Chapter 17. Prenatal Diagnosis
BS. Nguyễn Hồng Anh
Prenatal diagnosis is the science o identiying congenital abnormalities and genetic conditions in the etus. It encompasses the
diagnosis o structural malormations with specialized ultrasound, screening tests or aneuploidy, carrier screening or
genetic diseases, and diagnostic tests perormed on chorionic
villi and amnionic uid.
Based on population-based registry data including births,
etal deaths, and pregnancy terminations, 4 per 1000 pregnancies have a chromosomal abnormality (Wellesley, 2012).
I chromosomal microarray analysis (CMA) is perormed on
chorionic villi or amnionic uid, an additional 4 per 1000 are
ound to have a pathogenic copy number variant, such as a
microdeletion or microduplication (Srebniak, 2017). It is
important or all pregnant women to be oered both screening
and diagnostic tests.
Te goal o prenatal diagnosis is to provide accurate inormation about short- and long-term prognosis, recurrence risk,
and potential therapy. Nondirective counseling and provision
o unbiased knowledge are paramount. Management o an
aected pregnancy that includes diagnostic testing, discussion
o potential etal therapy options and postnatal care, and decisions related to expectant management or pregnancy termination are all incorporated into counseling (Flessel, 2011). Fetal
imaging o congenital anomalies is discussed in Chapter 15,
etal therapy in Chapter 19, and pregnancy termination in
Chapter 11.
HISTORICAL PERSPECTIVE
Nearly 50 years ago, Brock (1972, 1973) observed that pregnancies complicated by neural-tube deects had higher levels
o alpha-etoprotein (AFP) in maternal serum and amnionic
uid. Widespread serum screening began in 1977, ater a collaborative trial rom the United Kingdom established the association between elevated maternal serum AFP levels (MSAFP)
and etal open neural-tube deects (Wald, 1977). Screening at
16 to 18 weeks’ gestation detected 90 percent o etuses with
anencephaly and 80 percent o those with open spina bida,
similar to current screening perormance (American College o
Obstetricians and Gynecologists, 2019c).
Te terms level I and level II ultrasound were coined in this
context. In the Caliornia MSAFP Screening Program o the
1980s and early 1990s, the rst step in evaluation o an abnormally elevated MSAFP was a level I ultrasound examination.
Tis screening examination demonstrated that one third o
pregnancies with MSAFP elevation had incorrect gestational
age, multietal gestation, or etal demise as the etiology (Filly,
1993). Amniocentesis was then oered to the remaining two
thirds. I the amnionic uid AFP concentration was elevated,
a level II ultrasound examination was perormed to detect and
characterize a etal abnormality.
Additionally, an elevated amnionic uid AFP level prompted
a concurrent assay or amnionic uid acetylcholinesterase.
Because acetylcholinesterase leaks rom the exposed neural
tissue into the amnionic uid, presence o both analytes was
used to conrm the diagnosis. However, other etal abnormalities are associated with elevated amnionic uid AFP and
positive assay results or acetylcholinesterase. Tese include
ventral wall deects, esophageal atresia, etal teratoma, cloacal exstrophy, and skin abnormalities such as epidermolysis
bullosa.
Most neural-tube deects are now detected with a standard
ultrasound examination, and the detailed ultrasound examination
is the preerred diagnostic test (American College o Obstetricians
and Gynecologists, 2019c; Dashe, 2006). Level II ultrasound is
no longer an appropriate synonym or detailed ultrasound examination, because the study now includes a much more comprehensive evaluation o etal anatomy (Chap. 14, p. 251).
As MSAFP screening was being adopted, the designation
“advanced maternal age” (AMA) became popular. A 1979
National Institutes o Health Consensus Development Con-
erence recommended advising pregnant women who were 35
years and older about the possibility o amniocentesis or etal
karyotyping. Te threshold was based on the greater risk or
selected etal chromosomal abnormalities with increasing maternal age (Table 17-1). Tere was also an assumption that the
loss rate attributable to amniocentesis was equivalent to the etal
Down syndrome risk at maternal age 35. Tis is no longer the
case.
In 1984, Merkatz and colleagues reported that midtrimester MSAFP levels were lower in pregnancies with trisomies
21 and 18 than in unaected pregnancies. Maternal age was
incorporated into the calculation, which enabled assignment o
a specic risk (DiMaio, 1987; New England Regional Genetics Group, 1989). Te MSAFP screen detected approximately
25 percent o cases o etal trisomy 21 when the threshold ratio
or a positive result was set at 1:270—the approximate secondtrimester risk at maternal age 35. Tis trisomy 21 risk threshold
and the associated 5-percent alse-positive rate became standards that remain in use in some laboratories.
ANEUPLOIDY SCREENING
Aneuploidy is the presence o one or more extra chromosomes,
usually resulting in trisomy, or loss o a chromosome—monosomy
(Chap. 16, p. 309). O recognized pregnancies with chromosomal abnormalities, trisomy 21 accounts or approximately hal
o cases; trisomy 18 accounts or 15 percent; trisomy 13, or
5 percent; and the sex chromosomal abnormalities—45,X,
47,XXX, 47,XXY, and 47,XYY—or approximately 12 percent.
Screening tests are available or some or all o these aneuploidies.
Te risk or etal trisomy increases with maternal age, particularly ater age 35 (Fig. 16-2, p. 310). Te positive predictive value o all aneuploidy screening tests is higher or
women aged 35 years and older. Such women account or
18 percent o deliveries in the United States (Fig. 17-1). At
Parkland Hospital, the majority o Down syndrome births
occur in this age group (Hussamy, 2019). Other important
aneuploidy risk actors include a numerical chromosomal
abnormality or structural chromosomal rearrangement in the
woman or her partner—such as a balanced translocation—or a
prior pregnancy with autosomal trisomy or triploidy.
TABLE 17-1. Estimated Risks for Fetal Trisomy 21 and Any Aneuploidy
According to Maternal Age and Timing of Diagnosis
Trisomy 21 Any Aneuploidy
Age
Chorionic Villus
Sampling Amniocentesis Delivery Amniocentesis Delivery
35 1/249 1/280 1/356 1/132 1/204
36 1/196 1/220 1/280 1/105 1/167
37 1/152 1/171 1/218 1/83 1/130
38 1/117 1/131 1/167 1/65 1/103
39 1/89 1/100 1/128 1/53 1/81
40 1/68 1/76 1/97 1/40 1/63
41 1/51 1/57 1/73 1/31 1/50
42 1/38 1/43 1/55 1/25 1/39
43 1/29 1/32 1/41 1/19 1/30
44 1/21 1/24 1/30 1/15 1/24
45 1/16 1/18 1/23 1/12 1/19
Adapted from Hook, 1983; Snidjers, 1999.
0
Percent
2
10
8 6 4
12
14
16
18
Year
2018
18.0
15.5
2010
14.2 14.3
2002
13.7
12.8
1994
11
8.8
1986
6.9
FIGURE 17-1 Trends in the percentage of births to women aged
35 to 44 years. (Data from the Centers for Disease Control and
Prevention, 2015, 2019).334 The Fetal Patient
Section 6
Broadly speaking, there are two types o aneuploidy screening tests, those that are analyte-based and those that are cell-
ree DNA-based. Cell-ree DNA (cDNA) screening is more
sensitive and specic than analyte-based screening, and it has a
much higher positive predictive value. One prospective screening study or trisomy 21 in 15,000 pregnancies ound that the
average positive predictive value was 80 percent or cDNA but
only 3 percent or rst-trimester screening (Norton, 2015).
However, analyte-based screening has the unusual benet that
an abnormal result may indicate a genetic abnormality or
which screening was not specically perormed (p. 337). Te
position o the American College o Obstetricians and Gynecologists (2020e) is that no one screening test is superior in all
circumstances. No aneuploidy screening test is diagnostic, and
prenatal diagnosis is strongly recommended beore acting upon
a result.
Tere are selected circumstances in which aneuploidy screening is not recommended. Following diagnosis o a major etal
abnormality, prenatal diagnosis with amniocentesis or chorionic villus sampling is advised. Te etal risk cannot be normalized with a negative aneuploidy screening result. Screening
results can be alsely negative, and major abnormalities coner
risk or genetic syndromes not identied through screening
tests. Analyte-based screening tests are not valid in the setting o an abnormality that aects the AFP component, such
as a neural-tube deect or ventral-wall deect. Additionally, all
aneuploidy screening methods are less accurate in twin gestations and are invalid in triplet gestations and higher-order
multiples.
Aneuploidy screening should be an inormed patient choice
that incorporates the patient’s clinical circumstances, values,
interests, and goals. Counseling should be individualized, with
the understanding that at least 20 percent o women preer
not to receive aneuploidy screening, even when nancial barriers are removed (Kuppermann, 2014). Elements o counseling prior to aneuploidy screening are listed in Table 17-2. Te
screening test(s) oered and elected may depend on actors
such as gestational age, provider location or practice setting,
and the patient’s out-o-pocket costs.
■ Test Characteristics
Validity characteristics convey inormation about how well an
aneuploidy screening test dierentiates aected rom unaected
pregnancies. Te sensitivity, also known as the detection rate, is the
proportion o etuses with a particular aneuploidy who are detected
by the screening test. Te converse is the alse-negative rate. I a
woman elects a rst-trimester aneuploidy screen with a sensitivity
o 80 percent or trisomy 21, the alse-negative rate is 20 percent,
so the test is anticipated to miss 1 in 5 aected pregnancies.
Specicity is the proportion o unaected pregnancies with
a negative screening result. Te converse o specicity is the
TABLE 17-2. Aneuploidy Screening Counseling Elements
All pregnant women have 3 options: screening, diagnostic testing, and no screening or testing.
The purpose of a screening test is to provide information, not to dictate a course of action.
Diagnostic testing is safe and effective and provides information that screening does not.
There are differences between a screening test and a diagnostic test.
Screening evaluates whether the pregnancy is at increased risk for specific conditions and estimates the degree of risk.
Screening does not provide information regarding all genetic abnormalities.
With a negative screening test result, the risk is decreased but not eliminated.
With a positive screening result, a diagnostic test is recommended if the patient wants to know whether the fetus is
affected.
Irreversible management decisions should not be based on screening test results.
Cell-free DNA screening does not always provide a result, and the aneuploidy risk is increased in such cases.
Basic information is provided regarding the conditions for which screening is performed and the limitations of the
screening test.
Information may include prevalence of a genetic condition, associated abnormalities, and prognosis.
Diagnosis may aid earlier identification of associated abnormalities.
In the case of trisomy 18 or 13, diagnosis may affect pregnancy management if complications arise such as growth
restriction or nonreassuring fetal heart rate.
All screening tests are less effective in multifetal gestations.
Phenotypic expression of sex chromosome aneuploidies varies widely.
Apriori risk for fetal aneuploidy may affect whether a woman elects a screening test or diagnostic test.
Age-related risk information may be found in reference tables.
If a patient has had a prior fetus with autosomal trisomy, robertsonian translocation, or other chromosomal abnormality,
additional evaluation and counseling are recommended.
If a major fetal abnormality has been identified with ultrasound, a diagnostic test is preferred, and aneuploidy screening
is not recommended.
American College of Obstetricians and Gynecologists, 2020e; Dashe, 2016.Prenatal Diagnosis 335
CHAPTER 17
alse-positive rate, those without aneuploidy who nonetheless
screen positive. Because aneuploidies are individually uncommon, the screen-positive rate, which is the overall proportion o
tests with a positive result, is usually similar to the alse-positive
rate. Aneuploidy screening tests generally have screen-positive
rates no higher than 5 percent.
Sensitivity and specicity do not convey any inormation
regarding individual risk. Te positive predictive value (PPV)
is the proportion with a positive screening result who have
an aneuploid etus. PPV may be viewed as the test result. It
is usually expressed as a 1:X ratio or as a percentage. Because
PPV is directly aected by disease prevalence, it is higher
in women aged 35 years and older than in younger women
(Table 17-3). Negative predictive value is the proportion o
those with a negative screening test result who have unaected
(euploid) etuses. Because the prevalence o aneuploidy is low,
the negative predictive value o all aneuploidy screening tests
generally exceeds 99 percent (Gil, 2017; Norton, 2015).
■ Cellfree DNA Screening
Tis screening test identies DNA ragments derived primarily
rom apoptotic trophoblasts, which are placental cells undergoing programmed cell death. Tus, the term cell-ree etal DNA is
a misnomer. Dierences between placental and etal DNA may
cause alse-positive and alse-negative results. Tere are three types
o assays: whole-genome sequencing, chromosome selective or
targeted sequencing, and single nucleotide polymorphism analysis. CDNA screening can be perormed at any time ater 9 to
10 weeks’ gestation. When it was rst introduced, cDNA screening was oered primarily to pregnancies with increased aneuploidy risk, but it has since become widely available (American
College o Obstetricians and Gynecologists, 2020e).
CDNA screening is most commonly used to screen or
autosomal trisomies—trisomy 21, trisomy 18, and trisomy
13. It may also be used to screen or 45,X (urner syndrome),
47,XXX, 47,XXY, and 47,XYY. A recent metaanalysis concluded that the number o reported sex aneuploidy cases is too
small or accurate assessment o screening perormance (Gil,
2017). Te prognosis or these sex chromosomal aneuploidies
diers considerably rom that o the autosomal trisomies (Chap.
16, p. 313). I screening or sex chromosomal abnormalities is
elected, counseling should include this inormation.
CDNA has the highest sensitivity and specicity o any aneuploidy screening test. In a metaanalysis o 35 studies o largely
high-risk pregnancies, the pooled sensitivity to detect trisomy 21
was 99.7 percent, and or trisomies 18 and 13, 98 percent and
99 percent, respectively (Gil, 2017). For each o these autosomal
trisomies, the specicity is 99.9 percent, and the combined alsepositive rate is less than 0.2 percent. I the screening platorm
includes other genetic conditions, each additional condition
increases the overall alse-positive rate or the test.
Te PPV o a positive cDNA result is provided in the report
or may be estimated rom an online calculator or reerence table
(see able 17-3). Because the risk estimate or each aneuploidy
depends on maternal age, such inormation may be included as
part o pretest counseling. Tis can help to avoid the misconception that a positive screen indicates aneuploidy—it does not.
Because cDNA screening has a PPV ar higher than analyte
screening, it may be oered as a secondary screening test or
women who wish to avoid amniocentesis. Such an approach
has important caveats. I cDNA is used as a secondary screening test and yields a negative result, the residual risk or a chromosomal abnormality may be as high as 2 percent (Norton,
2014). And, i the cDNA result is positive, the delay in denitive diagnosis may aect management options.
In addition to the aorementioned aneuploidies, cDNA
screening is available or other genetic conditions. Tese
include aneuploidies such as trisomies 16 and 22, specic
microdeletion syndromes, and large copy number changes
throughout the genome. Currently, the American College
o Obstetricians and Gynecologists (2020e) does not recommend cDNA screening or these other conditions. Sensitivity and specicity o cDNA screening have not been
established or them, and screening accuracy has not been
validated clinically.
Limitations of cfDNA Screening
In 2 to 4 percent o pregnancies screened with cDNA, no result
is obtained. Tese cases are termed “no-call,” or indeterminate.
TABLE 17-3. Positive Predictive Value of Cell-Free DNA Screening for Autosomal
Trisomies and Sex Chromosome Abnormalities, According to Maternal
Age at Term
Maternal Age Trisomy 21 Trisomy 18 Trisomy 13 45,X 47,XXX 47,XXY 47,XYY
20 48% 14% 6% 41% 27% 29% 25%
25 51% 15% 7% 41% 27% 29% 25%
30 61% 21% 10% 41% 27% 29% 25%
35 79% 39% 21% 41% 28% 30% 25%
40 93% 69% 50% 41% 45% 52% 25%
45 98% 90% NA 41% 73% 77% NA
NA = not available.
Positive predictive values were obtained using the Cell Free DNA Screening Predictive
Value Calculator from the Perinatal Quality Foundation, 2020.
Calculations based on prevalence at 16 weeks’ gestation using sensitivities and specificities
from Gil, 2015.336 The Fetal Patient
Section 6
With a no-call result, the etal aneuploidy risk is as high as
4 percent (Norton, 2015). As shown in Table 17-4, this result
is similar to the average risk conerred by a positive analyte
screening test result.
Low etal raction is a common reason or no-call results.
Te etal raction is the proportion o cDNA in the maternal
circulation that is placental rather than maternal in origin.
Te etal raction approximates 10 percent in the late rst
trimester, increasing slightly thereater (Ashoor, 2013; Scott,
2018). Low etal raction is usually dened as <4 percent
o the total, and it is more prevalent beore 10 weeks’ gestation and in the setting o maternal obesity (Ashoor, 2013;
Juul, 2020; Scott, 2018). Te no-call rate may exceed 10 to
20 percent in women whose body mass index is 40 kg/m2 or
greater (Juul, 2020). Low etal raction is also more common
in the setting o aneuploidy, particularly trisomies 18 and 13
(Norton, 2015; Pergament, 2014). Te American College
o Medical Genetics and Genomics recommends that the
etal raction be included in cDNA reports because it assists
with interpretation o results (Gregg, 2016). Pretest counseling should include the possibility o a no-call result and its
clinical signicance (see able 17-2).
I the cDNA screening test is positive or yields a no-call
result, additional genetic counseling is indicated, and amniocentesis should be oered. I cDNA screening is repeated,
the risk or repeat screen ailure approximates 40 percent
(Dar, 2014; Quezada, 2015; Rolnik, 2018). Detailed ultrasound examination may be oered, but it is not a substitute
or amniocentesis, and the residual aneuploidy risk ollowing
normal detailed ultrasound examination is unknown.
It is important to remember that cDNA results may not
reect the etal DNA complement. Rather, a positive result
may be caused by conned placental mosaicism or early demise
o an aneuploid co-twin. Alternately, it may indicate maternal
aneuploidy, mosaicism, or even occult maternal malignancy
(Bianchi, 2015; Curnow, 2015; Grati, 2014; Wang, 2014). I
ultrasound identies an empty second gestational sac, cDNA
screening is not recommended. Te patient should be inormed
o such limitations prior to screening.
■ Analytebased Aneuploidy Screening
Te three categories o multiple marker-serum analyte screening are rst-trimester screens, second-trimester screens, and
combined rst- and second-trimester screens. Te serum concentration o each analyte is converted to a multiple o the
median (MoM) o the unaected population by adjusting or
maternal age, maternal weight, and gestational age. Te serum
AFP concentration is urther adjusted or maternal race and
or presence o diabetes, which aect calculation o neural-tube
deect risk (Greene, 1988; Huttly, 2004).
Te analyte-based screening result is based on a composite
likelihood ratio, and the maternal age-related risk is multiplied
by this ratio. Adjustment normalizes the distribution o analyte
levels and permits comparison o results rom dierent laboratories and populations. Each woman is provided with a specic
risk or trisomy 21 and or trisomy 18—or in the rst trimester,
or trisomy 18 or 13 in some cases. Each screening test has
a predetermined value at or above which it is deemed “positive” or abnormal. For second-trimester tests, this threshold
has traditionally been set at the risk or etal Down syndrome
at a maternal age o 35 years—approximately 1 in 270 in the
second trimester. Te trisomy 21 risk may be urther modied
based on selected ultrasound markers (p. 340).
TABLE 17-4. Characteristics of Screening Tests for Trisomy 21 in Singletons
Screening Test
Detection
Rate
FalsePositive
Rate
Positive Predictive
Valuea
Quadruple screen:
AFP, hCG, estriol, inhibin 80–82% 5% 3%
Firsttrimester screen:
NT, hCG, PAPP-A
First-trimester NT alone
80–84%
64–70%
5%
5%
3–4%
Integrated screening 94–96% 5% 5%
Sequential screening:
Stepwise
Contingent
92–97%
91–95%
5.1%
4.5%
5%
5%
Cellfree DNA screening:
Positive result
Low fetal fraction or no result
99%
—
0.1%
2–4%
Table 17-3
3–4%
aThe positive predictive value represents the overall population studied and cannot
be applied to any individual patient.
AFP = alpha-fetoprotein; hCG = human chorionic gonadotropin; NT = nuchal
translucency; PAPP-A = pregnancy-associated plasma protein A.
Data from Aldred, 2017; Baer, 2015; Dashe, 2016; Gil, 2015; Malone, 2005b; Norton,
2015; Pergament, 2014; Quezada, 2015.Prenatal Diagnosis 337
CHAPTER 17
A benet o analyte screening is that pregnancies with
abnormal results may have genetic abnormalities other than
those or which screening was perormed. For example, in a
review o more than 2500 pregnancies with genetic abnormalities rom the Caliornia Prenatal Screening Program, combined
rst- and second-trimester screening results were abnormal in
93 percent with trisomies 21 and 18 (Norton, 2016). However,
results were also abnormal in 80 percent with trisomy 13, in
80 percent with 45,X, in nearly 60 percent with other sex
chromosomal abnormalities, and in more than 50 percent with
other genetic abnormalities. Alamillo and associates (2013)
similarly identied an abnormal karyotype other than trisomies
21, 18, or 13 in 2 percent o pregnancies with abnormal rsttrimester screening results, accounting or 30 percent o abnormal karyotypes in the series.
Women younger than 35 are at lower risk or the specic
autosomal trisomies or which cDNA screening is typically
perormed. Tus, i the goal is to select a screening test that
will identiy the highest proportion o etuses with any chromosomal abnormality, the yield may be comparable or even
slightly higher with integrated or sequential screening than
with current cDNA screening (Baer, 2015; Norton, 2014).
First-trimester Aneuploidy Screening
Tis test includes human chorionic gonadotropin (hCG),
pregnancy-associated plasma protein A (PAPP-A), and ultrasound measurement o the nuchal translucency (N). It is per-
ormed between 11 and 14 weeks’ gestation. Pregnancies with
etal trisomy 21 are characterized by higher levels o hCG and
lower PAPP-A. With trisomy 18 and trisomy 13, levels o both
analytes are lower (Cuckle, 2000; Malone, 2005b).
Nuchal Translucency. Tis is the maximum thickness o the
subcutaneous translucent area between the skin and sot tissue
overlying the etal spine at the back o the neck (Fig. 17-2). It is
measured in the sagittal plane and is valid when the crown-rump
length (CRL) measurement is between 38 to 45 mm and 84 mm,
with the lower limit varying according to the laboratory. Measurement criteria are listed in able 14–4 (p. 250). Te N
should be imaged and measured with a high degree o precision
or aneuploidy detection to be accurate. Tis has led to standardized training, certication, and ongoing quality review programs.
An increased N is not a etal abnormality but rather a
marker that coners increased risk. It is helpul to dierentiate rom cystic hygroma, which appears sonographically as
a septated hypoechoic space behind the neck that extends
down the back (Fig. 15-30, p. 286). Te etal aneuploidy risk
is ve times greater with rst-trimester cystic hygroma than
with increased N (Malone, 2005a). An increased N measurement is associated with other genetic syndromes and various birth deects, especially etal cardiac abnormalities (Clur
2008; Simpson, 2007). I the N measurement is at least 3
mm or reaches the 99th percentile or CRL, a detailed ultrasound examination should be oered. Similarly, etal echocardiography may be considered i the N measurement is
at least 3 mm and is recommended i the N measurement
is 3.5 mm or greater (American College o Obstetricians and
Gynecologists, 2020e; American Institute o Ultrasound in
Medicine, 2019).
As an isolated marker, N detects only 70 percent o trisomy 21 etuses, and or this reason it is not used to screen singleton pregnancies (Alldred, 2017; Malone, 2005b). I cDNA
screening has already been perormed, the N measurement
does not improve etal aneuploidy detection and is not recommended or this purpose (American College o Obstetricians
and Gynecologists, 2020e; Rei, 2016). N evaluation may be
helpul in multietal gestations because serum screening is less
accurate, may not be available, and cannot provide inormation
specic to each etus. Te N distribution is similar in twins
and singletons (Cleary-Goldman, 2005).
Efficacy of First-trimester Screening. Beore rst-trimester
screening became widely adopted, our large prospective trials
were conducted, together including more than 100,000 pregnancies (Reddy, 2006). When the screen-positive rate was set
at 5 percent, trisomy 21 detection was 84 percent. A recent
metaanalysis also reported detection o 87 percent o trisomy
21 etuses (Alldred, 2017). First-trimester screening similarly
detects approximately 80 percent o etuses with trisomy 18
and 50 percent with trisomy 13 (Norton, 2015).
Maternal age aects the perormance o rst-trimester
aneuploidy screening. In women younger than age 35 at delivery, etal trisomy 21 detection ranges rom 67 to 75 percent
(Malone, 2005b; Wapner, 2003). Among women older than
35 at delivery, detection is as high as 90 to 95 percent, albeit at
a higher alse-positive rate o 15 to 22 percent.
In twin pregnancies, serum ree β-hCG and PAPP-A levels
are approximately double the singleton values (Vink, 2012).
Even with specic curves, a normal dichorionic co-twin will
tend to normalize screening results. Tus the aneuploidy detection rate is at least 15-percent lower (Bush, 2005).
Analyte abnormalities are termed unexplained i prenatal
diagnostic testing results are normal. Tere is a signicant
FIGURE 17-2 Sagittal image of a normal, 12-week fetus shows
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, also are
shown. (Reproduced with permission from Dr. Michael Zaretsky.)338 The Fetal Patient
Section 6
association between serum PAPP-A levels below the 5th percentile and preterm birth, growth restriction, preeclampsia, and
etal demise (Cignini, 2016; Dugo, 2004; Jellie-Pawlowski,
2015). Similarly, low levels o ree β-hCG have been associated
with etal demise (Goetzl, 2004). Te sensitivity and PPVs o
these markers are too low to make them clinically useul as
screening tests (American College o Obstetricians and Gynecologists, 2020c).
Second-trimester Aneuploidy Screening
Te quadruple marker or “quad” screening test is perormed
rom 15 through 20 or 21 weeks’ gestation depending on the
laboratory. Pregnancies with etal trisomy 21 are characterized
by lower MSAFP, higher hCG, lower unconjugated estriol,
and higher dimeric inhibin levels. In large prospective trials, trisomy 21 detection was 81 to 83 percent at a 5-percent
screen-positive rate (Malone, 2005b; Wald, 1996, 2003). In
cases o trisomy 18, levels o MSAFP, hCG, and unconjugated
estriol are all decreased, and inhibin is not part o the calculation. risomy 18 detection is similar to that or trisomy 21, but
the alse-positive rate is just 0.5 percent (Benn, 1999). As with
rst-trimester screening, aneuploidy detection rates are lower
in younger women and higher in women older than 35 years
at delivery. I second-trimester serum screening is used in twin
pregnancies, aneuploidy detection rates are signicantly lower
(Vink, 2012).
Quadruple-marker screening oers no benet over rsttrimester screening rom the standpoint o trisomy 21 or trisomy
18 detection. As a stand-alone test, it is generally used i women
do not begin care until the second trimester or i rst-trimester
screening and cDNA screening are not available. In 2019, women
who initiated prenatal care beyond the rst trimester made up
more than 20 percent o pregnancies in the United States (Martin, 2020). As subsequently discussed, combining rst- and second-trimester screening yields greater aneuploidy detection.
Maternal Serum AFP Elevation: Neural-tube Defect Screening. AFP is the major protein in etal serum, analogous to
albumin in a child or adult. Deects in etal integument, such
as neural-tube and ventral-wall deects, permit AFP to leak
into the amnionic uid and result in dramatically increased
maternal serum levels. Accurate gestational age assessment
is imperative because the AFP value rises by approximately
15 percent per week during the screening window (Knight,
1992).
Using an MSAFP level o 2.5 MoM as the upper limit o
normal, the neural-tube deect detection rate approximates
95 percent or anencephaly and 80 percent or spina bida,
with a screen-positive rate o 1 to 3 percent (American College
o Obstetricians and Gynecologists, 2019c; Palomaki, 2019).
Te overall PPV approximates just 2 percent. Higher screening threshold values are used in twin pregnancies, which have
twice the AFP level o singletons (Cuckle, 1990). Importantly,
because nearly all cases o anencephaly and most with spina
bida are detected during the standard second-trimester ultrasound examination, MSAFP screening is considered optional
(American College o Obstetricians and Gynecologists, 2019c;
Dashe, 2006; Norem, 2005).
I the serum AFP is elevated, a detailed ultrasound examination is recommended, and characteristic sonographic ndings
are diagnostic (Chap. 15, p. 276). Other abnormalities and
conditions also may result in MSAFP elevation (Table 17-5).
Amniocentesis or measurement o amnionic uid AFP and acetylcholinesterase levels can help to diagnose whether a myelomeningocele is open or closed, which is relevant when considering
etal surgical repair (Chap. 19, p. 372).
Most pregnancies with MSAFP elevation have no etal
abnormality. Te patient should receive counseling about the
benets and limitations o detailed ultrasound examination or
the diagnosis o neural-tube deects and other potential etiologies. Tese include other etal abnormalities, placental abnormalities, and selected adverse pregnancy outcomes (able 17-5).
Te likelihood o any abnormality or adverse outcome increases
in proportion to the AFP level.
Tere are similarly signicant associations between secondtrimester elevation o either hCG or dimeric inhibin alpha
levels and adverse pregnancy outcomes. Te likelihood o
adverse outcome is greater i multiple analytes are elevated
(Dugo, 2005). Te sensitivity and positive predictive values
o these markers are considered too low to be useul or screening or management. No specic program o maternal or etal
surveillance has been ound to avorably aect pregnancy outcomes (Dugo, 2010).
Low Maternal Serum Estriol Level. A maternal serum estriol
level less than 0.25 MoM has been associated with two uncommon but important conditions. Smith-Lemli-Opitz syndrome is
TABLE 17-5. Conditions Associated with MSAFP Level
Elevation
Underestimated gestational age
Multifetal gestation
Fetal death
Neural-tube defect
Gastroschisis
Omphalocele
Cystic hygroma
Esophageal or intestinal obstruction
Liver necrosis
Renal anomalies—polycystic kidneys, renal agenesis,
congenital nephrosis, urinary tract obstruction
Cloacal exstrophy
Osteogenesis imperfecta
Sacrococcygeal teratoma
Congenital skin abnormality
Pilonidal cyst
Chorioangioma of placenta
Placenta intervillous thrombosis
Placental abruption
Oligohydramnios
Preeclampsia
Fetal-growth restriction
Maternal hepatoma or teratoma
MSAFP = maternal serum alpha-fetoprotein.Prenatal Diagnosis 339
CHAPTER 17
an autosomal recessive condition resulting rom mutations in
the 7-dehydrocholesterol reductase gene. It is characterized by
abnormalities o the central nervous system, heart, kidney, and
extremities; with ambiguous genitalia; and with etal-growth
restriction. Detailed sonography should be oered i an unconjugated estriol level is <0.25 MoM (American Institute o
Ultrasound in Medicine, 2019; Dugo, 2010). An elevated
amnionic uid 7-dehydrocholesterol level can conrm the
diagnosis.
Te second condition is steroid sulatase defciency, also known
as X-linked ichthyosis. It is typically an isolated condition, but it
may also occur in the setting o a contiguous gene deletion syndrome (Chap. 16, p. 315). In such cases, it may be associated
with Kallmann syndrome, chondrodysplasia punctata, and/or
mental retardation (Langlois, 2009). I the estriol level is <0.25
MoM and the etus appears to be male, chromosomal microarray analysis or uorescence in situ hybridization to assess the
steroid sulatase locus on the X-chromosome may be considered.
Integrated and Sequential Screening
I rst-trimester screening is combined with second-trimester
screening, aneuploidy detection is signicantly improved.
Combined screening test options require that specimens are
obtained at the correct gestational age and sent to the same
laboratory. Te rst- and second-trimester components cannot be perormed independently, because the alse-positive rate
would be higher and providing accurate risk assessment would
be problematic.
Tree types o screening strategies are available:
1. Integrated screening combines results o rst- and secondtrimester tests. Tis includes a combined measurement o
etal N and serum analyte levels at 11 to 14 weeks’ gestation
plus quadruple markers at approximately 15 to 21 weeks. risomy 21 detection is 94 to 96 percent (see able 17-4).
2. Sequential screening involves inorming the patient o the
results ater rst-trimester screening, with plan to oer prenatal
diagnostic testing i the calculated risk value lies above a speci-
ed threshold. Tere are two testing strategies in this category:
• With stepwise sequential screening, women whose rsttrimester risk is at or below the threshold receive second-trimester screening. Using data rom the First- and
Second-rimester Evaluation o Risk trial, when the
rst-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 trisomy 21 detection
rate (Cuckle, 2008). Te alse-positive rate remained 5
percent (see able 17-4).
• With contingent sequential screening, women are divided
into high-, moderate-, and low-risk groups. Tose at highest risk or trisomy 21—or example, risk >1:30, are counseled and oered diagnostic testing. Women at moderate
risk, between 1:30 and 1:1500, undergo second-trimester
screening, whereas those at lowest risk o <1:1500 receive
negative screening test results and have no urther testing
(Cuckle, 2008). Using this strategy, more than 75 percent
o those screened are provided with reassuring results almost
immediately. Assuming that women accept diagnostic
testing when inormed that the risk is elevated, detection
approaches 91 percent.
■ Ultrasound Screening
Ultrasound can improve aneuploidy screening by providing
accurate gestational age assessment, by detecting multietal gestations, and by identiying major structural abnormalities and
minor aneuploidy markers. With rare exception, the aneuploidy
risk associated with any major abnormality is high enough to
recommend prenatal diagnosis with chromosomal microarray
analysis. We do not recommend aneuploidy screening ater
TABLE 17-6. Aneuploidy Risk Associated with Selected Major Fetal Anomalies
Abnormality Birth Prevalence
Aneuploidy
Risk (%) Common Aneuploidiesa
Cystic hygroma 1/5000 50–70 45,X; 21; 18; 13; triploidy
Nonimmune hydrops 1/1500–4000 10–20 21; 18; 13; 45,X, triploidy
Ventriculomegaly 1/1000–2000 5–25 13; 18; 21; triploidy
Holoprosencephaly 1/10,000–15,000 30–40 13; 18; 22; triploidy
Dandy-Walker malformation 1/12,000 40 18; 13; 21; triploidy
Cleft lip/palate 1/1000 5–15 18; 13
Cardiac defects 5–8/1000 10–30 21; 18; 13; 45,X; 22q11.2 microdeletion
Diaphragmatic hernia 1/3000–5000 5–15 18; 13; 21
Esophageal atresia 1/4000 10 18; 21
Duodenal atresia 1/10,000 30 21
Gastroschisis 1/2000 No increase
Omphalocele 1/3000–5000 30–50 18; 13; 21; triploidy
Clubfoot 1/1000 5–30 18; 13
aNumbers indicate autosomal trisomies except where indicated. For example, 45,X indicates Turner syndrome.
Data from Best, 2012; Canfield, 2006; Colvin, 2005; Cragan, 2009; Dolk, 2010; Ecker, 2000; Gallot, 2007;
Long, 2006; Orioli, 2010; Pavone, 2018; Pedersen, 2012; Sharma, 2011; Solomon, 2010; Walker, 2001.340 The Fetal Patient
Section 6
detection o a major abnormality, because o the many genetic
conditions are not detectable with screening tests. Counseling
should include the association o the abnormality with aneuploidy (Table 17-6). A caveat is that etal abnormalities are
requently not isolated, and associated-but-undetectable abnormalities may greatly aect prognosis.
Importantly, etuses with trisomy 21 and other genetic syndromes may not have obvious ultrasound abnormalities. Just
one third o trisomy 21 etuses have a major abnormality identied with second-trimester ultrasound evaluations (Bromley,
2002; Hussamy, 2019; Vintileos, 1995). I major anomalies
and minor aneuploidy markers are considered, 50 to 75 percent o pregnancies aected by Down syndrome are ound
to have a sonographic abnormality. Tis is not unexpected,
because nearly 40 percent o inants with trisomy 21 do not
have major organ system abnormalities identied in the newborn period (Hussamy, 2019). In contrast, the vast majority
o etuses with trisomies 18 and 13 and with triploidy do have
ultrasound abnormalities detectable in the second trimester.
Aneuploidy Markers
For more than three decades, investigators have recognized
that ultrasound detection o aneuploidy, particularly trisomy
21, may be improved by minor markers that are collectively
reerred to as “sot signs.” Minor markers are normal variants
rather than etal abnormalities, and in the absence o aneuploidy or an associated abnormality, they do not signicantly
aect prognosis. One or more markers are present in at least
10 percent o pregnancies (Bromley, 2002; Nyberg, 2003).
Examples are listed in Table 17-7 and depicted in Figure 17-3.
TABLE 17-7. First- and Second-Trimester
Ultrasound Markers Associated with Fetal
Trisomy 21
First trimester
Ductus venosus with A-wave absence or flow reversal
Nasal bone absence or hypoplasia
Nuchal translucency enlargement
Tricuspid valve regurgitation
Second trimester
Brachycephaly or shortened frontal lobes
Clinodactyly (hypoplastic middle phalanx of fifth digit)
Echogenic bowel
Flat-appearing facies
Echogenic intracardiac focus
Nasal bone absence or hypoplasia
Nuchal fold thickening
Renal pelvis dilatation
“Sandal gap” between first and second toes
Short ear length
Single transverse palmar crease
Single umbilical artery
Short femur
Short humerus
Subclavian artery aberrant (right side)
Widened iliac angle
Markers listed alphabetically.
B C
D E F
A
FIGURE 17-3 Minor sonographic markers that are associated with increased risk for fetal Down syndrome. A. Nuchal skinfold thickening
(bracket). B. Echogenic intracardiac focus (arrow). C. Mild renal pelvis dilatation (pyelectasis) (arrows). D. Echogenic bowel (arrow). E. Clinodactyly—hypoplasia of the 5th finger middle phalanx creates an inward curvature (arrow). F. “Sandal-gap” (arrow).Prenatal Diagnosis 341
CHAPTER 17
A minor marker is an indication or a detailed survey o etal
anatomy to determine i it is isolated (American Institute o
Ultrasound in Medicine, 2019).
I an isolated marker is identied and aneuploidy screening
has not yet been perormed, it should be oered. A benet o
cDNA screening is that isolated aneuploidy markers no longer
actor into the aneuploidy risk, because the negative predictive value o a normal cDNA test is so high. Conversely, i a
cDNA result is abnormal, the absence o minor markers is not
considered reassuring.
First-trimester Markers. Detection o rst-trimester ndings
associated with aneuploidy requires specialized imaging. N
evaluation is discussed earlier (p. 337). Other rst-trimester
markers are not routinely used in the United States but may
be available at specialized centers. Te Perinatal Quality Foundation’s Nuchal ranslucency Quality Review Program oers
an education program in rst-trimester nasal bone assessment.
Te Fetal Medicine Foundation also provides online instruction and certication in rst-trimester assessment o nasal bone,
ductus venosus ow, and tricuspid ow.
Tere are additional benets o rst-trimester ultrasound
evaluation in women who elect aneuploidy screening, including accurate assessment o gestational age and early detection o multietal gestation or etal demise. As discussed in
Chapter 14 (p. 249), standard rst-trimester sonography may
identiy selected major anomalies associated with aneuploidy,
such as cystic hygroma.
Second-trimester Markers. I analyte-based aneuploidy screening has been perormed, minor markers may be used to modiy
the risk using likelihood ratios (Table 17-8). Te risk increases
with the number o markers identied. Risk modication
should ollow a protocol that species criteria or each marker
(Reddy, 2014).
Te nuchal skinold is imaged in the transcerebellar view o
the head and is measured rom the outer edge o the skull to
the outer border o the skin (see Fig. 17-3A). Te skinold is
considered thickened i it measures ≥6 mm at 15 to 20 weeks
(Benacerra, 1985). Tis nding is seen in approximately 1 per
200 pregnancies and coners more than tenold risk or trisomy
21 (Bromley, 2002; Nyberg, 2001; Smith-Bindman, 2001).
An echogenic intracardiac ocus is a ocal papillary muscle
calcication that is neither a structural nor unctional cardiac
abnormality (see Fig. 17-3B). It is present in 4 to 7 percent
o normal etuses. Te etal trisomy 21 risk is approximately
doubled. Te nding is more commonly identied in Asian
individuals (Shipp, 2000). Fetuses with trisomy 13 may have
bilateral echogenic oci (Nyberg, 2001).
Mild renal pelvis dilatation is usually transient or physiologic
(Chap. 15, p. 298). Te renal pelves are measured anteriorto-posterior in a transverse image o the kidneys, with calipers
placed at the inner borders o the uid collection (see Fig.
17-3C). A measurement ≥4 mm is ound in about 2 percent o etuses and approximately doubles the risk or trisomy
21. Te degree o pelvic dilatation beyond 4 mm correlates
with the likelihood o an underlying renal abnormality, and
third-trimester sonography is recommended at approximately
32 weeks.
Echogenic etal bowel is dened as bowel that is sonographically as bright as etal bone (see Fig. 17-3D). It is identied in
approximately 0.5 percent o pregnancies and oten represents
a small amount o swallowed blood, not inrequently in the setting o MSAFP elevation. Te etal trisomy 21 risk is increased
approximately sixold. Echogenic bowel is also associated with
etal cytomegalovirus inection and cystic brosis—representing inspissated meconium in the latter. Follow-up ultrasound
evaluation is suggested at 32 weeks’ gestation to evaluate etal
growth (American College o Obstetricians and Gynecologists,
2020e).
For the purpose o Down syndrome screening, the etal
emur and humerus are considered short i below the 2.5th percentile or gestational age (American College o Obstetricians
and Gynecologists, 2020e). Tird-trimester ultrasound evaluation is suggested to assess etal growth. Fetuses with trisomy
21 and short emur are not more likely to be growth restricted,
however (Herrera, 2019).
Te etal nasal bone is absent in 30 percent o second-trimester etuses with trisomy 21 and hypoplastic in more than
50 percent (Moreno-Cid, 2014). Absent nasal bone occurs in
approximately 1 in 200 euploid etuses (American College o
Obstetricians and Gynecologists, 2020e; Viora, 2005). Te
nding represents delayed ossication rather than true absence.
Te nasal bone is measured between 15 and 22 weeks’ gestation. Hypoplasia may be dened as shorter than 2.5 mm, as >2
standard deviations below the mean, or based on a ratio to the
biparietal diameter (Cicero, 2003). Te etal prole and nasal
bone are components o the detailed etal anatomic survey,
but they are not assessed as part o the standard examination
and thus not used or routine screening (American Institute o
Ultrasound in Medicine, 2019).
One or more choroid plexus cysts are seen in 1 to 2 percent o
euploid pregnancies (Fig. 16-5, p. 312). Te cyst is a benign,
TABLE 17-8. Likelihood Ratios and False-Positive Rates
for Isolated Second-Trimester Markers
Used in Trisomy 21 Screening Protocols
Sonographic Marker
Likelihood
Ratio
Prevalence in
Unaffected
Fetuses (%)
Nuchal skinfold thickening 11–17 0.5
Renal pelvis dilation 1.5–1.9 2.0–2.2
Echogenic intracardiac
focus
1.4–2.8 3.8–3.9a
Echogenic bowel 6.1–6.7 0.5–0.7
Short femur 1.2–2.7 3.7–3.9
Short humerus 5.1–7.5 0.4
Any one marker 1.9–2.0 10.0–11.3
Two markers 6.2–9.7 1.6–2.0
Three or more 80–115 0.1–0.3
aHigher in Asian individuals.
Data from Bromley, 2002; Nyberg, 2001; Smith-Bindman,
2001.342 The Fetal Patient
Section 6
2. Te condition should have a well-dened phenotype, lead to a
detrimental eect on quality o lie, cause cognitive or physical
impairment, or require surgical or medical intervention.
3. Conditions with adult onset are not recommended or
inclusion.
Tere is a long-held principle that genetic carrier screening
or a particular condition not be repeated in a subsequent pregnancy. Te number o mutations or which testing is available
has greatly increased in recent years, but the additional yield
o rescreening is usually low. Consultation with a provider
with genetic expertise should be considered beore screening
is repeated (American College o Obstetricians and Gynecologists, 2020a).
■ Cystic Fibrosis
Tis disorder is caused by a mutation in the cystic fbrosis conductance transmembrane regulator (CFR) gene, which is located
on the long arm o chromosome 7 and encodes a chloridechannel protein. Although the most common CFR mutation
associated with classic cystic brosis (CF) is the ΔF508 mutation, more than 2100 mutations have been identied (Cystic
Fibrosis Mutation Database, 2020). CF may develop rom
either homozygosity or compound heterozygosity or mutations in
the CFR gene. One mutation must be present in each copy o
the gene, but they need not be the same mutation. Tere is a
wide range o clinical disease severity. Median survival length is
42 years, but approximately 15 percent have milder disease and
can survive or decades longer. Care o the pregnant woman
with CF is discussed in Chapter 54 (p. 968).
For more than two decades, the American College o Obstetricians and Gynecologists (2020a) has recommended that all
women who are pregnant or considering pregnancy be oered
CF carrier screening. Such screening should include, at minimum, a panel o 23 panethnic gene mutations, selected because
they are present in at least 0.1 percent o patients with classic CF. Te CF-mutation carrier requency approximates 1:25
in Ashkenazi Jews and those o non-Hispanic white ethnicity,
1:60 in Arican Americans and those o Hispanic white ethnicity, and 1:95 Asian Americans. Using the 23-mutation panel,
the residual carrier risk ater a negative test result was roughly
1:380 or Ashkenazi Jews and 1:170 to 1:200 or other ethnicities (American College o Obstetricians and Gynecologists,
2020a).
More recently however, expanded panels that use gene
sequencing have been reported to improve detection o at-risk
couples by 30 percent compared with the 23-mutation panel
(Beauchamp, 2019). Te American College o Medical Genetics now recommends either a targeted approach to carrier
screening, in which the standard mutation panel may be used,
or a more comprehensive approach in which specic variant
classications may be reported (Deignan, 2020).
CF is included in all newborn screening panels but is not a
substitute or carrier screening because it only identies aected
individuals. I the patient carries a mutation, her partner should
be oered screening. I both parents are carriers, genetic counseling is indicated, and prenatal diagnostic testing should be
oered.
normal variant o no clinical consequence. I associated with
structural abnormalities however, the trisomy 18 risk is
increased (Reddy, 2014).
CARRIER SCREENING FOR GENETIC
DISORDERS
Te goal o genetic carrier screening is to provide individuals with meaningul inormation to guide pregnancy planning
according to their personal values (American College o Obstetricians and Gynecologists, 2020b). Tere are three approaches,
each o which is an acceptable strategy: ethnicity-based carrier
screening, panethnic screening (perormed regardless o ethnicity), and expanded carrier screening—which is a type o panethnic screening perormed or a large number o conditions. All
carrier screening is optional and should be an inormed choice.
Carrier screening is ideally perormed prior to conception
so that both partners can have the opportunity to consider the
results and pursue options such as preimplantation genetic testing (p. 348) or donor gametes. I the screen detects a genetic
condition, partner screening should be oered. No screening
test will identiy all carriers o a condition. Genetic counseling
should include the residual risk or the condition i the partner’s screen is negative, as well as prenatal diagnosis options
(American College o Obstetricians and Gynecologists, 2020b).
Prenatal diagnosis is increasingly available i disease-causing
mutation or mutations are known. Te Genetic esting Registry o the National Institutes o Health contains detailed inormation regarding more than 18,000 genetic conditions and
78,000 genetic tests (www.ncbi.nlm.nih.gov/gtr/). In the setting o consanguinity, additional genetic counseling is indicated.
Ethnicity-based carrier screening is oered or selected autosomal recessive conditions that are ound in greater requency in
specic racial or ethnic groups. Te ounder eect occurs when
an otherwise rare gene is ound with greater requency in a
certain population and can be traced back to a single amily
member or small group o ancestors. It may develop when generations o individuals procreate only within their own groups
because o religious or ethnic prohibitions or geographical isolation. Because assignment o a single ethnicity is oten difcult, panethnic screening is increasingly preerred.
Expanded carrier screening has the advantage o screening or many conditions simultaneously, ranging rom a ew
dozen to more than 1000 (Chokshvilli, 2018). An unortunate
result is that much o the population—more than 50 percent
o those screened–may be identied to be carriers or at least 1
condition. Some conditions are so rare that inormation about
detection and residual risk are limited. Counseling regarding
phenotypic expression o dierent mutations and determination o whether variants have clinical signicance has become
increasingly complex. Tus, expanded carrier screening can
cause anxiety or amilies and may pose challenges to alreadystrained genetic counseling resources. Te American College
o Obstetricians and Gynecologists (2020b) has the ollowing
guidelines or conditions included in expanded panels:
1. Te carrier requency should be at least 1:100, which corresponds to a population prevalence o 1:40,000.Prenatal Diagnosis 343
CHAPTER 17
or β-thalassemia. Hemoglobin S is also more common among
individuals o Mediterranean, Middle Eastern, and Asian
Indian descent (Davies, 2000). I a couple is at risk to have
a child with a sickle hemoglobinopathy, genetic counseling
should be oered. Prenatal diagnosis can be perormed with
either chorionic villus sampling or amniocentesis.
Thalassemias
Tese are the most common single-gene disorders worldwide.
Talassemias are characterized as alpha (α) or beta (β) depending on whether α- or β-hemoglobin chains are aected. In general, α-thalassemia is more likely to be caused by deletions o
α-globin chains, whereas β-thalassemia more oten stems rom
mutations in β-globin chains.
In α-thalassemia, the number o α-globin genes that are
deleted may range rom one to all our. I two α-globin genes
are deleted, both may be deleted rom the same chromosome—
cis conguration (αα/--), or one may be deleted rom each
chromosome—trans conguration (α–/α–). Te cis conguration is more prevalent among Southeast Asians, whereas those
o Arican descent are more likely to inherit the trans conguration. Alpha-thalassemia trait results in mild anemia. However,
i the patient and her partner both carry cis deletions, ospring
are at risk or an absence o α-hemoglobin, called Hb Barts
disease. Tis can lead to hydrops and etal loss (Chap. 18, p.
360). Hemoglobin electrophoresis cannot detect α-thalassemia
or α-thalassemia trait. Molecular genetic testing should be
considered i there is microcytic anemia in the absence o iron
deciency and i the hemoglobin electrophoresis is normal,
particularly among individuals o Southeast Asian descent
(American College o Obstetricians and Gynecologists, 2019a).
In β-thalassemia, β-globin genes may cause reduced or
absent production o β-globin chains. I the mutation aects
one gene, it results in β-thalassemia minor. I both copies
are aected, the result is either β-thalassemia major—termed
Cooley anemia—or β-thalassemia intermedia. Hemoglobin
electrophoresis demonstrates elevated levels o hemoglobins
that do not contain β-chains, which are hemoglobins F and A2.
β-Talassemia minor should be considered i an individual o
one or more o the aorementioned ethnicities is ound to have
microcytic anemia in the absence o iron deciency. A hemoglobin A2 level exceeding 3.5 percent conrms the diagnosis.
■ Recessive Diseases in Ashkenazi
Jewish Individuals
Among Jewish individuals o Eastern and Central European
(Ashkenazi) descent, there are three severe diseases or which
the carrier requency and detection rate are high enough that
screening should be oered: ay-Sachs disease, Canavan disease,
and amilial dysautonomia (American College o Obstetricians
and Gynecologists 2020a). Te carrier rate approximates 1 in
30 or ay-Sachs disease, 1 in 40 or Canavan disease, and 1
in 32 or amilial dysautonomia. For each, the detection rate is
at least 98 percent. As or other genetic conditions, screening is
ideally perormed prior to conception or during early pregnancy.
Tere are several other autosomal recessive conditions
or which the College also recommends that screening be
■ Spinal Muscular Atrophy
Tis autosomal recessive disorder results in spinal cord motor
neuron degeneration that leads to skeletal muscle atrophy and
generalized weakness. Tere is currently no eective treatment.
Te prevalence o spinal muscular atrophy (SMA) is 1 in 6000 to
10,000 live births. ypes I, II, III, and IV are caused by mutations
in the survival motor neuron (SMN1) gene, which is located on
the long arm o chromosome 5 (5q13.2) and encodes the SMN
protein. ypes I and II account or 80 percent o cases and are
both lethal (American College o Obstetricians and Gynecologists, 2020a). SMA type I, known as Werdnig-Homann disease,
is the most severe. Disease onset is within the rst 6 months, and
aected children die o respiratory ailure by age 2 years. ype
II generally has onset beore age 2 years, and the age at death
can range rom 2 years to the third decade o lie. ype III also
presents beore age 2 years, with disease severity that is milder and
more variable. ype IV does not present until adulthood.
Te American College o Obstetricians and Gynecologists
(2020a) recommends that carrier screening or SMA be oered
to all women who are considering pregnancy or are currently
pregnant. Prior to screening, the potential spectrum o severity, carrier requency, and detection rate should be provided.
Posttest counseling should include the residual risk ater a
negative result, which diers according to ethnicity and also
according to the number o SMN1 copies detected. Te SMA
carrier requency approximates 1:35 in those o non-Hispanic
white ethnicity, 1:41 in Ashkenazi Jews, 1:53 in Asians, 1:66 in
Arican Americans, and 1:117 in those o Hispanic white ethnicity (Hendrickson, 2009). Carrier detection rates range rom
90 to 95 percent or each ethnicity except Arican Americans,
in whom it approximates 70 percent (MacDonald, 2014).
Although there is usually one copy o the SMN1 gene on
each chromosome, approximately 3 to 4 percent o carriers have no copies o this gene on one chromosome and two
copies on the other. Because such individuals have two copies in total, they are not detected i a quantitative polymerase
chain reaction test is used. Carrier screening is less sensitive in
Arican American individuals, because they are more likely to
have this genetic variation. Additionally, some individuals have
three copies o the gene and are at even lower risk. I the patient
or her partner has a amily history o SMA, or i carrier screening is positive, genetic counseling is indicated.
■ Hemoglobinopathies
Tese include the sickle hemoglobinopathies—sickle cell anemia and sickle cell hemoglobin C disease, thalassemias, and
sickle cell–β-thalassemia. Teir pathophysiology and management are discussed in Chapter 59 (p. 1053). Te risk is
increased in women who are o Arican or Arican-American,
Mediterranean, Middle Eastern, or Southeast Asian ethnicity. In such individuals, hemoglobin electrophoresis should be
oered prenatally or prior to conception (American College o
Obstetricians and Gynecologists, 2019a).
Sickle Hemoglobinopathies
Approximately 1 in 12 Arican Americans has sickle-cell trait,
1 in 40 carries hemoglobin C, and 1 in 40 carries the trait344 The Fetal Patient
Section 6
considered. Tese include Bloom syndrome, amilial 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 diers rom the
other conditions listed because it has a wide range in phenotype
and because treatment is available in the orm o enzyme repletion and substrate reduction therapy.
Tay-Sachs Disease
Tis lysosomal-storage disease causes progressive neurodegeneration and death in early childhood. It is characterized by
deciency o the enzyme hexosaminidase A, which results in
accumulation o GM2 gangliosides in the central nervous system. Aected individuals have almost complete absence o the
enzyme, whereas carriers are asymptomatic but have less than
55-percent hexosaminidase A activity. An international aySachs carrier screening campaign was initiated in the 1970s
and met with unprecedented success in the Ashkenazi Jewish
population. Te incidence o ay-Sachs disease subsequently
declined more than 90 percent (Kaback, 1993). Other groups
at increased risk or ay-Sachs disease include those o FrenchCanadian and Cajun descent.
Carrier screening or ay-Sachs disease is perormed with
DNA-based mutation analysis or hexosaminidase activity testing. DNA-based mutation analysis is the preerred test in Ashkenazi Jewish individuals and other high-risk groups but has
a lower detection rate in the general population. Tereore,
hexosaminidase activity testing is recommended or screening
in individuals rom lower-risk ethnicities. I a woman is pregnant or taking oral contraceptives, a hexosaminidase activity
test should be perormed on leukocytes to avoid a high alsepositive rate (American College o Obstetricians and Gynecologists, 2020a). I both partners are ound to be carriers, prenatal
diagnostic testing should be oered. Hexosaminidase activity
may be measured rom chorionic villi or amnionic uid.
PRENATAL AND PREIMPLANTATION
DIAGNOSTIC TESTS
Diagnostic procedures used in prenatal diagnosis include
amniocentesis and chorionic villus sampling (CVS). Fetal
blood sampling is rarely perormed solely or genetic diagnosis
but may be used in conjunction with intrauterine transusion.
Preimplantation genetic tests or aneuploidies, structural chromosomal rearrangements, and monogenic disorders are available or couples undergoing in vitro ertilization (IVF).
In the setting o a etal structural abnormality, chromosomal
microarray analysis (CMA) is recommended. CMA is preerred
to cytogenetic analysis (karyotyping) because it can detect clinically signicant chromosomal abnormalities in approximately
6.5 percent o etuses with normal karyotype (Callaway, 2013;
de Wit, 2014, Wapner, 2012). And, in the absence o a etal
structural abnormality, CMA has detected additional chromosomal abnormalities (pathogenic copy number variants) in up
to 1 percent o those with normal karyotype. CMA is thereore
made available whenever a prenatal diagnostic procedure is per-
ormed (American College o Obstetricians and Gynecologists,
2018; Callaway, 2013). ypes o CMA platorms and their
benets and limitations are reviewed in Chapter 16 (p. 326).
Karyotyping identies aneuploidy and polyploidy. Additionally, it can identiy balanced chromosomal rearrangements that
are currently undetectable with CMA. For example, karyotyping
perormed when a couple has experienced recurrent pregnancy
loss may reveal that one parent carries a balanced robertsonian
translocation (Chap. 16, p. 315). An additional indication is
the etus with a structural abnormality that strongly suggests
a particular aneuploidy—such as endocardial cushion deect in
trisomy 21 or holoprosencephaly in trisomy 13.
I rapid identication o a specic chromosomal abnormality, such as trisomy 21, 18, or 13, is needed, uorescence in
situ hybridization (FISH) may be used in conjunction with
CMA or karyotyping. Decision-making based on FISH should
incorporate clinical inormation consistent with the suspected
diagnosis, such as a screening result or ultrasound nding
(American College o Obstetricians and Gynecologists, 2018).
Because o improvements in aneuploidy screening tests, there
has been a dramatic drop in the number o prenatal diagnostic
procedures. Larion and coworkers (2014) reported a 70-percent
decline in CVS and a nearly 50-percent drop in amniocentesis procedures since the introduction o cDNA screening in
2012. Tis urther amplied the decrease in amniocentesis procedures that occurred with the advent o rst-trimester screening (Warso, 2015). Unortunately, ewer than 40 percent o
women with a positive screening result elect prenatal diagnosis
(Dar, 2014). I prenatal diagnosis is elected, however, the
percent with an abnormal result has increased signicantly
(Awomolo, 2018).
■ Amniocentesis
Because o its established saety and efcacy, amniocentesis is
oered to all pregnant women (American College o Obstetricians and Gynecologists, 2020e). Amnionic uid is withdrawn
transabdominally under direct ultrasound guidance. Although
typically perormed between 15 and 20 weeks’ gestation,
amniocentesis may be done at any point later in gestation. ests
include those or genetic conditions, congenital inections, and
alloimmunization. Fetal lung maturity is no longer routinely
assessed, based on recommendations against using it to assist
with delivery timing (American College o Obstetricians and
Gynecologists, 2019b).
Technique
Amniocentesis is perormed with a 20- or 22-gauge needle
using aseptic technique (Fig. 17-4). A standard spinal needle is
9 cm long. A longer needle may be required or obese patients.
o aid in needle selection, we nd it helpul to measure the
distance rom skin to amnionic uid with ultrasound prior to
the procedure. Ater the patient empties her bladder, the skin is
prepared with an antiseptic such as povidone-iodine. Shellsh
allergy is not a contraindication to the povidone-iodine antiseptic (Westermann-Clark, 2015).
All aspects o the procedure are perormed under direct
ultrasound guidance. A pocket o amnionic uid close to the
midline is selected, avoiding etal parts and being cognizantPrenatal Diagnosis 345
CHAPTER 17
o uterine size and shape. Te needle is inserted perpendicular
to the skin, gently puncturing the chorioamnion rather than
pushing or “tenting” it away rom the uterine wall. Te amnion
usually uses with the underlying chorion by 16 weeks’ gestation, and the procedure is generally deerred until ater chorioamnion usion. Discomort rom the procedure is considered
minor and is related more to ocal myometrial contraction than
needle discomort at the skin. Local anesthetic has not been
ound to be benecial (Mujezinovic, 2011).
Te amnionic uid should be clear and colorless or pale
yellow. Blood-tinged uid is more requent with transplacental
passage o the needle and generally clears with continued aspiration. Needle passage through the placenta occurs in approximately 60 percent o cases with anterior placentation and is
not associated with pregnancy loss (Bombard, 1995, Marthin,
1997). Dark brown or greenish uid may represent a past episode o intraamnionic bleeding.
Te volume o uid generally needed or commonly per-
ormed analyses is shown in Table 17-9. Because the initial 1 to
2 mL o uid aspirate may be contaminated with maternal cells,
it is oten discarded. Approximately 20 to 30 mL o uid is then
collected or either etal CMA or karyotyping beore the needle
is withdrawn. Te uterine puncture site is observed or bleeding,
and etal cardiac motion is documented ollowing the procedure.
I the patient is Rh D-negative and unsensitized, anti-D immune
globulin is administered ater the procedure (Chap. 18, p. 356).
Multifetal Pregnancy. When perorming the procedure in a
dichorionic or monochorionic diamnionic twin gestation, care-
ul attention is paid to the location o each sac and the dividing
membrane. Until recently, a small quantity o dilute indigo carmine dye was oten injected beore removing the needle rom
the rst sac, with return o clear amnionic uid anticipated ollowing needle placement into the second sac. Because o widespread shortages o indigo carmine dye, experienced providers
typically oer amniocentesis without dye injection. Methylene
blue dye is contraindicated because it has been associated with
jejunal atresia and neonatal methemoglobinemia (van der Pol,
1992).
Complications
Midtrimester losses attributable to amniocentesis have decreased
with improvements in imaging technology. When perormed
by an experienced provider, the procedure-related loss rate is
estimated to be 0.1 to 0.3 percent, or about 1 per 500 procedures (American College o Obstetricians and Gynecologists,
2018; Odibo, 2008, Salomon, 2019). Te loss rate may be
doubled in women whose body mass index exceeds 40 kg/m2
(Harper, 2012). In twin pregnancies, Cahill and coworkers
(2009) reported a loss rate o 1.8 percent attributable to amniocentesis.
A B
FIGURE 17-4 A. Amniocentesis. B. The amniocentesis needle
is seen in the upper right portion of this sonogram. (Figures 17-4,
17-5, and 17-7 are reproduced with permission from Mastrobattista
JM, Espinoza J: Invasive prenatal diagnostic procedures. In Yeomans
ER, Hoffman BL, Gilstrap LC III, et al (eds): Cunningham and Gilstrap’s
Operative Obstetrics, 3rd ed. New York, NY: McGraw Hill; 2017.)
TABLE 17-9. Selected Tests Performed on Amnionic
Fluid and Volume of Fluid Required
Test Volume (mL)a
Chromosomal microarray analysis 20
Fetal karyotype 20
FISHb 10
Genotype studies (alloimmunization) 20
PCR tests for cytomegalovirus,
toxoplasmosis, or parvovirus
1–2 each test
Cytomegalovirus culture 2–3
Tests no longer commonly used:
Fetal lung maturity tests 10
Delta OD 450 (bilirubin analysis) 2–3
Alpha-fetoprotein 2
aThe volume of fluid needed for each test may vary according to individual laboratory specifications.
bFluorescence in situ hybridization (FISH) is typically performed for chromosomes 21, 18, 13, X, and Y.
PCR = polymerase chain reaction.346 The Fetal Patient
Section 6
When counseling regarding etal loss ollowing a procedure, it can be helpul to explain the dierence between overall losses and procedure-related losses. In a recent metaanalysis
that included more than 60,000 amniocentesis procedures and
300,000 control pregnancies, the loss rate in the absence o
a procedure was 0.6 percent, approximately twice the rate o
losses attributable to procedures (Salomon, 2019). Te indication or the procedure is also relevant, because severe etal
abnormalities and hydrops can signicantly increase the loss
rate, regardless o a procedure.
Other complications o amniocentesis include amnionic
uid leakage or transient vaginal spotting in 1 to 2 percent.
Leakage o amnionic uid generally occurs within 48 hours o
the procedure. Leakage o uid typically resolves ater a ew
days, and etal survival exceeds 90 percent (Borgida, 2000).
Management recommendations include pelvic rest and avoidance o strenuous activity. Needle injuries to the etus are rare.
I karyotyping is perormed, amnionic uid culture is successul
in more than 99 percent o cases, although cells are less likely to
grow i the etus is abnormal (Persutte, 1995).
Early Amniocentesis
Amniocentesis was ormerly oered between 11 and 14 weeks’
gestation. Te technique is the same as or traditional amniocentesis, but the procedure may be more challenging because
o lack o membrane usion to the uterine wall. Approximately
1 mL is withdrawn or each week o gestation. Early amniocentesis is associated with signicantly higher rates o etal loss
and other procedure-related complications than other procedures. Complications include development o talipes equinovarus (cluboot) and amnionic uid leakage (Canadian Early
and Mid-rimester Amniocentesis rial, 1998; Philip, 2004).
For these reasons, early amniocentesis is not recommended
(American College o Obstetricians and Gynecologists, 2018).
■ Chorionic Villus Sampling
Biopsy o chorionic villi is typically perormed between 10
and 13 weeks’ gestation. As with amniocentesis, the specimen
is usually sent or CMA or karyotype analysis. Te primary
advantage o CVS is that results are available earlier in pregnancy, permitting more time or decision-making and saer
pregnancy termination, i desired.
Technique
CVS is perormed transcervically or transabdominally. Both
approaches are equally sae and eective (American College
o Obstetricians and Gynecologists, 2018). Te transcervical
approach uses a exible polyethylene catheter that contains
a blunt-tipped, malleable stylet. ransabdominal sampling is
perormed with an 18- or 20-gauge spinal needle. Both use
aseptic technique and are perormed under direct transabdominal ultrasound guidance. Te catheter or needle is inserted into
the early placenta—chorion rondosum, and villi are aspirated
into a syringe that contains tissue culture media (Fig. 17-5).
Relative contraindications include vaginal bleeding or spotting, active genital tract inection, extreme uterine ante- or
retroexion, or body habitus precluding adequate visualization. I the patient is Rh D-negative and unsensitized, anti-D
immune globulin is administered ater the procedure.
Complications
Te overall etal loss rate ollowing CVS is higher than that with
midtrimester amniocentesis. Tis is because o losses that would
have occurred between the rst and second trimester in the absence
o a procedure. Te procedure-related loss rate is comparable to
that with amniocentesis, approximately 0.1 to 0.3 percent (American College o Obstetricians and Gynecologists, 2018). In act, a
recent metaanalysis identied no signicant losses rom CVS when
controls with similar risk proles were selected (Salomon, 2019).
As with amniocentesis, CVS indication will aect the loss rate. For
example, etuses with increased N thickness have a greater likelihood o demise. Tere is also a learning curve associated with sae
perormance o CVS (Silver, 1990; Wijnberger, 2003).
An early problem with CVS was its association with limbreduction deects and oromandibular limb hypogenesis, shown
in Figure 17-6 (Firth, 1991, 1994; Hsieh, 1995). Tese were
A B
FIGURE 17-5 A. Transcervical chorionic villus sampling. B. Catheter entering the placenta is marked and labeled.Prenatal Diagnosis 347
CHAPTER 17
subsequently ound to be associated with procedures perormed
at 7 weeks’ gestation (Holmes, 1993). When perormed at or
ater 10 weeks’ gestation, the incidence o limb deects does not
exceed the background population rate o approximately 1 case
per 1000 (Evans, 2005; Kuliev, 1996).
Vaginal spotting is not uncommon ollowing transcervical
sampling, but it is sel-limited and not associated with pregnancy loss. Te incidence o inection or leakage o amnionic
uid is less than 0.5 percent (American College o Obstetricians and Gynecologists, 2018).
A limitation o CVS is that chromosomal mosaicism is identied in up to 2 percent o specimens (Malvestiti, 2015). In
most cases, the mosaicism reects con-
ned placental mosaicism rather than
a true second cell line within the etus.
Tis is discussed in Chapter 16 (p. 318).
Amniocentesis should be oered, and
i the result is normal, the mosaicism is
usually presumed to be conned to the
placenta. Conned placental mosaicism
is associated with etal-growth restriction
(Baero, 2012; outain, 2018).
■ Fetal Blood Sampling
Tis procedure is also called cordocentesis
or percutaneous umbilical blood sampling
(Fig. 17-7). It was initially described or
etal transusion o red blood cells in the
setting o anemia rom alloimmunization,
and etal anemia assessment remains the
most common indication (Chap. 18, p.
356). Other indications include assessment and treatment o platelet alloimmunization and or etal
karyotype determination, particularly in cases o mosaicism
identied ollowing amniocentesis or CVS. Improvements in
testing using an amniocentesis specimen have eliminated the
need or etal blood sampling in most cases (Society or Maternal-Fetal Medicine, 2013).
Technique
Using aseptic technique, a 22- or 23-gauge spinal needle is
introduced into the umbilical vein under direct ultrasound
guidance, and blood is slowly withdrawn into a heparinized
syringe. Precise visualization o the needle is essential. As with
A B
FIGURE 17-6 Oromandibular limb hypogenesis is characterized by transverse limb
deficiency and absence or hypoplasia of the tongue or mandible. This is hypothesized to
result from vascular disruption with subsequent loss of tissue. A. Sonogram obtained at
25 weeks’ gestation demonstrates a fetal limb reduction defect involving the right hand.
B. Photograph of the right extremity of the same newborn. Chorionic villus sampling was
not performed in this pregnancy. (Reproduced with permission from Dr. Jamie Morgan.)
Uterine
wall
Placenta
Umbilical
cord
Ultrasound
transducer
A
B
FIGURE 17-7 Fetal blood sampling. A. 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. B. Sonogram shows an anterior placenta
with transplacental needle passage into the umbilical vein (UV).348 The Fetal Patient
Section 6
Te American Society or Reproductive Medicine (2018) has
concluded that the utility o routine perormance o PG-A or
all IVF pregnancies has yet to be determined.
PG-M may be perormed when there is increased risk or a
particular single-gene disorder or a hereditary cancer syndrome
(American College o Obstetricians and Gynecologists, 2020d).
It is a test or a specic condition rather than a panel. In selected
cases, this technique may also be used identiy a suitable uture
donor o umbilical cord blood or an aected amily member.
PG-SR is oered i one o the parents is a carrier o such
a rearrangement, such as a duplication, deletion, translocation,
insertion, or inversion. Te test will identiy an unbalanced
chromosomal rearrangement but will not detect a balanced carrier. Counseling should thereore include this limitation
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