the blastomeres may not reflect the chromosomal complement of the developing embryo (American Society for Reproductive Medicine, 2008). In addition, the implantation rate of normal embryos is slightly lower following this technique.

3. Trophectoderm biopsy involves removal of 5 to 7 cells from a 5- to 6-day blastocyst. 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.

Preimplantation Genetic Diagnosis

A genetic abnormality—rather than infertility—may be a reason for a couple to elect IVF. With a known carrier(s) of a specific genetic disease or a balanced chromosomal rearrangement, PGD may be performed to determine if an oocyte or embryo has the defect. Only embryos without the abnormality would be implanted.

This procedure has numerous 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 greater later cancer risk; and to match human leukocyte antigens for umbilical cord stem cell transplantation for a sibling (de Wert, 2007; Fragouli, 2007; Grewal, 2004; Rund, 2005; Xu, 2004).

Because typically only one or two cells are available for analysis and because 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

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 PGS has obvious theoretical advantages, it has faced challenges in practice.

Mosaicism is common in cleavage-stage embryo blastomeres. However, it may not be clinically significant because it often does not reflect the actual embryonic chromosomal complement. In addition, among women 35 years or older, pregnancy rates following PGS with FISH are significantly lower than rates observed following IVF without PGS (Mastenbroek, 2007, 2011). Because the number of chromosome pairs per cell nucleus that can be evaluated with FISH is limited, more recent efforts have focused on the use of chromosomal comprehensive screening with CMA (Dahdouh, 2015).