PRENATAL SCREENING AND

DIAGNOSIS
Prenatal screening
SOGC Clinical Practice Guidelines – Prenatal Screening for Fetal Aneuploidy
Obstetrics: Normal and Problem Pregnancies (Gabbe) – Prenatal Genetic Diagnosis
Williams Obstetrics: Prenatal Diagnosis and Fetal Therapy

• Screening: use of specific marker(s) to identify individuals in a population at highest risk for particular disorder
◦ Condition being screened for should be severe, and results of screening should affect our management of
the pregnancy or the infant’s care after birth
◦ An accurate diagnostic test for all individuals who screen positive must be in place, in addition to
accessible intervention
◦ Markers must be maximally sensitive and specific (i.e. able to identify significant proportion of individuals
with condition and minimal number of individuals without the condition)
• Prenatal non-invasive screening tests for Down syndrome, trisomy 18, and open neural tube defects (OTNDs)
must be offered to all women for risk stratification prior to consideration of invasive diagnostic methods
• Women >40 years of age (at estimated date of delivery) can opt to undergo invasive diagnostic methods such
as CVS and amniocentesis based on age alone, without prior screening (benefits > risks)
Screening: surveying the population to identify individuals at higher risk of having a certain condition

Diagnosis: determining whether an individual has a certain condition

 Applies to Outcome

Screening Population Risk of disease

Diagnosis Individual Diagnosis of disease

First trimester screening for aneuploidy

Screening in the first trimester involves: (i) ultrasound (nuchal translucency) and (ii) a group of serum biochemical
markers called the first trimester screen (FTS).

• Nuchal translucency (NT): sonographic visualization of increased thickness of subcutaneous fluid behind the
fetal neck above 95th percentile for fetal age; increased size of translucency from 11-14 weeks gestational age
associated with increased risk of Down syndrome, as well as other trisomies, Turner syndrome, and congenital
cardiac defects.
• Taiwan J Obstet Gynecol. 2010 Jun;49(2):133-8.
◦ Mechanism of NTrelates to:
▪ Abnormal collagen synthesis: trisomies 13, 18, 21, and Turner syndrome have altered formation of
extracellular matrix proteins such as collagen, which results in increased NT.
▪ Defects in lymphatics formation: hypothesized to be central defect leading to both NT and cardiac
defects. An underlying endothelial differentiation defect causes abnormal lymphatic vessel
formation, which in turn leads to accumulation of fluid behind neck. Cardiovascular defects may also
stem from this underlying endothelial defect.
• FTS serum biochemical markers: pregnancy-associated plasma protein A (PAPP-A) and free b-hCG
◦ Decreased PAPP-A and increased free b-hCG in patients carrying fetuses with Down syndrome
• Consideration of maternal age, nuchal translucency, and serum levels of PAPP-A and b-hCG detects 83% of
cases of Down syndrome (with 5% false positive rate)
Second trimester screening for aneuploidy
Quad screen: Maternal serum alpha fetoprotein (AFP), unconjugated estriol (uE3), human chorionic gonadotropin
(hCG), dimeric inhibin-A (DIA) measured at 15-20 weeks gestation

• Detects 77% fetuses with Down syndrome with 5.2% false positive rate
Production Down syndrome Other relations

Alpha fetoprotein Glycoprotein produced by Decreased: mechanism Increased in any body

(AFP) yolk sac and subsequently by
liver and gastrointestinal tract.
It is the major protein
constituent of fetal serum; its
role is analogous to albumin
serum.
related to impaired renal
excretion and impaired
membrane/placental diffusion;
not from decreased production
of AFP.
wall defect leads to
leakage of AFP from fetus
to amniotic fluid, which is
reflected in maternal

in adult circulation. AFP is

excreted by the kidneys to the Prenat Diagn. 1991 Aug;11(8): • Abdominal wall
amniotic fluid, and 625-8. defects:
subsequently transfers to gastroschisis,
maternal serum by omphalocele

transplacental and • Open neural tube

transamniotic diffusion. defects

• Fetal demise: fetal

blood reaching
maternal circulation

• Increased in
maternal smoking
Unconjugated Synthesized from conversion Decreased: mechanism • Decreased with
estriol of fetal hepatic substrates in unclear but maybe due to maternal type 1
placenta: fetal immaturity of fetal adrenal diabetes and obesity
dihydroepiandrosterone cortex, fetal liver, or placenta
sulfate (DHEAS) originating
from adrenal cortex
undergoes hydroxylation in
liver → converted to estriol in
placenta → diffuses into
maternal circulation

Human chorionic Glycoprotein synthesized in Increased: hypothesized to • Decreased with

gonadotropin trophoblast with primary role involve abnormal development maternal type 1
(hCG): increasing progesterone of lymphatic vessels resulting diabetes, obesity, and
production by corpus luteum
in obstructed lymphatic smoking
drainage → reduced hCG
• Levels rise from inhibitor factor transfer to fetal
implantation to 8 weeks circulation and ultimately
gestational age, plateau placenta → increased hCG
until 12 weeks, production by trophoblast
decrease until 18
weeks, plateau until
term
Inhibin A Synthesized in trophoblast,
decidua, and fetal
membranes during
pregnancy; acts to inhibit FSH
release from anterior pituitary
Increased: hypothesized to
originate from increased post-
translational processing of
inhibin α precursor protein to
dimeric inhibin A in Down
syndrome pregnancies

Ultrasound screening for gross structural anomalies

• Occurs at 18-20 weeks gestational age
• Aneuploidy (see Aneuploidy chapter) commonly associated with significant anatomical anomalies
• “Soft markers” on ultrasound in second trimester confer increased risk of chromosomal abnormalities, including
nasal bone hypoplasia, brachycephaly, and clinodactyly

Screening for open neural tube defects

• Open neural tube defects: anencephaly, spina bifida, cephalocele, rare forms of spinal fusion anomalies
◦ Risk factors: family history, autosomal recessive syndromes (e.g. Joubert, Roberts syndromes),
aneuploidy, maternal hyperglycemia, medications (e.g. valproic acid, carbamazepine)
• Maternal serum AFP at 15-20 weeks > 2.0 or 2.5 multiples of the median (MoM) of unaffected pregnancies
associated with neural tube defects
• Specialized sonography (ultrasound) findings:
◦ “Lemon sign” (frontal bone scalloping) and “banana sign” (cerebellar bowing with cisterna magna
effacement) associated with open spina bifida; seen in second trimester fetuses
◦ Visualization of spine in transverse and sagittal orientations typically sufficient to describe location and
size of spinal defects
• Amniocentesis should be offered to all patients with positive screen, regardless of ultrasound findings
◦ Amniocentesis to analyze AFP and acetylcholinesterase (AchE): detection of AchE suggests open neural
tube defect or other structural defects
Summary of first and second trimester screening options
• Ministry of Health and Long-Term Care – Ontario Prenatal Screening (website)
• Williams Obstetrics – Prenatal Diagnosis and Fetal Therapy
In Ontario, different screening modalities are offered before 14 weeks gestation (early prenatal screening) and after
14 weeks gestation (late prenatal screening) for Down syndrome, trisomy 18, and open neural tube defects.

Early Prenatal Screening Modalities (adapted from MOHLTC website)

Procedure First trimester Integrated prenatal Serum integrated prenatal
screening screening (IPS) screening (SIPS)

Blood sample 1 11-14 weeks 11-14 weeks 11-14 weeks

Nuchal translucency ultrasound 11-14 weeks 11-14 weeks N/A

Blood sample 2 N/A 15-20 weeks 15-20 weeks

Results available 12-15 weeks 16-20 weeks 16-20 weeks

Detection rate for Down syndrome 80-85% (3-9%) 85-90% (2-4%) 80-90% (2-7%)
(false positive rate)

Diagnostic test if screen positive CVS at 13-15 Amniocentesis at Amniocentesis at 15-22

weeks 15-22 weeks weeks
Late Prenatal Screening Modalities (adapted from MOHLTC website)

Triple screening Quadruple screening

Blood sample 15-20 weeks 15-20 weeks

Results available 16-21 weeks 16-21 weeks

Detection rate (false positive rate) 70% (7%) 75-85% (5-10%)

Diagnostic test if screen positive Amniocentesis at 15-22 weeks Amniocentesis at 15-22 weeks
Patients in Ontario can elect to pursue integrated prenatal screening or triple/quadruple screening independently (if
prenatal screening is not elected to be done until after 14 weeks). In most centers, quadruple screening has replaced
triple screening. Stepwise and contingent sequential screenings are not options available in Ontario.

Integrated prenatal screen (nondisclosure)

• Highest detection rate achieved if screening tests conducted in both first and second trimesters – first trimester
results not disclosed until second trimester results available
• Nondisclosure approach increases detection rate by 5% in comparison to first trimester screen alone; however
ethical concerns regarding delayed notification of first trimester results and thus increased maternal risk with
delayed termination options
Stepwise sequential screening (disclosure)
• High risk cohort based on first trimester screening results offered invasive prenatal diagnostic investigations
• Moderate and low risk cohorts all receive second trimester screening
Contingent sequential screening (disclosure)
• Risk stratification based on first trimester screening results – high risk cohort (risk above 1/30) to undergo CVS;
low risk cohort (less than 1/1500) does not receive further investigations; moderate risk cohort (risk between
1/1500 and 1/30) to undergo second trimester serum testing
• Most cost effective among 3 variations of screening schedules since approximately 85% of patients will not
undergo second trimester screening

Prenatal diagnosis
SOGC Clinical Practice Guidelines – Canadian Guidelines for Prenatal Diagnosis: Genetic Indications for Prenatal Diagnosis
Obstetrics: Normal and Problem Pregnancies (Gabbe)
Prenatal genetic diagnosis: involves an invasive procedure (amniocentesis, chorionic villus sampling, or fetal blood
sampling) to conduct subsequent cell culture and chromosomal evaluation

Indications for offering invasive prenatal genetic diagnosis

• Results from first or second trimester maternal serum screening which suggest aneuploidy (see Prenatal
screening above)
• All women with previous abortus, stillbirth, or livebirth with trisomy or other chromosomal abnormality (an
exception is Turner syndrome)
• Woman or partner mosaic for chromosomal anomaly or has chromosomal rearrangement
• Parents who are carriers/affected individuals with X-linked disorders
• 2+ relatives with Down syndrome
• Exposure to therapeutic radiation in males: associated with numerical and structural abnormalities in sperm
which persist many years after treatment
• Pregnancies conceived by intracytoplasmic sperm injection
• At-risk individuals for microdeletion/microduplication syndromes, such as DiGeorge (22q deletion), Beckwith-
Wiedemann (11p duplication), Prader-Willi/Angelman (15q deletion) syndromes
• Abnormal sonographic findings
◦ Presence of major fetal anomalies: e.g. multiple congenital anomalies, neural tube defects, cystic
hygroma, limb abnormalities, omphalocele, duodenal stenosis/atresia, significant ventriculomegaly, facial
abnormalities; or in association with IUGR or variations in amniotic fluid volume
◦ Presence of minor fetal anomalies (“soft signs”) during the second trimester associated with chromosomal
abnormalities (see Prenatal screening above)
Timing and overview Procedure Complications

Amniocentesis • Conducted in second • Ultrasound-guided • Common: transient vaginal

trimester after 15 aspiration of amniotic spotting, minimal amniotic fluid
weeks gestation
fluid (approximately leakage

◦ Timing: the 20 mL) and cells • Less common: amnionitis,

amnion following hemorrhage, sepsis

must fuse percutaneous • Fetal loss following

with the insertion of needle amniocentesis estimated to be
chorion to into amniotic sac 1:200 (ranges from 1:100 to
successfull 1:600)

y pass the ◦ 1:200 risk of Down

needle syndrome at maternal
through age 35; risk of DS
both layers outweighs risk of
into the amniocentesis after
amniotic age 35

fluid; fusion • Early amniocentesis (11-14

happens by weeks) associated with
15 weeks increased risk of talipes
equinovarus (clubfoot)
Chorionic villus • Performed during • Transcervical • Additional 1-2% risk of fetal loss,
sampling (CVS) first trimester, ideally approach involves in contrast to 0.5-1% for
10-12 weeks if inserting flexible amniocentesis

transcervical catheter through ◦ This figure increases

approach. cervix with ultrasound with history of vaginal
Transabdominal guidance toward bleeding before
approach can be trophoblast tissue; procedure, number of
performed at any aspirates 10-25mg of attempts at acquiring
time throughout villi
chorionic villi (should
pregnancy.
• Transabdominal be limited to 2
• After first trimester, approach involves attempts)

CVS known as insertion of needle • Increased risk of limb or facial

placental biopsy
into long axis of anomalies if CVS performed
• Analysis of chorionic placenta; villi is prior to 10 weeks, up to 1 in
villi and amniotic fluid aspirated 3000 fetuses

cells reveals same ◦ Placental biopsy

genetic information, frequently used in
however some place of cordocentesis
specific screening for rapid karyotyping
tests such as AFP due to lower rates of
levels require associated
evaluation from complications and less
amniocentesis procedural skill
sample required

• Relative contraindications:
vaginal bleeding, extreme uterine
anteflexion or retroflexion,
genital tract infection

• Prerequisites: requires prior

sonographic evaluation of
Fetal blood • Performed after 18 • Needle inserted in • Prerequisites: Requires prior
sampling weeks gestation
placental cord root ultrasound to determine fetal
(cordocentesis) • Typically used when into umbilical vein
viability, position of the fetus,
results from • Small volume of blood presence of fetal or placental
amniocentesis and aspirated to confirm abnormalities, and location of
CVS samples the sample contains placenta and umbilical cord.
are equivocal
fetal blood.
Doppler imaging with colour flow
• Also used to evaluate • Once confirmed, to assess umbilical cord and
anemia (Rh or ABO sample sent for rapid placenta
isoimmunization), karyotyping
coagulopathy
(Hemophilia A/B, von
Willebrand disease),
bacterial or viral
cultures, PCR,
metabolic or
hematologic work-up