Practical Genetic Counseling For The Laboratory - (7. Prenatal Screening Technologies and Test Issues)

7
Prenatal Screening Technologies

and Test Issues
DANIELLE L AGRAVE, PATRICIA L. DEVERS WINTERS, AND GERALYN
L A M B E R T -M E S S E R L I A N
Prenatal screening began in 1977 (Wald et al. 1977) with the measurement of mater-
nal serum alpha fetoprotein (AFP) in the second trimester to evaluate the risk of open
neural tube defects (ONTDs). An observation followed soon thereafter that maternal
serum AFP levels were low in pregnancies affected with fetal trisomy. This led to the
implementation of routine, serum-based prenatal screening for Down syndrome (tri-
somy 21 [T21]). Markers in addition to AFP emerged rapidly, and today the second-
trimester Quad test, which includes unconjugated estriol (uE3), human chorionic
gonadotropin (hCG), and inhibin A (inhA), is the most common Down syndrome
screening test performed worldwide.
Improvements in serum marker screening for Down syndrome included first-
trimester combined and Integrated tests. In first-trimester combined screening (com-
monly referred to as “first-trimester screening”), results are available early in pregnancy
(11–13 completed weeks). The markers used are pregnancy-associated plasma protein
A (PAPP-A), hCG, and an ultrasound measurement taken at the back of the baby’s neck,
Copyright © 2017. Oxford University Press, Incorporated. All rights reserved.
called nuchal translucency. The Integrated test uses both first-and second-trimester
markers to give a patient risk result. The results of the Integrated test are not available
until the second trimester of pregnancy; however, the test performance is enhanced,
providing up to 95% detection of trisomy 21 with a 5% false-positive rate as opposed
to an 85% detection rate and 5% false-positive rate associated with the Quad screen or
Combined test (Wald et al. 2003; Malone et al. 2005). The Combined and Integrated
tests are less widely used than the Quad, in part due to the specialized sonographer
training required for nuchal translucency measurement.
A new technology has recently emerged to screen pregnancies for Down syndrome
and other common fetal aneuploidies, using cell-free DNA in maternal plasma. This
prenatal screening test can detect >98% of pregnancies affected with Down syndrome
with a false-positive rate below 1% (Palomaki et al. 2011). The optimal implementa-
tion of cell-free DNA screening is currently debated among professional societies.
The option of prenatal screening is recommended for all pregnant women
(American College of Obstetricians and Gynecologists 2016). This chapter will
177
Practical Genetic Counseling for the Laboratory, edited by McKinsey L. Goodenberger, et al., Oxford University Press, Incorporated,
2017. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/cityuhk/detail.action?docID=4828920.
Created from cityuhk on 2023-08-10 03:43:14.
178 P ractical Genetic Counseling for the L aboratory
Box 7.1 Common Screening Terminology

sensitivity or detection rate: the percentage of affected pregnancies correctly given
a screen-positive test result
specificity: the percentage of unaffected pregnancies correctly given a screen-
negative test result
false positive rate: the percentage of unaffected pregnancies incorrectly given a
screen-positive result
positive predictive value: the number of affected pregnancies with a screen-positive
result divided by the number of all (affected and unaffected) pregnancies with a
screen-positive result
describe the available screening tests for aneuploidy and their performance charac-
teristics, strengths, and limitations. Each pregnant woman is expected to make an
informed choice about screening based on her age, pregnancy history, culture, and
personal preferences (Hill et al. 2016).
Box 7.1 lists common screening terminology.
Maternal Serum Screening

Traditional serum screening strives to identify pregnancies at high risk for any of sev-
eral disorders: ONTDs, T21, and trisomy 18 (T18).
MARKERS USED IN SCREENING

Prenatal serum screening is accomplished by measuring various markers in the first
and/or second trimesters. PAPP-A and hCG are the most common first-trimester
markers, while AFP, uE3, hCG, and inhA are the second-trimester markers. These
marker measurements can be determined using several available automated immu-
noassay systems.
AFP
AFP is a protein initially made by the yolk sac in pregnancy and then by the fetal liver
as pregnancy progresses and the yolk sac degenerates. AFP is excreted in amniotic
fluid through fetal urine, at levels about one-hundredth (~10,000 ng/mL) of that mea-
sured in fetal serum (~1,000,000 ng/mL) in the second trimester. From the amniotic
fluid, AFP diffuses across the placenta into maternal serum. Maternal serum levels
are first detected at about 10 weeks of gestation, at concentrations that are much
lower (~50 ng/mL) than those in amniotic fluid. The biologic role of AFP is unknown,
as some pregnancies have occurred without measurable levels in serum and without
adverse consequences (Greenberg et al. 1992).
Pren a ta l Scre e n in g Te ch n o l o gie s 179
uE3
The synthesis of uE3 also relies on the fetus. The steroidogenic pathway converts
cholesterol to dehydroepiandrosterone sulphate (DHEAS) in the fetal adrenal gland.
DHEAS circulates to the fetal liver, where a unique enzyme, 16α-hydroxylase, converts
it to a hydroxylated form. In the placenta, further enzyme processing results in the
production of estriol that is secreted into maternal serum. About 10% of estriol circu-
lates in the unconjugated form, while the majority of estriol is conjugated by sulfation
or glucuronidation in the maternal liver for renal clearance (Penney and Klenke 1980).
In addition to its functions as an estrogen in pregnancy, levels of estriol in maternal
serum can indicate errors in the fetal and placental pathway required for its synthesis.
hCG
hCG is glycoprotein, synthesized by trophoblast cells in the placenta. The alpha and
beta subunit proteins of hCG dimerize for biologic activity. Nonetheless, there are
various dynamic forms of hCG during the course of pregnancy, including free alpha
and beta subunits of various sizes. In the early stages of pregnancy, a large-molecular-
weight hCG protein that is heavily glycosylated (hyperglycosylated hCG) predomi-
nates in serum (reviewed in Fournier et al. 2015). The initial, and critical, function of
hCG is to sustain the corpus luteum and its secretion of progesterone, which is neces-
sary to support early pregnancy. At about 8 to 10 weeks of gestation, the placenta has
grown sufficiently and provides enough progesterone to sustain the pregnancy. Levels
of hCG decline in pregnancy with this transition.
inhA
InhA is a dimeric glycoprotein synthesized, like hCG, in the placenta. The inhibin alpha
subunit is bound to one of several beta subunit gene products (βA subunit) in this
family to make the specific bioactive dimeric inhibin A protein. Inhibin A is initially
produced by the corpus luteum, and then by the placenta in a developing pregnancy.
One of its roles in pregnancy is the suppression of pituitary hormones to prevent fol-
licle recruitment and development.
PAPP-A
The placenta is the source of PAPP-A in pregnancy. This very large protein circulates in
maternal serum as a heterotetramer, comprising two subunits of PAPP-A covalently
linked to two subunits of major basic protein (pro MBP) (Boldt and Conover 2007).
The pro MBP subunits are inactivating. A dimer of two PAPP-A proteins provides
biologic activity, cleaving the insulin-like growth factor binding protein. In this way,
PAPP-A facilitates the activity of the insulin-like growth factors and promotes fetal
growth in pregnancy.
PAT H O P H Y S I O L O G Y O F A LT E R E D S E R U M M A R K E R
LEVELS
There is no known biologic explanation for altered marker levels in aneuploidies, such
as Down syndrome. None of the genes for these markers are expressed on chromosome
21, eliminating a simple dosage effect as an explanation. One prevailing hypothesis for
the altered maternal serum marker levels observed in Down syndrome is that there
is fetal hyposecretion and placental hypersecretion (Newby et al. 1997) in the second
trimester of affected pregnancies. This hypothesis is consistent with the observation
of reduced levels of markers originating from the fetus (AFP and uE3) and elevated
levels of placental markers (hCG and inhA) in second trimester maternal serum.
G E S TAT I O N A L A G E E F F E C T S O N M A R K E R L E V E L S I N
PREGNANCY
All maternal serum maker levels are affected by gestational age. In the second trimes-
ter (15–22 weeks), levels of AFP increase by 15% per week. Unconjugated estriol levels
also rise in the second trimester, at a rate of 20% to 25% per week. In contrast, mater-
nal serum levels of hCG decline during the second trimester. InhA levels are minimally
affected by gestational age, with a slight decline at 17 weeks. In the first trimester of
pregnancy (10–13 weeks), levels of hCG fall rapidly, while maternal serum PAPP-A
levels increase dramatically, on average 50% per week.
Since maternal serum marker levels depend on gestational age, correct dating
must be provided at the time of screening. Errors in estimation of gestational age
can contribute to false-positive or -negative results. When an ultrasound estimate
of gestational age is found to differ by seven or more days from an initial estimate of
gestational age based on the last menstrual period, revision of the screening report
should be considered.
M U LT I P L E O F T H E M E D I A N
The effects of gestational age on maternal serum marker levels can be accounted for in
prenatal screening by the use of the normalizing unit called the multiple of the median
(MoM). This unit is calculated by first establishing normative median data for each
serum marker in the pregnant population. Each laboratory must establish its own
median data because of differences in values based on test method, testing laboratory,
and patient population. The optimal process by which to establish median data is for
the testing laboratory to run 300 to 500 patient serum samples from its own popula-
tion of pregnant women, selected in the appropriate screening window, either 10 to
13 or 15 to 22 weeks of gestation. The resulting marker levels are then analyzed with
respect to gestational age by weighted regression analysis to establish the median
equation for each marker.
Once reliable and laboratory-specific median data are in place, patient results for
each marker are evaluated with respect to the appropriate median. An individual
patient result is divided by the median value expected for that marker at her particular
gestational age (week or day). For example, if a woman has an AFP value of 60 ng/mL
at 17 weeks of gestation, this result would be divided by the laboratory median of 40
ng/mL, giving her an AFP MoM of 1.5. The marker level, adjusted for gestational age
and expressed as an MoM value, is then used for interpretation of the patient-specific
risk for Down syndrome.
Pre na ta l Scre e n in g Te ch n o l o gie s 181
ADJUSTMENT OF MARKER LEVELS

Some demographic variables are known to influence marker levels (Wald et al. 1997;
Lambert-Messerlian et al. 2009). Adjustment of serum markers for these variables
results in improved screening performance.
Maternal Weight
Maternal weight is inversely correlated with serum marker levels. This effect is
observed for all markers because the larger blood volume of heavier women provides
for greater dilution. The relationship between body weight and serum marker levels
can be characterized by a median equation, as done for gestational age. Serum marker
levels are measured in women of varying maternal weights and regression analysis is
performed. The gestational age–specific MoM value for each marker can be further
corrected in each woman using the appropriate median for her weight. As might be
expected, weight adjustment is particularly important in very light or heavy women.
Racial/Ethnic Group
For unknown biologic reasons, some serum marker levels are altered in women from
certain racial or ethnic groups. The most widely published data are from African
American as compared to Caucasian women. Levels of AFP and hCG tend to be higher
in African American than Caucasian women, by about 10% to 15%, and inhA levels
are lower by about 8%. First-trimester levels of PAPP-A (~30–50% higher) and hCG
(~10–20% higher) are also relatively high in African American women (Spencer et al.
2005). Other effects of race on marker levels have been described, but routine adjust-
ment is not common in the United States.
Adjustment for race can again be accomplished by generating race-specific median
data for both gestational age and maternal weight. In the absence of a large racially
diverse pregnancy population, the gestation-and weight-corrected MoM value can
be adjusted by a correction factor. For example, an AFP MoM of 1.8 in an African
American woman that was derived from a Caucasian median equation would be
adjusted by a correction factor of 1.1 to 1.6 (1.8/1.1) MoM.
Diabetes
In women with insulin-dependent diabetes, maternal serum levels of AFP (20% lower)
and uE3 (5% lower) are reduced in comparison to pregnant women without diabe-
tes. Adjustment of these marker levels by an appropriate correction factor is com-
monly performed by screening laboratories. However, controversy exists regarding
whether or not adjustment for diabetes is needed when weight adjustment has been
performed, whether marker levels are affected in women with non–insulin-dependent
diabetes, and if the extent of glycemic control during pregnancy affects the extent of
marker alterations (Baumgarten and Robinson 1988).
Assisted Reproductive Technology

Pregnancies achieved by assisted reproductive technologies, in particular in vitro fer-
tilization (IVF), have altered marker levels as compared to those conceived spontane-
ously. This issue was brought to light by a publication (Frishman et al. 1997) showing
that the second-trimester screen-positive rate in singleton IVF pregnancies was twice
as high as that in age-matched spontaneous pregnancies. This could be explained by
increased (~10–20% higher) hCG and inhA levels and reduced (10% lower) uE3 levels
after IVF conception. Marker levels must be adjusted for IVF pregnancies to avoid a
high false-positive rate and unnecessary diagnostic procedures.
Recent studies (Lambert-Messerlian et al. 2006) have shown that pregnancies
achieved by other technologies, such as ovulation induction or intrauterine insemi-
nation, also have altered serum screening marker levels. However, these procedures
are not commonly reported to screening laboratories. Of interest, pregnancies con-
ceived by assisted reproductive technologies that involve egg donation have a unique
marker pattern, with significantly increased maternal serum AFP and inhA levels.
Since these markers have opposing effects on Down syndrome risk, correction is less
important.
First-trimester markers are also affected by IVF, but generally to a lesser degree
than the second-trimester markers (Lambert-Messerlian et al. 2006). The marker
alterations, slightly reduced PAPP-A levels (10%) and increased hCG levels (10%),
have opposite effects on risk and result in very little impact on the screen-positive
rate for Down syndrome.
Smoking
Cigarette smoking dramatically increases second-trimester levels of inhA (40%) and
reduces hCG levels (20%) relative to nonsmokers. Levels of uE3 in the second trimes-
ter are also slightly reduced (4%). Adjustment for smoking is recommended since this
pattern of markers in women who smoke would lead to an increased screen-positive
rate (Lambert-Messerlian et al. 2006).
In contrast, first-trimester screening results for Down syndrome are minimally
affected by smoking. First-trimester levels of PAPP-A (10%) and hCG (20%) are both
reduced in smokers versus nonsmokers and have opposite effects on risk. Screening
for T18 in the first trimester or Integrated test, however, is significantly affected by
smoking. The reduction in PAPP-A and hCG leads to increased T18 screen-positive
rates, and adjustment for smoking is recommended. A recent study suggests that
adjustment of markers is not needed in women who are former smokers (Lambert-
Messerlian et al. 2015).
Twin Pregnancy
The levels of serum markers in twin pregnancies are approximately double that seen
in singletons. Risk analysis is performed by dividing each MoM by the appropriate
correction factor (AFP and inhA = 2.0, uE3 = 1.7, hCG = 1.9, PAPP-A = 1.8 MoM)
and evaluating the pregnancy as if it were a singleton. Chorionicity is taken into
account for risk calculation. It is impossible to know the contribution of each fetus
to maternal serum marker levels, so screening is less effective in twin than singleton
pregnancies.
Other variables have been reported with a weak association to serum marker levels
but are not considered in routine screening. Some of these include fetal gender, parity,
and vaginal bleeding.
Pre n ata l Scre e n in g Te ch n o l o gie s 183
M E T H O D O F C A L C U L AT I N G PAT I E N T- S P E C I F I C R I S K
A patient-specific risk for Down syndrome is generated in prenatal screening using
the woman’s a priori risk in combination with her serum marker results. After conver-
sion of each marker result to an adjusted MoM value, a likelihood ratio can be deter-
mined. The likelihood ratio is obtained from published reports of the distribution
of marker MoM levels in Down syndrome and unaffected pregnancies. The a priori
risk is multiplied by the likelihood ratio for each marker MoM value to give a final
pregnancy-specific risk.
Prenatal screening results are evaluated by comparing the patient-specific risk to
a predetermined test cutoff risk. For example, if the second-trimester test cutoff is
set at a midtrimester risk of 1 in 270 and the patient-specific risk is 1 in 100, this
woman would be considered screen positive. The performance of each screening test
is determined in part by the test cutoff. The test cutoff is selected by the screening pro-
gram using information about the maternal age distribution of the population being
screened and the targeted, acceptable screen-positive rate in their community. At a
second-trimester risk cutoff of 1 in 270, the Quad test provides an 80% detection rate
for Down syndrome with 5% screening positive. If the risk cutoff is raised to above 1
in 270 (i.e., midtrimester risk of 1 in 190), both the detection rate and false-positive
rate will decrease (74% detection, 4% screen positive), and vice versa.
D I S O R D E R S D E T E C T E D B Y M AT E R N A L
SERUM SCREENING
NTDs
NTDs occur when there is a failure of closure at any point along the neural tube at
approximately 19 to 27 days after conception. Failure of the neural tube to close cor-
rectly leads to malformation of the spinal column, spinal cord, skull, and/or brain.
ONTDs expose the neural tissue, either brain or spinal cord, through a defect in the
skull or vertebrae and can lead to disability or death. ONTDs are not covered by skin,
although there may be a very thin membrane over the defect. Closed NTDs are rarer
than ONTDs and are covered by skin. For this reason, only ONTDs are likely to be
identified by biochemical screening methods.
About two-thirds of ONTDs fall under the category of a spina bifida (literally, “split
spine”). These can be further defined as a meningocele (the membranes that cover the
spinal cord protrude through a defect in the vertebrae) or a myelomeningocele (both
the membranes and part of the spinal cord protrude from a vertebral defect). Spina
bifida may lead to an Arnold-Chiari malformation, which occurs when there is a dis-
placement of part of the cerebellum through the opening at the base of the skull, and in
turn may cause hydrocephalus (an accumulation of cerebrospinal fluid in the ventricles
of the brain) in up to 80% of affected infants. Children with spina bifida have a range
of clinical findings depending on the size and location of the lesion, the presence and
degree of hydrocephaly, and the type of defect (open or closed, and if the nerves are
protruding or not). These can include difficulty with bladder and bowel control, muscle
weakness and/or paralysis of the lower extremities, and intellectual disabilities.
Anencephaly, in which the bones of the skull are missing or malformed and large
parts of the brain are missing, makes up the majority of the remaining one-third of
ONTD cases. Anencephaly is more common in females, with a female:male ratio of
2:1 (Robinson and Linden 1993, p. 127). Due to the severity of the condition, and the
absence of much of the brain tissue, very few infants born with this condition survive
more than a few days (Medical Task Force on Anencephaly 1990).
Because ONTDs are “open” to the amniotic fluid, the finding of fetal proteins that
are not normally found in amniotic fluid, or that are normally present, but in much
smaller amounts, can be used to screen for the presence of an ONTD. AFP is found
normally in fetal blood, amniotic fluid, and maternal blood and is used to screen for
the presence of ONTDs. While some AFP is found normally in amniotic fluid, when
there is an ONTD, additional AFP “leaks” into the amniotic fluid, which increases the
concentration to a level that is often significantly higher than expected. This increases
the amount of AFP that crosses the placenta into the maternal bloodstream, which
allows the laboratory to screen for the presence of an ONTD by testing for AFP levels
in maternal serum.
NTDs occur in approximately 7 in every 10,000 live births in the United States
(Williams et al. 2015), although the incidence varies by geographic location, race, fam-
ily history, and weight. NTDs are more common in the eastern half of the United
States and Canada and less common in the West. Migration studies have shown that
when people move from Britain (where the risk of NTD is high) to an area with a
lower risk, they assume the risk of their new location. This indicates that something
other than ethnicity is affecting risk (Frey and Hauser 2003). However, race may still
have some effect; anencephaly is more common in babies born to Hispanic mothers
(Canfield et al. 2014), although the reasons for this are not well understood.
There is also a genetic component to the development of NTDs. They can be syn-
dromic due to the presence of a chromosome or single gene disorder, and there is also
some genetic influence affecting the risk of having a child with a nonsyndromic NTD.
It has been noted that once a woman has a child with an NTD, her chance of having
another is approximately 4%, and if she has two affected children, the recurrence risk
rises to 11.1% (Frey and Hauser 2003). Folic acid supplementation taken before con-
ception and during the first four weeks of pregnancy can significantly reduce the rate
of NTD in a population. In the United States, it is recommended that every woman
of childbearing age take a multivitamin that includes 0.4 mg of folic acid each day.
Women who have had a previous child with an NTD are recommended to take a sup-
plement that has 4 mg of folic acid. Adherence to such supplementation has shown
as much as a 75% reduction in recurrence of NTDs (Frey and Hauser 2003). In an
effort to ensure that all women of childbearing age ingest sufficient folate, that vita-
min has been added to cereal grains in the United States and Canada since 1998 and
has reduced the overall incidence of NTDs by about 28% (Williams et al. 2015).
Down Syndrome
Down syndrome (T21) is the most common genetic cause of intellectual disability.
It is caused by the presence of an extra chromosome 21. Thus, most individuals with
T21 have 47 chromosomes instead of the usual 46. The exceptions are those who have
T21 due to a translocation between one chromosome 21 and another acrocentric
chromosome. Down syndrome is due to the presence of a Robertsonian translocation

in approximately 3% to 4% of affected individuals. The phenotype of these individuals
is not different than the majority of people with T21, but the recurrence risk for the
parents of a child with “translocation Down syndrome” to have additional affected
children is as high as 15% (Gardner et al. 2012, p. 151), much greater than that of
parents with a child who does not have an unbalanced translocation. Therefore, it
is important to identify if a child with Down syndrome has a translocation by per-
forming a chromosome analysis by karyotype on a sample from the affected child; if a
translocation is found, a chromosome analysis should be performed on the parents to
determine if one of them is a carrier for the translocation in question.
Women carrying a fetus with T21 may have normal ultrasound exams, as approxi-
mately 45% of fetuses have few to no obvious physical anomalies to be identified upon
sonographic examination (Smith-Bindman et al. 2007). However, some ultrasound
findings are relatively common in fetuses with T21 and, if observed, should raise the
concern that the fetus may be affected. These can include increased nuchal translu-
cency (first trimester), thickened nuchal fold (second trimester), short femurs, pyelec-
tasis, and the “double bubble” sign indicating the presence of duodenal atresia. Some
minor malformations (soft markers) may be visible on ultrasound as well, such as
sandal-gap toe, short hands/fingers, flat face, and large tongue.
T21, like other autosomal aneuploidies, shows an increased prevalence in older
mothers. As the median age of the pregnant population in the United States has
increased, so has the prevalence of T21 (Resta 2005). The birth prevalence is approxi-
mately 1 in 691 (Parker et al. 2010). However, the prevalence may be significantly
higher at screening depending on the gestational age, as approximately 43% of fetuses
identified with T21 in the first trimester and 23% of those identified in the second
trimester are expected to miscarry (Resta 2005).
T18
T18 is much less common than Down syndrome at birth. Like T21, T18 shows an
increased prevalence in older mothers. Due to the very high fetal loss rate (~72%
between 12 weeks gestation and birth), the birth prevalence is approximately 1 in

5,000 to 6,000 live births (Savva et al. 2010; Cereda and Carey 2012). T18 is caused by
a nondisjunction event that leads to the presence of an extra chromosome 18 in the
fetus. Infants with T18 are significantly more impaired than those with T21.
Infants with T18 are usually very small at birth, even when delivered at full term.
Common malformations include heart defects that are generally more severe than
those seen in T21, intrauterine growth retardation (IUGR), “strawberry-shaped”
skull, clenched fists with a unique pattern of overlapping fingers, and rocker-bottom
feet. Affected babies may also have spina bifida, eye problems, hearing loss, kidney
problems, cryptorchidism, and feeding problems. Most infants with T18 will not sur-
vive their first month due to the severity of their medical problems, but a small subset
of infants with T18 will survive their first year. Severe intellectual disability is present
in the few children with this disorder who survive infancy (Cereda and Carey 2012).
Ultrasound findings may include increased nuchal translucency, cardiac malforma-
tions, abnormal head shape, clenched fists and rocker-bottom or clubbed feet, and
IUGR. Spina bifida or omphalocele may also be present, and choroid plexus cyst is
seen in approximately 50% of affected fetuses (Cereda and Carey 2012). As IUGR is a
common finding (seen in 28% of second-trimester fetuses and 87% of third-trimester
fetuses [Cereda and Carey 2012]), care should be taken when contemplating recalcula-
tion of a maternal serum screening test that is positive for T18 due to dating discrep-
ancies between the last menstrual period–derived due date and a due date calculated
based on second-or third-trimester ultrasound measurements. It may be inadvisable
to redate a pregnancy at risk for T18 based on the fetus being smaller than expected,
as this may represent the observation of IUGR, a symptom of the disorder, and not
incorrect dating.
OTHER DISORDERS OF INTEREST

Trisomy 13
Trisomy 13 (T13) is a rare finding, as compared to even T18, and the birth preva-
lence is estimated to be 1 in 10,000 (Savva et al. 2010). Most affected pregnancies
with T13 are identified using multiple marker screening screen positive for either T21
or T18. Adding a T13-specific algorithm does increase detection for the Integrated
test, although there is no material effect on the detection for this disorder when
using either the Combined or Quad screen (Bestwick et al. 2013). Despite the lack of
specificity of serum screening for T13, the majority of affected pregnancies would be
identified by either the Combined or Integrated (with nuchal translucency) screens.
Second-trimester screening alone is a poor test for this condition (Bestwick et al.
2013). Ultrasound and cell-free DNA (cfDNA) testing may be better specific screening
tools for this particular aneuploidy, especially in the second trimester.
Triploidy
While screening for triploidy is not a specific goal of maternal serum screening, spe-
cific patterns of the second-trimester analytes are observed in patients with triploid
pregnancies (Table 7.1). Women carrying affected fetuses may screen positive for T21,
T18, ONTD, or more than one anomaly. While this disorder is not a primary target
Table 7.1 Second-Trimester Marker Patterns
AFP hGC uE3 DIA Associated with:

↓ ↑ ↓ ↑ Down syndrome; overestimated gestational age
↓ ↓ ↓ - Trisomy 18
↑ ↓ ↓ ↓ Open neural tube defect; maternal-fetal
hemorrhage; ventral wall defect
↑ N ↓ N Anencephaly; fetal demise
↓ ↓ ↓↓ N X-linked ichthyosis; Smith-Lemli-Opitz syndrome
↑ ↑↑ ↓/N ↑/↑↑ Diandric/paternal triploidy
N ↓ ↓/N ↓ Digynic/maternal triploidy
↓ = Low, ↑ = High, N = Normal, ↑↑ = Very high, ↓ ↓ = Very low, -= not relevant
for any maternal serum screening program, there are two distinct patterns of second-
trimester markers that can be seen in affected pregnancies, primarily distinguished
from each other by the levels of hCG and dimeric inhibin A (DIA) (Huang et al. 2005).
High, or very high, levels of hCG/DIA are associated with diandric triploidy, and low
levels of hCG/DIA are associated with digynic triploidy (see Table 7.1). Huang et al.
(2005) reported that second-trimester screening would detect 90% of triploidy cases.
Despite unique marker patterns in the second trimester, no specific test algorithm to
identify triploidy pregnancies has been published.
Triploidy is common in early pregnancy, making up 1% to 3% of recognized preg-
nancies (Gardner et al. 2012, p. 288), but greater than 99% are lost in the first and
second trimester, with digynic triploids mostly aborting early in the first trimester
and diandric triploids aborting in both the first and second trimesters (Zaragoza
et al. 2000). Identifying pregnancies at an increased risk of triploidy is important as
diandric triploidy can be associated with partial hydatidiform moles, which may have
maternal health consequences, such as development of trophoblastic hyperplasia
and, rarely, conversion to choriocarcinoma, a form of cancer (Zaragoza et al. 2000).
Second-trimester serum screening markers can suggest which cases may be at highest
risk for partial mole (Benn et al. 2001).
Smith-Lemli-Opitz-Syndrome
Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive defect leading to defi-
cient cholesterol biosynthesis. Children born with SLOS present with mental delay
and impaired growth and function of the skeletal, genital, cardiac, pulmonary, and
renal systems. Maternal serum screening for SLOS can be implemented using second-
trimester levels of AFP, uE3, and hCG in an algorithm that provides for 83% detection
of SLOS with a very low (0.3%) screen-positive rate. Although SLOS is rare, occurring
in only 1 out of every 130,000 pregnancies, a second-trimester screening program
also identifies pregnancies at high risk for other abnormalities, such as steroid sulfa-
tase deficiency, neuroanatomic defects, premature birth, IUGR, or fetal demise (Craig
et al. 2006). Implementation of SLOS screening requires genetic counseling to deter-
mine appropriate follow-up testing. SLOS diagnosis is achieved by measurement of

7-dehydrocholesterol levels in amniotic fluid or molecular genetic testing.
Prenatal Screening with Circulating Cell

Free DNA cfDNA
Use of cfDNA in maternal blood, also referred to as noninvasive prenatal screening,
noninvasive prenatal testing, or cell-free fetal DNA screening, is the latest advance
in the field of aneuploidy screening. Since its introduction in 2011, there has been a
drastic uptake of cfDNA screening and an ever-expanding menu of screening options.
cfDNA screening first became clinically available in the United States for T21, followed
shortly by the addition of T13 and T18. The test then evolved to include options for
sex chromosome screening, at first for monosomy X and then including sex chromo-
some trisomies. The testing options then expanded to include triploidy, additional
aneuploidies (such as 9, 16, or 22), select microdeletions, and genome-wide coverage

of any chromosomal gain or loss ≥7 Mb. Given the rapid evolution already seen and
the potential for further expansion of screening panels, it is likely that more con-
ditions and chromosomal gains or losses at even higher resolutions will continue to
become available. cfDNA testing for single gene disorders is available outside of the
United States and is being used in a diagnostic, rather than screening, manner. This
use is outside the scope of this chapter, which will focus only on cfDNA screening for
fetal chromosomal imbalances.
BIOLOGICAL BASIS OF CFDNA SCREENING

Circulating cfDNA comprises short fragments of DNA in the bloodstream. cfDNA is
present in the blood of all people and is typically the result of cells undergoing apop-
tosis as part of the natural cell cycle. In 1997, Lo et al. identified fetal cfDNA in the
plasma of pregnant women. It is now realized that the “fetal” cfDNA is of placental
origin and is released into the maternal circulation after apoptosis of cytotropho-
blast cells of the placenta. cfDNA is an ideal candidate for next-generation sequencing
(NGS), as it is found naturally in 150 to 200 bp fragments and is reliably detected in
maternal blood as early as seven weeks gestation. Importantly, cfDNA from a preg-
nancy is cleared from maternal blood within hours after delivery, which ensures that
any testing performed on cfDNA reflects the current state of cfDNA, not that of a
previous pregnancy.
The techniques that allow screening for aneuploidy are based on two important
premises. First, the entire genome of the baby and the mother is represented in the
cfDNA; second, the amount of cfDNA from each chromosome circulates as a fixed
percentage of the genome. In the case of aneuploidy, there is a higher percentage of
cfDNA than expected from the chromosome of interest. Specialized algorithms and
bioinformatics allow for the differentiation between trisomic and disomic (euploid)
states.
CFDNA TEST METHODS

Aneuploidy screening with cfDNA relies on determining the proportion of cfDNA
fragments originating from chromosomes of interest (for example, 13, 18, or 21).
Pregnant women carrying a fetus with aneuploidy will have subtle differences in the
proportion of fragments from the aneuploid chromosome. For example, in a euploid
pregnancy, the contribution of cfDNA from chromosome 21 is about 1.3% of the frag-
ments sequenced from the genome, for both the mother and the baby. If, however, the
fetus is affected with T21, more DNA is contributed from chromosome 21 because of
the extra copy of this chromosome. The mother continues to have 1.3% of her cfDNA
originate from chromosome 21, but the total percentage is higher because of the extra
fetal contribution.
Four different options for testing cfDNA in maternal plasma have been used com-
mercially. Massively parallel shotgun sequencing relies on sequencing large numbers
of unique DNA fragments in the plasma and aligning the fragments to a reference
genome to determine their chromosome of origin. The sequence reads from each chro-
mosome are then counted to determine if there is an increase in the proportion of
reads from one of the chromosomes of interest, suggesting aneuploidy. The differ-
ence between aneuploidy and euploidy is small; therefore, large numbers of sequenced
fragments are required, which can be costly. However, because the entire genome is
being sequenced, analysis of other chromosomes or subchromosomal regions can eas-
ily be introduced into the test algorithm.
Targeted sequencing also employs massively parallel sequencing but adds preampli-
fication of specific loci from select chromosomes of interest (currently chromosomes
13, 18, 21, X, and Y). The benefit of targeted sequencing is a reduction in the number
of sequenced fragments needed for analysis, which can reduce costs associated with
sequencing. One drawback is that chromosomal abnormalities beyond the targeted
chromosomes cannot be incorporated into the testing paradigm without changing the
assay because sequencing information from other, nontargeted chromosomes is not
obtained.
Single nucleotide polymorphism (SNP)-based sequencing uses targeted polymerase
chain reaction (PCR) amplification and sequencing of specific nucleotides (rather than
nonpolymorphic loci) on chromosomes of interest. By focusing on polymorphic loci,
the number and identity of each allele are measured in each sequence read. Aneuploidy
risk is computed by using a Bayesian-based maximum likelihood estimation, which
compares the expected and observed allele ratios. SNP-based cfDNA screening can
detect the presence of additional fetal haplotypes, which indicates a twin pregnancy
(ongoing or vanishing twin) or triploidy. In addition, SNP-based cfDNA screening can
detect stretches of loss of heterozygosity because all SNPs in a certain region appear
homozygous. This can be due to consanguinity or uniparental disomy. However,
interpretation of SNP-based cfDNA screening relies heavily on extremely complex
bioinformatics.
Most recently, cfDNA screening using a targeted quantitative microarray platform
has been introduced. Compared to NGS technologies, microarray technologies may
reduce turnaround time and capital cost. However, there is limited published informa-
tion regarding the performance of cfDNA testing with this methodology.
D ATA A N A LY S I S
Regardless of the testing method, screening for aneuploidy with cfDNA generates a
large amount of data. This requires sophisticated software and algorithms for analysis.
There are various approaches (Table 7.2).
F E TA L F R A C T I O N
Fetal fraction refers to the proportion of the total cfDNA that is placental in origin.
For example, if 10% of the total cfDNA originates from the placenta, the fetal frac-
tion of that sample is 10%. Fetal fraction is an important parameter that affects
the performance of cfDNA aneuploidy screening. Studies have suggested that the
median fetal fraction is around 10% (Ashoor et al. 2013), but with a wide variation.
Table 7.2 Approaches to Data Analysis for cfDNA Aneuploidy Screening
Counting Method Brief description

Z-score Calculates the ratio of sequences originating
from a chromosome of interest to sequences
originating from all chromosomes to determine
if there is an increased contribution from the
chromosome of interest
Normalized chromosome value Similar to a Z-score, but compares the
chromosome of interest to a customized set of
reference chromosomes to determine if there is
an increased contribution from the chromosome
of interest
Multiple hypothesis theory Multiple hypothetical monosomic, disomic,
and trisomic fetal genotypes are created in
silico and compared to what the sequencing
data is expected to look like for each scenario.
The hypothesis with the maximum likelihood is
determined.
False-negative results can occur if the fetal fraction is not sufficient (too low) to
allow for discrimination between euploid and aneuploid states. The fetal fraction
increases with gestational age, staying relatively stable from 10 to 22 weeks (with
only ~0.1% increase each week during that period) and then increasing more drasti-
cally during the third trimester (by about 1% per week) (Wang et al. 2013). Maternal
weight or BMI has consistently been shown to be inversely correlated with fetal
fraction. In addition, some fetal aneuploidies, most notably T18 and T13, have been
shown to be associated with decreased fetal fraction (Ashoor et al. 2013; Wang et al.
2013; Dar et al. 2014; Rava et al. 2014). Conversely, most studies have not shown
maternal age or a priori aneuploidy risk to be correlated with fetal fraction (Rava
et al. 2014).
Some laboratories estimate fetal fraction for every sample and employ a lower
fetal fraction threshold, historically 4% (Palomaki et al. 2012), below which the test
is canceled and no results are provided. Other laboratories estimate the fetal fraction
and incorporate that measurement into the bioinformatics algorithm as part of the
calculated risk figure; these labs typically still employ a fetal fraction threshold below
which the test is canceled. Fetal fraction measurements are imprecise, allowing some
samples with fetal fraction levels below the threshold to be erroneously accepted for
testing, possibly leading to false-negative calls. Another approach is to design the test-
ing assay in a way that allows for the ability to distinguish aneuploidy from euploidy at
fetal fraction levels below the typical threshold and obviate the need to measure fetal
fraction. Fewer samples will be canceled; however, a small number of samples with
fetal fraction levels below that required to distinguish aneuploidy (for that particular
test) will be processed, possibly leading to false-negative calls.
The most common reason for technical cancellation is fetal fraction below the lab-
oratory’s threshold. The technical cancellation rate is laboratory-specific and ranges
from 0.1% to 8% (McCullough et al. 2014; Pergament et al. 2014; Taneja et al. 2016).
Given that certain aneuploidies are known to be associated with low fetal fraction and
that samples with low fetal fraction are canceled, these “failed samples” or “no-calls”
have a higher rate of aneuploidy, as high as 22% in some reports (Pergament et al.
2014). The failed samples are generally not taken into account when calculating test
performance, resulting in a falsely elevated sensitivity.
Fetal fraction can be determined either with a separate test or directly from the
sequencing data. Several methods for determining fetal fraction have been described,
including using Y chromosome markers from a male fetus, paternally inherited alleles
absent from the maternal genome, or, more commonly, fetal-specific methylation pat-
terns, in which regions that are hypermethylated in the placenta relative to mater-
nal blood are analyzed to determine the proportion originating from the placenta
(Nygren et al. 2010). SNP allele frequencies are also commonly used, in which maxi-
mum likelihood estimates of the most likely fetal fraction are based on measurements
from several loci at which the fetal genotype and the maternal genotype differ (Sparks
et al. 2012). New techniques continue to be studied; for example, recent publications
have suggested that the overall fragment length distribution can be used to estimate
fetal fraction by capitalizing on the shorter length of placental cfDNA fragments com-
pared to maternal (Yu et al. 2014). Drawbacks of fetal fraction estimation methods
can include the additional laboratory work required and the need to split the sample
in two (one aliquot for the actual sequencing and the other for fetal fraction analysis),
depending on the method used to determine fetal fraction.
Total fetal fraction is higher in twin gestations than in singletons but is not dou-
ble. Therefore, the fetal fraction contribution per fetus is less than that of a singleton
fetus. Monozygotic twins are expected to have concordant karyotypes and can be con-
sidered as a singleton pregnancy for the purposes of cfDNA screening. However, dizy-
gotic twins may have discordant karyotypes. Given that the per-fetus fetal fraction
contribution is lower than in singletons, the concern is that false-negative results may
be more likely to occur. The data for the performance of cfDNA aneuploidy screening
in gestations with twins or higher-order multiples are more limited than those for
singleton gestations but are promising (Canick et al. 2012; del Mar Gil et al. 2014;
Bevilacqua et al. 2015; Gil et al. 2015; Fosler et al. 2016).
R E S U LT R E P O R T I N G
Laboratories report results in varying formats. Some laboratories employ a single
threshold approach in which there is one cutoff to differentiate between results above
and below a threshold, leading to results that are reported in a bimodal format, such
as “screen positive” or “screen negative.” Others use a double-threshold approach in
which there are two cutoffs. Results above the highest cutoff are considered screen-
positive results and results below the lower cutoff are considered screen-negative
results. The area between the two cutoffs is the area of overlap between euploid and
aneuploid samples. These results are screen positive and require follow-up; however,
they are more likely to represent a false-positive result than are those results that are
above the highest cutoff. These results can be reported descriptively (e.g., aneuploidy
detected, aneuploidy suspected, and no aneuploidy detected) or as risk figures (e.g.,
>99%, between 0.01% and 99%, and <0.01%), with the vast majority of results falling
into either of the two extremes. Unlike maternal serum screening, where individual-
ized patient-specific risks take into account maternal age, history, and other pertinent
factors, the risk figures provided with cfDNA screening do not take these factors into
account and are not an indication of a patient’s specific risk for aneuploidy.
TEST PERFORMANCE
Initial validation studies of cfDNA analysis for aneuploidy screening showed very
high sensitivity and specificity, particularly for Down syndrome (Palomaki et al. 2011;
Bianchi, Platt, Goldberg, et al. 2012; Palomaki et al. 2012). A 2015 meta-analysis (Gil
et al. 2015) of 37 relevant publications showed that the sensitivity and specificity of
cfDNA screening for Down syndrome was 99.2% and 99.91%, respectively. For T18
and T13, the sensitivity was 96.3% and 91.0%, respectively, and the specificity was
99.87% for each. As mentioned previously, published performance statistics do not
take into account the test failure rate, which falsely elevates detection rate. The differ-
ences in detection rates among the chromosomal conditions being screened are due
both to biologic and technical factors. First, T18 and T13 are less common than T21,
so fewer affected pregnancies have been studied. This leads to wider confidence inter-
vals and a greater impact on performance from one discordant result. Second, the
guanine-cytosine (GC) content of chromosome 13 and 18 is higher than that of chro-
mosome 21, resulting in decreased efficiency of sequencing and, therefore, increased
difficulty differentiating between euploid and aneuploid results. Third, T18 and T13
have been associated with lower levels of fetal cfDNA in maternal blood, thought to be
due to the decreased placental mass often seen in these aneuploid pregnancies, which
makes differentiation of aneuploidy more difficult. Finally, increased rates of confined
placental mosaicism have been seen for T13; confined placental mosaicism and the
presence of a “vanishing twin,” which may have failed to survive due to the presence of
aneuploidy, are two of the possible biologic explanations for T13 false-positive results.
The same meta-analysis showed a sensitivity and specificity of 90.3% and 99.77% for
monosomy X screening, and 93.0% and 99.86% for sex chromosome aneuploidies
other than monosomy X. For some laboratories, the failure rate for sex chromosome
analysis is considerably higher than that for the autosomal trisomies.
The limited amount of published data on expanded screening panels (microdele-
tions, additional trisomies, and genome-wide imbalances) precludes accurate test per-
formance estimates at the time of this writing. For this reason, many professional
societies, including the American College of Obstetricians and Gynecologists (2016),
do not recommend the use of cfDNA screening for these types of chromosomal
aberrations.
It is important to remember that cfDNA screening will not screen for all genetic
or chromosome conditions, or all causes of birth defects, intellectual disability, or
other developmental concerns such as autism. Also, even with these high sensitivities
and specificities, because cfDNA screening is a screening test false-positive and false-
negative results will occur. As with all screening, women receiving positive results
should be offered confirmatory testing with a diagnostic procedure. Given that cfDNA
results are typically thought to reflect placental chromosome status, rather than fetal
status directly, there have been discussions regarding whether chorionic villus sam-
pling is an appropriate option for confirmatory testing following a positive cfDNA
screening result. If chorionic villus sampling is being considered, it is important to
take into consideration the risk for confined placental mosaicism and the possible
need for follow-up amniocentesis.
The majority of studies validating cfDNA aneuploidy screening were performed in
populations of women who were at an increased risk for fetal aneuploidy, based on
maternal age, previous serum screening results, ultrasound findings, personal history
of an aneuploid pregnancy, or known parental Robertsonian translocation carriers.
More recent publications have studied cfDNA aneuploidy screening in women of aver-
age aneuploidy risk (see “cfDNA Aneuploidy Screening in Average-Risk Pregnancies”
below for additional information). While the sensitivity and specificity of aneuploidy
screening do not appear to be correlated with prior aneuploidy risk, because the preva-
lence of these disorders is lower, the positive predictive value is reduced. However,
the positive and negative predictive values of cfDNA aneuploidy screening far exceed
that of conventional aneuploidy screening options (Table 7.3) (Bianchi, Parker,
Wentworth, et al. 2014). Positive predictive values should not be calculated based on
maternal age-related risks in isolation if other clinical information (e.g., ultrasound
findings or previous serum screen results) is available.
FA L S E P O S I T I V E S
False-positive results (results in which cfDNA screening suggests aneuploidy when the
fetus is found to have a normal karyotype) occur in ~0.72% of tests when screening
Table 7.3 Test Performance Metrics for Aneuploidy Screening
25 years of age 40 years of age

Reference Trisomy Sensitivity Specificity Prevalence PPV Prevalence PPV

(%) (%) (%) (%)
Quad 21 81 95 1/933a 2 1/67a 20
screening
First- 21 87 95 1/712b 2 1/51b 26
trimester
screening
Cell-free 21 99.2 99.91 1/712b 61 1/51b 96
DNA 18 96.3 99.87 1/1765b 30 1/126b 86
screening 13 91.0 99.87 1/5621b 11 1/401b 64
a
risk at 16 weeks; brisk at 10 weeks
PPV = positive predictive value
Sources: Snijders et al. 1995; Malone et al. 2005; Gil et al. 2015
for trisomies 21, 18, 13, and sex chromosome aneuploidies (Gil et al.2015). There are
several explanations for this.
Statistical False-Positive Result

cfDNA testing is a screening test, and there is known overlap between euploid and
aneuploid samples. Statistically, a certain number of false-positive results is expected
based solely on the design and performance of the test.
Confined Placental Mosaicism

Confined placental mosaicism has been reported in 1% to 2% of all chorionic villus
samples (Gardner et al. 2012, p. 56; Malvestiti et al. 2015). Since the fetal cfDNA
in maternal blood is of placental origin, in some cases cfDNA screening will detect
confined placental mosaicism when present. Since the fetus is unaffected in these
cases, this leads to a false-positive cfDNA result with a biologic explanation. Confined
placental mosaicism can be associated with adverse pregnancy outcomes, including
IUGR, so knowledge of the presence of confined placental mosaicism may aid in preg-
nancy management.
Maternal Chromosome Abnormality

Given that the vast majority of cfDNA present in maternal blood is of maternal ori-
gin, a chromosome abnormality, even a relatively small copy number variant or very
low level of mosaicism for chromosomal aneuploidy or segmental aneuploidy, in a
pregnant woman may lead to a positive aneuploidy result on cfDNA screening. This is
of particular importance when screening for sex chromosomal aneuploidies, such as
monosomy X, XXX, or XXY. In the former condition, the normal age-related loss of one
X chromosome in female cells can lead to a higher rate of false-positive monosomy X
results. In the latter two, maternal XXX karyotype that may have gone undiagnosed
may present as a false-positive XXX or XXY (in combination with a male fetus) cfDNA
screening result.
Suspected maternal chromosomal abnormalities can be reported differently by dif-
ferent labs; some may report a positive result (without indicating that it is suspected
to be of maternal origin), some may issue an uninterpretable result and provide verbal
or written interpretation regarding the origin of the imbalance, and some may issue a
report stating that the imbalance is suspected to be of maternal origin. It is important
to understand the policies of the specific laboratory when interpreting results.
Vanishing Twin
Although cfDNA is cleared from maternal blood within hours after delivery, it is not
clear how long cfDNA will persist in maternal blood following a fetal demise. Given that
the placental tissue remains in the uterus in the case of a co-twin demise or vanishing
twin and that those cells are likely undergoing apoptosis, it is biologically plausible
for cfDNA to persist in maternal blood. In fact, Curnow et al. (2015) demonstrated
the presence of cfDNA in maternal blood at least eight weeks after a fetal demise.
Given the increased risk for fetal demise in aneuploid pregnancies, a positive cfDNA
screening result in a pregnancy with known vanishing twin may reflect the genotype
of the demised twin rather than the ongoing twin, leading to a false-positive cfDNA
result. However, not all aneuploid pregnancies end in miscarriage, and aneuploidy in
the ongoing twin cannot be excluded without further testing. For this reason, confir-
mation of a positive cfDNA result with diagnostic testing is strongly recommended,
even when a twin demise has been observed. In addition, in the case of a demise of a
male co-twin and a female ongoing twin, cfDNA screening may detect the presence of
Y chromosome material and falsely report XY sex chromosome status.
SNP-based cfDNA screening can detect the presence of an additional fetal “DNA
signature” but cannot distinguish twins (ongoing or vanishing) from paternal trip-
loidy. When this pattern is identified, a report indicating the possibility of triploidy
will be generated, and appropriate clinical follow-up would then be recommended.
Abnormality in Non-test Chromosome

Some cfDNA screening algorithms rely on ratios of test chromosome (i.e., the chromo-
some of interest) to non-test chromosomes (i.e., chromosomes other than the chro-
mosome of interest). If there is an aneuploidy in one of these non-test chromosomes,
the ratio can be disturbed and the results can be interpreted as an abnormality in the
test chromosome. For example, if chromosome 21 is being compared to chromosome
17, and there is partial monosomy of chromosome 17, it would appear as if there
were more than the expected number of sequences from chromosome 21 (when in fact
there were actually less than the expected number of sequences from chromosome
17). The use of multiple non-test chromosomes as reference mediates, but does not
completely obviate, this issue.
When diagnostic testing detects a false-positive cfDNA screen, there are currently
no generally agreed upon clinical management recommendations. Since many false
positives will be due to confined placental mosaicism, and confined placental mosa-
icism may be associated with adverse pregnancy outcome, follow-up ultrasound to
assess fetal growth has been advocated by some.
FA L S E N E G AT I V E S
False-negative results refer to cfDNA results that are screen negative when, in fact, the
fetus is affected. The false-negative rate of cfDNA aneuploidy screening is lower than
previously available serum aneuploidy screening, particularly for T21. The underly-
ing cause for the majority of false-negative results is generally accepted to be insuf-
ficient fetal fraction (for more information see the “Fetal Fraction” section), although
affected fetuses with euploid or mosaic placentas have been documented as a rare
cause of false-negative screening results (Cao et al. 2016).
D I S C O R DA N T S E X
Many laboratories offer the option of sex chromosome analysis. Discordant sex calls
(referring to XX result in a male fetus or XY result in a female fetus) can have mul-
tiple causes for discordance; some are technical and some biologic in etiology. This
may be due to technical limitations of testing. The difficulty of sex chromosome
analysis is amplified by the relative shortness of the Y chromosome and areas of
homology between the two sex chromosomes. There are fewer cfDNA reads available
from the Y chromosome because of its small size, many of which cannot be uniquely
mapped due to homology with other chromosomes, leading to a smaller number of
reads available for interpretation. In addition, there are several clinical scenarios
that may lead to discordant sex chromosome calls, including co-twin demise (see
the “Vanishing Twin” section above) or maternal history of blood transfusion or
organ transplant recipient (see “Maternal History of Recent Blood Transfusion or
Organ Transplant Recipient” section below). Finally, a disorder of sex differentia-
tion may be present, but this is one of the least likely explanations for apparent sex
discordance. However, if all other potential etiologies have been excluded, and the
fetal karyotype confirms discordance with ultrasound appearance of genitalia, fur-
ther testing for disorders of sexual differentiation can be considered. Bianchi, Parsa,
Bhatt, et al. (2015b) proposed clinical management for cases in which there is dis-
cordance between cfDNA results and ultrasound examination for fetal sex.
T E S T FA I L U R E S
Samples sent for cfDNA aneuploidy screening may result in a cancellation for either
administrative or technical reasons. Administrative cancellations occur prior to ini-
tiation of the testing process and can be due to reasons such as mislabeling, incor-
rect or defective tube, or receipt beyond stability. Technical cancellations, on the
other hand, are due to issues that occur once the sample has been accepted for acces-
sioning and laboratory handling has begun. Most technical cancellations are due to
fetal fraction below the laboratory’s threshold (see “Fetal Fraction section” above
for more details).
Following a failed result, options typically include no further testing, redraw for
cfDNA, other serum screening options, or diagnostic testing. The redraw success rate
varies but can be less than 40% in a real-world setting (Dar et al. 2014). Certainly other
clinical factors, in particular gestational age, may affect patients’ preference following
a failed result. Given the potential increased risk for aneuploidy in failed samples, pro-
fessional societies recommend that women have additional genetic counseling and be
offered ultrasound and diagnostic testing following a technical cancellation (American

College of Obstetricians and Gynecologists 2016). One of the important benefits of
cfDNA aneuploidy screening is the avoidance of unnecessary diagnostic testing pro-
cedures in euploid pregnancies; offering diagnostic testing to the up to 8% of women
whose cfDNA screen fails offsets one of the benefits of cfDNA screening compared to
maternal serum screening.
I N C I D E N TA L F I N D I N G S A N D O T H E R C O N S I D E R AT I O N S
Laboratories have different policies regarding how and whether to report incidental
or secondary findings, such as suspected chromosomal abnormalities in a non-test
chromosome. The presence of secondary findings may lead to a test cancellation, an
abnormal test result, or a normal test result with additional findings reported either
verbally or on the written test report. One should be familiar with the specific labora-
tory’s policies regarding reporting of incidental findings.
Maternal Malignancy
Several publications have identified an association between maternal malignancy or
leiomyoma (Osborne et al. 2013; McCullough et al. 2014; Bianchi, Chudova, Sehnert,
et al. 2015a) and abnormal cfDNA screening results. The reason for the abnormal
cfDNA result is presumably that the screening is detecting cfDNA derived from the
tumor (circulating tumor DNA) rather than from the pregnancy. In most cases, the
cfDNA results are highly unusual, resulting in aneuploidy for multiple chromosomes.
Pregnancies Conceived via Egg Donor or Gestational Carrier

cfDNA screening via SNP-based sequencing requires a biologic relationship between
the developing fetus and the gestating woman. For this reason, laboratories perform-
ing cfDNA screening with this methodology cannot interpret results for pregnancies
in which the fetus and the mother do not have a biologic (i.e., genetic) mother–
offspring relationship.
Consanguinity
Similarly, the presence of consanguinity (or areas of homozygosity) can affect the
interpretation of aneuploidy screening by SNP-based cfDNA sequencing and may
result in test cancellation.
Maternal History of Recent Blood Transfusion or Organ Transplant Recipient

Studies have documented that donor DNA is detectable following transfusion and
persists for days, sometimes longer. For this reason, if whole blood was transfused,
deferring blood draw for cfDNA screening would be appropriate. However, donor cells
may linger in maternal blood, so a cfDNA screening result may reflect not the patient
and pregnancy but rather the donor. If packed red blood cells are transfused, the risk
for donor cfDNA is reduced, but not excluded.
Transplanted solid organs continue to undergo natural cell cycle apoptosis, thereby
releasing cfDNA into maternal circulation. Unless the organ donor had an unbalanced
chromosomal abnormality, the presence of this “foreign” cfDNA would not inter-
fere with aneuploidy screening. However, because the sex chromosome status of the
organ donor may differ from the pregnant woman (e.g., the donor may be male (XY)
while the pregnant woman is female (XX)), sex chromosome analysis may be inac-
curate. Of note, because of the required biologic (genetic) relationship between the
pregnant woman and the fetus for SNP-based cfDNA screening, women who have
received a bone marrow transplant are not eligible for aneuploidy screening via that
methodology.
G U I D E L I N E S F O R C L I N I C A L I M P L E M E N TAT I O N O F
SCREENING WITH CFDNA
As cfDNA aneuploidy screening has evolved, so too have professional medical soci-
ety recommendations for appropriate use. Aneuploidy screening using cfDNA has
been accepted as an option in standard-of-care aneuploidy screening. Table 7.4 pro-
vides an overview of the recommendations from major societies, current at the time
Table 7.4 Professional Recommendations for cfDNA Aneuploidy Screening
ACOG/ SMFM1 NSGC2 ISPD3 ACMG4

Endorsed for high-risk Yes Yes Yes Yes
population
Endorsed for low-risk Yes No Yes Yes
population
Endorsed for sex Yes No Yes Yes
chromosome
aneuploidies
Endorsed for additional No No No No
aneuploidies
(i.e., 9, 16, 22)
Endorsed for No No Yes No
microdeletions
Endorsed for multifetal No Not Yes for Dependent
pregnancies discussed twins on specific
laboratory
Endorsed for genome- No No No No
wide abnormalities
1
American College of Obstetricians and Gynecologists/ Society for Maternal- Fetal Medicine
(American College of Obstetricians and Gynecologists 2016); 2National Society of Genetic Counselors
(Devers et al. 2013); 3International Society for Prenatal Diagnosis (Benn et al. 2015); 4American
College of Medical Genetics and Genomics (Gregg et al. 2013)
of writing. Issues of insurance coverage and test cost are paramount in decision
making about cfDNA at this time.
C F D N A A N E U P L O I D Y S C R E E N I N G I N AV E R A G E - R
ISK
PREGNANCIES
The original validation studies (Palomaki et al. 2011; Bianchi et al. 2012; Palomaki
et al. 2012) demonstrating the performance of cfDNA aneuploidy screening were per-
formed in populations of women with an increased risk for aneuploidy. There is no
biologic reason that the test would be expected to perform differently in an average-
risk population, but the lack of published data (and the higher cost of cfDNA com-
pared to other aneuploidy screening options) led to a restriction of use to the high-risk
population. More recent studies (Bianchi et al. 2014; Dar et al. 2014; Norton et al.
2015) show that the sensitivity and specificity of cfDNA aneuploidy screening in an
average-risk population is similar to that of a high-risk population. However, posi-
tive and negative predictive value are highly influenced by the prevalence of a disor-
der. Therefore, the chance that a result, once obtained, is correct varies based on the
population being studied. Since the prevalence of autosomal aneuploidy is lower in
an average-risk population, a cfDNA result that is positive for T21, T18, or T13 is
less likely to represent a true positive than a positive screen in a high-risk popula-
tion. Conversely, a negative cfDNA result is more likely to represent a true negative
when reported to a low-or average-risk woman than one reported to a woman who
is considered to be at high risk. Given recent updates to professional medical society
statements and an increase in the number of third-party payers covering cfDNA aneu-
ploidy screening in the average-risk population, it is likely that its use will become
more widespread in the very near future.
CONCURRENT ANEUPLOIDY SCREENING IN PREGNANCY

In general, parallel or simultaneous aneuploidy screening with different methodolo-
gies is not recommended because of the cost and additive false-positive rates, which
then exceed that of either test individually.
Maternal Serum Screening and cfDNA

cfDNA aneuploidy screening can be used as a second-tier screen for women who have
a positive result on a traditional serum or ultrasound screen, but traditional serum
screening for fetal aneuploidy should not be performed following cfDNA screen-
ing (American College of Obstetricians and Gynecologists 2016). However, cfDNA
screening does not provide information regarding the risk of ONTDs. For this reason,
second-trimester maternal serum AFP screening and/or a level II ultrasound is recom-
mended in addition to the cfDNA screen.
Nuchal Translucency and cfDNA

A nuchal translucency measurement should not be used in isolation for aneuploidy
screening (Benn et al. 2015; American College of Obstetricians and Gynecologists
2016). Therefore, measurement of nuchal translucency is unnecessary if cfDNA is
being used for aneuploidy screening. However, other information can be confirmed
from first-trimester ultrasound evaluation, including viability, number and chorion-
icity of fetuses, presence/absence of vanishing twin, gestational age, and presence/

absence of some major anomalies. This information can ensure that cfDNA is being
ordered appropriately and may also aid in a patient’s decision-making process regard-
ing options; for example, if a cystic hygroma is noted on first-trimester ultrasound, a
patient may elect to proceed with diagnostic testing rather than aneuploidy screening.
Role of a Genetic Counselor

Genetic counselors (GCs) working in the performing laboratory often serve as liaisons
between the lab and ordering providers. With regard to prenatal testing, some of the
more easily defined duties include calling out abnormal or unusual results, obtaining
clinical information as needed before testing, and obtaining outcome information for
quality assurance purposes. The GC is often called upon to address questions from
providers both before and after the test to help them to understand the best way to
use new technology, such as cfDNA testing; to understand when one method may be
a better choice for a particular patient versus another; or to provide specific informa-
tion to allow the provider to counsel the patient appropriately. GCs in these laborato-
ries may work closely with laboratory directors and bioinformatics team members on
report design and comments, gather outcome data to help refine performance statis-
tics, and relay feedback from providers to allow for test development in directions that
those providers would find most helpful.
In addition to consulting with providers and addressing their questions, GCs are
often called upon to provide consultation directly to patients who have received
results or are considering testing. This particular service may vary from laboratory to
laboratory and is likely to be found more often in labs that specialize in cfDNA testing
versus previously existing genetics or general labs that are integrating this new tech-
nology into their existing service framework.
Lastly, the GC working with the cfDNA laboratory will spend a significant amount
of time educating providers and patients, as this is a very new technology. There is
quite a bit of confusion in the medical community regarding the testing and how it
may best be used, especially in circumstances outside of the more common “high risk”
for aneuploidy scenario.
The skill set of GCs positions them to integrate into areas outside of laboratory
operations, such as research and development (new product development; contrib-
uting to abstracts, publications, and conference presentations), marketing (creating
brochures and other collateral materials, organizing and participating in branded edu-
cational efforts, conducting market research), sales (direct customer sales, providing
internal training for sales team, serving as an expert resource for sales team), regula-
tory (serving as a liaison between the laboratory and regulatory governing bodies,
ensuring compliance with regulations and recommendations from regulatory bodies),
and medical affairs (serving as an expert resource, creating unbranded physician and
patient education tools).
Future Directions
In the short time that cfDNA screening has been available clinically, we have already
witnessed a fast-paced evolution in the technologies being used and the conditions
and populations being screened. It is likely that in the coming years, as the potential
for cfDNA becomes further realized, we will continue to see dramatic changes in how
the technology is used in reproductive medicine.
Because there is no one screening test that is superior to others in all areas, algo-
rithms combining the results of various screening methodologies may be developed.
This has already occurred with the development of first-trimester combined screen-
ing, in which ultrasound markers and serum analytes were combined. In the future,
protocols may be developed that incorporate cfDNA screening for aneuploidy with
first-trimester analyte screening for adverse pregnancy outcome; or reflex panels may
begin with serum screening and reflex to cfDNA testing if the risk for aneuploidy is
above a specified cutoff. Alternatively, if cfDNA cannot be interpreted due to low fetal
fraction, testing might reflex to a trimester-appropriate serum screen to try to refine

the risk for women in this group.
Whole genome analysis for chromosomal gains or losses ≥7 Mb is already clini-
cally available. It is likely that technology improvements will enable further refine-
ment and identification of copy number variants at increasingly higher resolutions.
This may even extend to the ability to identify single nucleotide changes, which might
allow mutation detection for single gene disorders, or whole exome or whole genome
sequencing at depths appropriate for identification of disease- causing variants.
Noninvasive prenatal diagnosis for certain single gene disorders is currently available
in the United Kingdom, and this will likely gain wider availability and acceptance in
the coming years.
Finally, cfDNA technology may become outdated and replaced by other sources
of genetic information about the pregnancy, such as cfRNA or whole fetal cells. The
ability to detect and isolate whole fetal cells would provide an opportunity for direct
fetal testing without the background of maternal DNA and may lead to a host of fully
noninvasive diagnostic tests.
References
American College of Obstetricians and Gynecologists (2016). Practice Bulletin No. 163: Screening
for fetal aneuploidy. Obstet Gynecol, 127(5), pp. e123–e137.
Ashoor, G., Syngelaki, A., Poon, L.C., Rezende, J.C., & Nicolaides, K.H. (2013). Fetal fraction in
maternal plasma cell-free DNA at 11–13 weeks’ gestation: relation to maternal and fetal
characteristics. Ultrasound Obstet Gynecol, 41(1), pp. 26–32.
Baumgarten, A., & Robinson, J. (1988). Prospective study of an inverse relationship between
maternal glycosylated hemoglobin and serum alpha-fetoprotein concentrations in preg-
nant women with diabetes. Am J Obstet Gynecol, 159(1), pp. 77–81.
Benn, P.A., Gainey, A., Ingardia, C.J., Rodis, J.F., & Egan, J.F. (2001). Second trimester maternal
serum analytes in triploid pregnancies: correlation with phenotype and sex chromosome
complement. Prenat Diagn, 21(8), pp. 680–686.
Benn, P., Borrell, A., Chiu, R.W., Cuckle, H., Dugoff, L., Faas, B., Gross, S., Huang, T., Johnson,
J., Maymon, R., Norton, M., Odibo, A., Schielen, P., Spencer, K., Wright, D., & Yaron, Y.
(2015). Position statement from the Chromosome Abnormality Screening Committee on
behalf of the Board of the International Society for Prenatal Diagnosis. Prenat Diagn, 35(8),
pp. 725–734.
Bestwick, J.P., Huttly, W.J., & Wald, N.J. (2013). Detection of trisomy 18 and trisomy 13 using
first and second trimester Down’s syndrome screening markers. J Med Screen, 20(2),
pp. 57–65.
Bevilacqua, E., Gil, M.M., Nicolaides, K.H., Ordonez, E., Cirigliano, V., Dierickx, H., Willems, P.J.,
& Jani, J.C. (2015). Performance of screening for aneuploidies by cell-free DNA analysis of
maternal blood in twin pregnancies. Ultrasound Obstet Gynecol, 45(1), pp. 61–66.
Bianchi, D.W., Chudova, D., Sehnert, A.J., Bhatt, S., Murray, K., Prosen, T.L., Garber, J.E., Wilkins-
Haug, L., Vora, N.L., Warsof, S., Goldberg, J., Ziainia, T., & Halks-Miller, M. (2015a).
Noninvasive Prenatal Testing and Incidental Detection of Occult Maternal Malignancies.
JAMA, 314(2), pp. 162–169.
Bianchi, D.W., Parker, R.L., Wentworth, J., Madankumar, R., Saffer, C., Das, A.F., Craig, J.A.,
Chudova, D.I., Devers, P.L., Jones, K.W., Oliver, K., Rava, R.P., & Sehnert, A.J.; CARE Study
Group (2014). DNA sequencing versus standard prenatal aneuploidy screening. N Engl J
Med, 370(9), pp. 799–808.
Bianchi, D.W., Parsa, S., Bhatt, S., Halks-Miller, M., Kurtzman, K., Sehnert, A.J., & Swanson, A.
(2015b). Fetal sex chromosome testing by maternal plasma DNA sequencing: clinical labo-
ratory experience and biology. Obstet Gynecol, 125(2), pp. 375–382.
Bianchi, D.W., Platt, L.D., Goldberg, J.D., Abuhamad, A.Z., Sehnert, A.J., & Rava, R.P.; MatErnal
BLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) Study Group (2012).
Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet
Gynecol, 119(5), pp. 890–901.
Boldt, H.B., & Conover, C.A. (2007). Pregnancy-associated plasma protein-A (PAPP-A): a local
regulator of IGF bioavailability through cleavage of IGFBPs. Growth Horm IGF Res, 17(1),
pp. 10–18.
Canfield, M.A., Mai, C.T., Wang, Y., O’Halloran, A., Marengo, L.K., Olney, R.S., Borger, C.L.,
Rutkowski, R., Fornoff, J., Irwin, N., Copeland, G., Flood, T.J., Meyer, R.E., Rickard, R.,
Alverson, C.J., Sweatlock, J., & Kirby, R.S., National Birth Defects Prevention Network
(2014). The association between race/ethnicity and major birth defects in the United
States, 1999–2007. Am J Public Health, 104(9), pp. e14–e23.
Canick, J.A., Kloza, E.M., Lambert-Messerlian, G.M., Haddow, J.E., Ehrich, M., van den Boom,
D., Bombard, A.T., Deciu, C., & Palomaki, G.E. (2012). DNA sequencing of maternal plasma
to identify Down syndrome and other trisomies in multiple gestations. Prenat Diagn, 32(8),
pp. 730–734.
Cao, Y., Hoppman, N.L., Kerr, S.E., Sattler, C.A., Borowski, K.S., Wick, M.J., Highsmith, W. E., &
Aypar, U. (2016). False negative cell-free DNA screening result in a newborn with trisomy
13. Case Rep Genet [Epub ahead of print, Feb 22; accessed 28 June 2016].
Cereda, A., & Carey, J.C. (2012). The trisomy 18 syndrome. Orphan J Rare Dis, 23(7), p. 81.
Craig, W.Y., Haddow, J.E., Palomaki, G.E., Kelley, R.I., Kratz, L.E., Shackleton, C.H., Marcos,
J., Stephen Tint, G., MacRae, A.R., Nowaczyk, M.J., Kloza, E.M., Irons, M.B., & Roberson,
M. (2006). Identifying Smith-Lemli-Opitz syndrome in conjunction with prenatal screening
for Down syndrome. Prenat Diagn, 26(9), pp. 842–849.
Curnow, K.J., Wilkins-Haug, L., Ryan, A., Kırkızlar, E., Stosic, M., Hall, M.P., Sigurjonsson, S.,
Demko, Z., Rabinowitz, M., & Gross, S.J. (2015). Detection of triploid, molar, and vanishing
twin pregnancies by a single-nucleotide polymorphism-based noninvasive prenatal test. Am
J Obstet Gynecol, 212(1), pp. 79.e1–e9.
Dar, P., Curnow, K., Gross, S.J., Hall, M.P., Stosic, M., Demko, Z., Zimmermann, B., Hill, M.,
Sigurjonsson, S., Ryan, A., Banjevic, M., Kolacki, P.L., Koch, S.W., Strom, C.M., Rabinowitz,
M., & Benn, P. (2014). Clinical experience and follow-up with large scale single-nucleotide
polymorphism-based noninvasive prenatal aneuploidy testing. Am J Obstet Gynecol, 211(5),
pp. 527.e1–e17.
del Mar Gil, M., Quezada, M.S., Bregant, B., Syngelaki, A., & Nicolaides, K.H. (2014). Cell-free
DNA analysis for trisomy risk assessment in first-trimester twin pregnancies. Fetal Diagn
Ther, 35(3), pp. 204–211.
Devers, P.L., Cronister, A., Ormond, K.E., Facio, F., Brasington, C.K., & Flodman, P. (2013).
Noninvasive prenatal testing/noninvasive prenatal diagnosis: the position of the National
Society of Genetic Counselors. J Genet Couns, 22(3), pp. 291–295.
Fosler, L., Winters, P., Jones, K.W., Curnow, K.J., Sehnert, A.J., Bhatt, S., & Platt, L.D. (2016).
Aneuploidy screening using noninvasive prenatal testing in twin pregnancies. Ultrasound
Obstet Gynecol [Epub ahead of print, May 19; accessed 30 June 2016].
Fournier, T., Guibourdenche, J., & Evain-Brion, D. (2015). Review: hCGs: different sources of
production, different glycoforms and functions. Placenta, 36(Suppl 1), pp. S60–65.
Frey, L., & Hauser, A. (2003). Epidemiology of neural tube defects. Epilepsia, 44(Suppl 3),
pp. 4–13.
Frishman, G.N., Canick, J.A., Hogan, J.W., Hackett, R.J., Kellner, L.H., & Saller, D.N.Jr. (1997).
Serum triple-marker screening in in vitro fertilization and naturally conceived pregnancies.
Obstet Gynecol, 90(1), pp. 98–101.
Gardner, R. J. M., Sutherland, G. R., & Shaffer, L.G. (2012). Chromosome Abnormalities and
Genetic Counseling, 4th ed. New York: Oxford University Press, Inc.
Gil, M.M., Quezada, M.S., Revello, R., Akolekar, R., & Nicolaides, K.H. (2015). Analysis of cell-
free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis.
Ultrasound Obstet Gynecol, 45(3), pp. 249–266.
Greenberg, F., Faucett, A., Rose, E., Bancalari, L., Kardon, N.B., Mizejewski, G., Haddow, J.E., &
Alpert, E. (1992). Congenital deficiency of alpha-fetoprotein. Am J Obstet Gynecol, 167(2),
pp. 509–511.
Gregg, A.R., Gross, S.J., Best, R.G., Monaghan, K.G., Bajaj, K., Skotko, B.G., Thompson, B.H., &
Watson, M.S. (2013). ACMG statement on noninvasive prenatal screening for fetal aneu-
ploidy. Genet Med, 15(5), pp. 395–398.
Hill, M., Johnson, J.A., Langlois, S., Lee, H., Winsor, S., Dineley, B., Horniachek, M., Lalatta,
F., Ronzoni, L., Barrett, A.N., Advani, H.V., Choolani, M., Rabinowitz, R., Pajkrt, E., van
Schendel, R.V., Henneman, L., Rommers, W., Bilardo, C.M., Rendeiro, P., Ribeiro, M.J.,
Rocha, J., Bay Lund, I.C., Petersen, O.B., Becher, N., Vogel, I., Stefánsdottir, V., Ingvarsdottir,
S., Gottfredsdottir, H., Morris, S., & Chitty, L.S. (2016). Preferences for prenatal tests for
Down syndrome: an international comparison of the views of pregnant women and health
professionals. Eur J Hum Genet, 24(7), pp. 968–975.
Huang, T., Alberman, E., Wald, N., & Summers, A.M. (2005). Triploidy identified through second-
trimester serum screening. Prenat Diagn, 25(3), pp. 229–233.
Lambert-Messerlian, G., Dugoff, L., Vidaver, J., Canick, J.A., Malone, F.D., Ball, R.H., Comstock,
C.H., Nyberg, D.A., Saade, G., Eddleman, K., Klugman, S., Craigo, S.D., Timor-Tritsch, I.E.,
Carr, S.R., Wolfe, H.M., & D’Alton, M.E. (2006). First-and second-trimester Down syn-
drome screening markers in pregnancies achieved through assisted reproductive technolo-
gies (ART): a FASTER trial study. Prenat Diagn, 26(8), pp. 672–678.
Lambert-Messerlian, G., Palomaki, G.E., & Canick, J.A. (2009). Adjustment of serum markers in
first trimester screening. J Med Screen, 16(2), pp. 102–103.
Lambert-Messerlian, G., & Palomaki, G.E. (2015). Prenatal serum screening markers may not
require adjustment in former smokers. Prenat Diagn, 35(13), pp. 1371–1373.
Lo, Y. M., Corbetta, N., Chamberlain, P. F., Rai, V., Sargent, I. L., Redman, C. W., & Wainscott,
J.S. (1997). Presence of fetal DNA in maternal plasma and serum. Lancet, 350(9076), pp.
485–487.
Malone, F.D., Canick, J.A., Ball, R.H., Nyberg, D.A., Comstock, C.H., Bukowski, R., Berkowitz,
R.L., Gross, S.J., Dugoff, L., Craigo, S.D., Timor-Tritsch, I.E., Carr, S.R., Wolfe, H.M.,
Dukes, K., Bianchi, D.W., Rudnicka, A.R., Hackshaw, A.K., Lambert-Messerlian, G., Wald,
N.J., & D’Alton, M.E.; First-and Second-Trimester Evaluation of Risk (FASTER) Research
Consortium (2005). First-trimester or second-trimester screening, or both, for Down’s syn-
drome. N Engl J Med, 353(19), pp. 2001–2011.
Malvestiti, F., Agrati, C., Grimi, B., Pompilii, E., Izzi, C., Martinoni, L., Gaetani, E., Liuti, M.R.,
Trotta, A., Maggi, F., Simoni, G., & Grati, F.R. (2015). Interpreting mosaicism in chorionic
villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amnio-
centesis. Prenat Diagn, 35(11), pp. 1117–1127.
McCullough, R.M., Almasri, E.A., Guan, X., Geis, J.A., Hicks, S.C., Mazloom, A.R., Deciu, C.,
Oeth, P., Bombard, A.T., Paxton, B., Dharajiya, N., & Saldivar, J.S. (2014). Non-invasive
prenatal chromosomal aneuploidy testing—clinical experience: 100,000 clinical samples.
PLoS ONE 9(10), pp. e109173. [online]. Available at: http://journals.plos.org/plosone/
article?id=10.1371/journal.pone.0109173 [Accessed 30 June 2016].
Medical Task Force on Anencephaly. (1990). The infant with anencephaly. N Engl J Med, 322(10),
pp. 669–674.
Newby, D., Aitken, D.A., Crossley, J.A., Howatson, A.G., Macri, J.N., & Connor, J.M. (1997).
Biochemical markers of trisomy 21 and the pathophysiology of Down’s syndrome pregnan-
cies. Prenat Diagn, 17(10), pp. 941–951.
Norton, M.E., Jacobsson, B., Swamy, G.K., Laurent, L.C., Ranzini, A.C., Brar, H., Tomlinson,
M.W., Pereira, L., Spitz, J.L., Hollemon, D., Cuckle, H., Musci, T.J., & Wapner, R.J. (2015).
Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med, 372(17), pp.
1589–1597.
Nygren, A.O., Dean, J., Jensen, T.J., Kruse, S., Kwong, W., van den Boom, D., & Ehrich, M. (2010).
Quantification of fetal DNA by use of methylation-based DNA discrimination. Clin Chem,
56(10), pp. 1627–1635.
Osborne, C.M., Hardisty, E., Devers, P., Kaiser-Rogers, K., Hayden, M.A., Goodnight, W., & Vora,
N.L. (2013). Discordant noninvasive prenatal testing results in a patient subsequently diag-
nosed with metastatic disease. Prenat Diagn, 33(6), pp. 609–611.
Palomaki, G.E., Deciu, C., Kloza, E.M., Lambert-Messerlian, G.M., Haddow, J.E., Neveux, L.M.,
Ehrich, M., van den Boom, D., Bombard, A.T., Grody, W.W., & Nelson, S.F., Canick, J.A.
(2012). DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy
13 as well as Down syndrome: an international collaborative study. Genet Med, 14(3), pp.
296–305.
Palomaki, G.E., Kloza, E.M., Lambert-Messerlian, G.M., Haddow, J.E., Neveux, L.M., Ehrich,
M., van den Boom, D., Bombard, A.T., Deciu, C., Grody, W.W., Nelson, S.F., & Canick, J.A.
(2011). DNA sequencing of maternal plasma to detect Down syndrome: an international
clinical validation study. Genet Med, 13(11), pp. 913–920.
Parker, S.E., Mai, C.T., Canfield, M.A., Rickard, R., Wang, Y., Meyer, R.E., Anderson, P., Mason,
C.A., Collins, J.S., Kirby, R.S., & Correa, A.; National Birth Defects Prevention Network
(2010). Updated national birth prevalence estimates for selected birth defects in the United
States, 2004–2006. Birth Defects Res A Clin Mol Teratol, 88(12), pp. 1008–1016.
Penney, L.L., & Klenke, W.J. (1980). Variability in unconjugated and total estriol in serum during
normal third trimester pregnancy. Clin Chem, 26(13), pp. 1800–1803.
Pergament, E., Cuckle, H., Zimmermann, B., Banjevic, M., Sigurjonsson, S., Ryan, A., Hall, M.P.,
Dodd, M., Lacroute, P., Stosic, M., Chopra, N., Hunkapiller, N., Prosen, D.E., McAdoo, S.,
Demko, Z., Siddiqui, A., Hill, M., & Rabinowitz, M. (2014). Single-nucleotide polymorphism-
based noninvasive prenatal screening in a high-risk and low-risk cohort. Obstet Gynecol,
124(2 Pt 1), pp. 210–218.
Rava, R.P., Srinivasan, A., Sehnert, A.J., & Bianchi, D.W. (2014). Circulating fetal cell-free DNA
fractions differ in autosomal aneuploidies and monosomy X. Clin Chem, 60(1), pp. 243–250.
Resta, R.G. (2005). Changing demographics of advanced maternal age (AMA) and the impact on
the predicted incidence of Down syndrome in the United States: implications for prenatal
screening and genetic counseling. Am J Med Genet A, 133A(1), pp. 31–36.
Robinson, A. & Linden, M.G. (1993). Clinical Genetics Handbook, 2nd ed. Boston: Blackwell
Scientific Publications, Inc., p. 127.
Savva, G.M., Walker, K., & Morris, J.K. (2010). The maternal age-specific live birth prevalence
of trisomies 13 and 18 compared to trisomy 21 (Down syndrome). Prenat Diagn, 30(1),
pp. 57–64.
Smith-Bindman, R., Chu, P., & Goldberg, J.D. (2007). Second trimester prenatal ultrasound for
the detection of pregnancies at increased risk of Down syndrome. Prenat Diagn, 27(6), pp.
535–544.
Snijders, R.J., Sebire, N.J., & Nicolaides, K.H. (1995). Maternal age and gestational age-specific
risk for chromosomal defects. Fetal Diagn Ther, 10(6), pp. 356–367.
Sparks, A.B., Struble, C.A., Wang, E.T., Song, K., & Oliphant, A. (2012). Noninvasive prenatal
detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation
for trisomy 21 and trisomy 18. Am J Obstet Gynecol, 206(4), pp. 319.e1–e9.
Spencer, K., Heath, V., El-Sheikhah, A., Ong, C.Y., & Nicolaides, K.H. (2005). Ethnicity and the
need for correction of biochemical and ultrasound markers of chromosomal anomalies
in the first trimester: a study of Oriental, Asian and Afro-Caribbean populations. Prenat
Diagn, 25(5), pp. 365–369.
Taneja, P.A., Snyder, H.L., De Feo, E., Kruglyak, K.M., Halks-Miller, M., Curnow, K.J., & Bhatt,
S. (2016). Noninvasive prenatal testing in the general obstetric population: clinical per-
formance and counseling considerations in over 85 000 cases. Prenat Diagn, 36(3), pp.
237–243.
Wald, N.J., Cuckle, H., Brock, J.H., Peto, R., Polani, P.E., & Woodford, F.P. (1977). Maternal
serum-alpha-fetoprotein measurement in antenatal screening for anencephaly and spina
bifida in early pregnancy. Report of U.K. collaborative study on alpha-fetoprotein in rela-

tion to neural-tube defects. Lancet, 1(8026), pp. 1323–1332.
Wald, N.J., Kennard, A., Hackshaw, A., & McGuire, A. (1997). Antenatal screening for Down’s
syndrome. J Med Screen, 4(4), pp. 181–246.
Wald, N.J., Rodeck, C., Hackshaw, A.K., Walters, J., Chitty, L., & Mackinson, A.M.; SURUSS
Research Group (2003). First and second trimester antenatal screening for Down’s syn-
drome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). Health
Technol Assess, 7(11), pp. 1–77.
Wang, E., Batey, A., Struble, C., Musci, T., Song, K., & Oliphant, A. (2013). Gestational age and
maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn, 33(7), pp.
662–666.
Williams, J., Mai, C.T., Mulinare, J., Isenburg, J., Flood, T.J., Ethen, M., Frohnert, B., & Kirby,
R.S.; Centers for Disease Control and Prevention. (2015). Updated estimates of neural
tube defects prevented by mandatory folic Acid fortification—United States, 1995–2011.
MMWR Morb Mort Wkly Rep, 64(1), pp,1–5.
Yu, S.C., Chan, K.C., Zheng, Y.W., Jiang, P., Liao, G.J., Sun, H., Akolekar, R., Leung, T.Y., Go,
A.T., van Vugt, J.M., Minekawa, R., Oudejans, C.B., Nicolaides, K.H., Chiu, R.W., & Lo, Y.M.
(2014). Size-based molecular diagnostics using plasma DNA for noninvasive prenatal test-
ing. Proc Natl Acad Sci U S A, 111(23), pp. 8583–8588.
Zaragoza, M.V., Surti, U., Redline, R.W., Millie, E., Chakravarti, A., & Hassold, T.J. (2000). Parental
origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and
association with the partial hydatidiform mole. Am J Hum Genet, 66(6), pp. 1807–1820.

Practical Genetic Counseling For The Laboratory - (7. Prenatal Screening Technologies and Test Issues)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Practical Genetic Counseling For The Laboratory - (7. Prenatal Screening Technologies and Test Issues)

Uploaded by

Copyright:

Available Formats

7

Prenatal Screening Technologies

Box 7.1 Common Screening Terminology

Maternal Serum Screening

MARKERS USED IN SCREENING

ADJUSTMENT OF MARKER LEVELS

Assisted Reproductive Technology

chromosome. Down syndrome is due to the presence of a Robertsonian translocation

between 12 weeks gestation and birth), the birth prevalence is approximately 1 in

OTHER DISORDERS OF INTEREST

Table 7.1 Second-​Trimester Marker Patterns

AFP hGC uE3 DIA Associated with:

mine appropriate follow-​up testing. SLOS diagnosis is achieved by measurement of

Prenatal Screening with Circulating Cell

aneuploidies (such as 9, 16, or 22), select microdeletions, and genome-​wide coverage

BIOLOGICAL BASIS OF CFDNA SCREENING

CFDNA TEST METHODS

tion regarding the performance of cfDNA testing with this methodology.

Table 7.2 Approaches to Data Analysis for cfDNA Aneuploidy Screening

Counting Method Brief description

Table 7.3 Test Performance Metrics for Aneuploidy Screening

25 years of age 40 years of age

Reference Trisomy Sensitivity Specificity Prevalence PPV Prevalence PPV

Statistical False-​Positive Result

Confined Placental Mosaicism

Maternal Chromosome Abnormality

Abnormality in Non-​test Chromosome

offered ultrasound and diagnostic testing following a technical cancellation (American

Pregnancies Conceived via Egg Donor or Gestational Carrier

Maternal History of Recent Blood Transfusion or Organ Transplant Recipient

Table 7.4 Professional Recommendations for cfDNA Aneuploidy Screening

ACOG/​ SMFM1 NSGC2 ISPD3 ACMG4

CONCURRENT ANEUPLOIDY SCREENING IN PREGNANCY

Maternal Serum Screening and cfDNA

Nuchal Translucency and cfDNA

icity of fetuses, presence/​absence of vanishing twin, gestational age, and presence/​

Role of a Genetic Counselor

fraction, testing might reflex to a trimester-​appropriate serum screen to try to refine

bifida in early pregnancy. Report of U.K. collaborative study on alpha-​fetoprotein in rela-

You might also like

Table 7.1 Second-Trimester Marker Patterns

mine appropriate follow-up testing. SLOS diagnosis is achieved by measurement of

aneuploidies (such as 9, 16, or 22), select microdeletions, and genome-wide coverage

Table 7.2 Approaches to Data Analysis for cfDNA Aneuploidy Screening

Table 7.3 Test Performance Metrics for Aneuploidy Screening

Statistical False-Positive Result

Abnormality in Non-test Chromosome

Table 7.4 Professional Recommendations for cfDNA Aneuploidy Screening

ACOG/ SMFM1 NSGC2 ISPD3 ACMG4

icity of fetuses, presence/absence of vanishing twin, gestational age, and presence/

fraction, testing might reflex to a trimester-appropriate serum screen to try to refine

bifida in early pregnancy. Report of U.K. collaborative study on alpha-fetoprotein in rela-