Seminar and Tutorial

Prenatal
Diagnosis

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: ..

Table of Contents

Prenatal Screening and Diagnosis Of Neural

Tube Defect
Prenatal Screening and Diagnosis Of Down
Syndrome and Aneuploidies
Prenatal and preimplantation diagnosis

Prenatal Screening and Diagnosis

Of Neural Tube Defect

Introduction

Neural Tube Defect < NTD> : second most

prevalent congenital anomaly, second to car
diac malformation.
Significant Roles to prevent NTDs:
Maternal screening programme
Sonographic imaging with or without the
AF testing
Folic acid as the prevention of NTDs

Incidence and Epidemiology

Highly variable as
Ethnics and geographic data
Folic acid supplement programme
Prenatal screening programme
Screening Maternal AFP with
folic supplement led to
decreased prevalence of
NTDs

 The Unites States < By 2006>

3: 10000
Englands and Wales < 1970-1997>
1: 10000
Higher in Young age, Hispanic and
White
The Highest in the United
kingdom China and Egypt

Etiology and Risk Factor

Multifactorial**

Environmental
Factors

Genetic
Factors**

Environmental Factors

Inadequate Folate*** : the nucleic formation

was disturbed then the cell turnover during th
e neural tube closure was blocked.
Drugs** : Disturb the folic acid metabolism :
valproate* and carbamazepine* , coumadin* ,t
halidomide* and efavirenz*

Insulin dependent DM : induce hypoxia

and high oxidative stress then disturb the sign

aling pathways led to embryopathy and apopt
osis of neural tube.

Environmental Factors

Hyperthermia: hot tub , sauna

Amniotic bands : physically disrupt the
normal tube development
Obesity
GeographicalUnited Kingdom, India,
China, Egypt, Mexico, Southern Appalachian
United States

Genetic Factors

Polymorphic variants in genes in the folate

pathway
MTHFR polymorphism : Genetic variant 677C
to T which is the thermolabile enzyme which
decrease the enzyme activity therefore the
homocysteine levels would be higher and th
e DNA synthesis will be incomplete.

Genetic Factors

Aneuploidy**trisomy 13 and 18, triploidy

Syndromes with autosomal recessive
disorder
Meckel Gruber, Roberts, Joubert,
JarchoLevin, HARDE (hydrocephalus-agyri
a-retinal dysplasia-encephalocele)

 95 percent of NTDs develop in the absence

of a family history.
Recurrence risk*****
3-5 percents if a couple has a previously
had a child with either anencephaly or spi
na bifida
10 percents if a couple has two affected
children.
5 percents if either parents was born with
NTDs

NTDs Screening

Candidates : All should be screened since

90-95 percent of cases occur in low risk
pregnancy , especially the one with risk fact
ors< previously affected fetus , positive fami
ly history , exposure to the associated medic
ation , GDM and obesity

NTDs Screening

Screening Test:
Ultrasonography < Primary screening
tool>
Maternal serum AFP < MSAFP>
Amniotic fluid AFP < AFAFP>
Fetal blood AFP

Ultrasound Examination

Primary screening test for NTD

Potentially detect NTDs more than
MSAFP
18-20 wks of GA to perform the screening
since the second trimester ultrasound increa
se the detection of spina bifida 92-95 percen
t and anencephaly to 100*** percent

second-trimester fetuses with open spina bifida

Frontal bone scallopingthe lemon sign****

Anterior curvature of the cerebellum with
effacement of the cisterna magnathe bana
na sign****

The lemon sign

Transventricular
plane
Frontal bone
scallopingthe lem
on sign with
ventriculomegaly.

The banana sign

******
Transcerebellar plane
Image of the fetal
head at the level of
the posterior fossa,
Anterior curvature of
the cerebellum with
effacement of the cis
terna magnathe ba
nana sign.

Sonographic Findings in
NTDs

AFP

Synthesized by fetal yolk sac and later

by GI tract and liver
Function: immunoregulation and
intravascular transport protein : analogo
us of albumin
normal concentration gradient between
fetal plasma and maternal serum 50,000
:1.
neural-tube and ventral wall defects,
permit AFP to leak into the amnionic flui
d, resulting in increased maternal serum
AFP levels

AFP

Maternal serum AFP Peaks at 28-32 wks

inconsequence of the high fetoplacental per
meability
AFAFP < Amniotic fluid AFP > secreted from
kidney to AF and peaks at GA 12-14 wks and
declines until undetectable level at term
Fetal plasma AFP peaks at 10-13 wks but no
clinical roles

MSAFP testing

MSAFP could be used at GA of 15 -20 wks

Result was expressed as multiple of median
for each gestational age which can be
compared with different lab and population
s
The value above 2-2.5 of MOMs ,
designated for abnormal result.
If the value elevation persist after a
repetition then a specialised ultrasound exa
mination is needed for assessment

MSAFP

2009 meta-analysis
The positive predictive value is only 2 to 6
percent
Sensitivity :
90 percents in anencephaly
80 percent for spina bifida

 The positive predictive value of MSAFP level

between 2.5-2.9 MOMs for NTDs is 1.45 perc
ents
MSAFP level greater than 7 MOMs ,the
positive predictive value of for NTDs is 13.5
percents

 false-positive rate of up to 5 percent (black

hatched area)
false-negative rates of up to 20 percent for
spina bifida (tan hatched area) and 10
percent for anencephaly (red hatched area).

Factors increasing MSAFP***

GA**: increasing by 15 percents per week
during second trimester , if BPD differs
more than 1 week comparing to the stated
age then recalculated MSAFP must be don
e.
Maternal weight **: vary in each women in
term of volume distribution
Diabetes mellitus**: lower the threshold to
1.5 MOMs

Factors increasing MSAFP

Fetal anomalies** : abdominal wall defect,
89 % in omphalocele and 100% in gastrosc
hisis
Multiple gestation** : the concentration is
proportional to the number of fetuses : the
upper limit for twins pregnancy is 4-5 MOM
s
Race: Black women

Factors increasing MSAFP

Fetal viability : fetal death** raise the MSAFP

Preeclampsia **
Fetal-growth restriction**
Maternal hepatoma or teratoma

Fetal anomalies increasing AFP***

Gastroschisis, Omphalocele **
Cystic hygroma
Esophageal or intestinal obstruction**
Liver necrosis **
Renal anomaliespolycystic kidneys, renal
agenesis, congenital nephrosis, urinary
tract**
Sacrococcygeal teratoma**

Fetal anomalies increasing AFP

Pilonidal cyst**
Chorioangioma of placenta **
Placenta intervillous thrombosis
Placental abruption
Oligohydramnios

AFAFP and AFAChE

AFAFP : Amniotic fluid AFP

AFAChE : Amniotic fluid acetylcholine
esterase
A primary biochemical test performed to
diagnose open NTDs when the MSAFP is posi
tive and ultrasound is equivocal or normal.

 An elevation of both AFP and AChE values in

amniotic fluid suggest an open fetal NTDs
with 98 percents accuracy and a false positi
ve rate of 0.4 percents
Fetal genetic study if amniocentesis is
performed .

Fetal MRI

Ultrasounds with limitations including the

reverberation causing suboptimal visualizati
on of the brain , maternal obesity and oligoh
ydramnios can hamper the accurate diagnos
is
Further studies are needed to evaluate the
diagnostic capability of MRI ,considered whe
n the ultrasound is not optimal

Prenatal diagnostic study

Targeted Ultrasound imaging

If ultrasound is equivocal, AFAFP and
AFAChE is performed as the diagnostic test
Amniocentesis for genetic study and AFAFP
and AFAChE

Additional Diagnostic study

Ultrasound Survey the other malformation

which is associated with NTD namely oral
cleft ,musculoskeletal renal cardiovascular w
ere the most commonly found as the associa
ted anomaly.
6.5 percents of NTD with anomalies
associated with chromosome and genetic ab
normality
< Trisomy 18 is the most common>***

Unexplained MSAFP Elevation

Normal specialized sonographic evaluation,

with or without amniocentesis
Increased risk for various adverse pregnancy
outcomes. < Not used as a screening test >
fetal-growth restriction
Oligohydramnios
placental abruption
preterm membrane rupture
preterm birth
fetal death.

Pregnancy management

ACOG recommends the route of delivery for the

fetus with spina bifida should be individualized.
cesarean section to reduce the chance of
mechanical trauma and spinal infection
Fetal surgery to improve the motor outcomes
but increasing the maternal and fetal risk.
Pregnancy termination
In 83 percents of anenchephaly
In 63 percents of spina bifida

Prevention
0.4 mg of folic acid orally every day before
conception and through the first trimester, to redu
ce the NTD risk by 80 percents in low risk women*
***
4 mg folic acid taken daily

1 month before ******conception and through

the first trimester in women increased risk for NTD
s
Increased risk for NTDs:
one or more prior affected children
either parents has such a defect

 Folic acid supplementation may not

decrease the risk for NTDs

valproic acid exposure

pregestational diabetes
first-trimester fever or hot tub exposure
defects associated with a genetic syndrome

Prenatal diagnosis of Down

Syndrome and other aneuploidies

Introduction
Down syndrome is the most common
chromosome abnormality
Moderate to severe learning disability, heart
defect, intestinal malformation, vision and hearin
g loss
High financial and psychological support to the
affected family
Aneuploid conceptuses
50 percent of first-trimester abortions
5 to 7 percent of all stillbirths and neonatal
deaths.

Prenatal diagnosis and screening test of aneuploidies.

Screening test<
noninvasive>
Diagnostic test
First trimester

Ultrasound < nuchal

translucency and CRL>
Maternal serum marker
< BhcG, PAPP-A>
Integrated test < Full>
Sequential test
Contingent test

Second trimester

Triple test
Quad test
Maternal serum free cell fetal
DNA

Invasive procedure

Chorionic villi sampling

Amniocentesis
Fetal blood sampling

Preimplantation
diagnostic testing

Candidates for invasive prenatal diagnosis

Maternal age : risk of having down
syndrome and any aneuploidies increased w
ith age from 35 years to 40 years by four fol
ds comparing to the young age and 10 fold i
ncreased risk at the age of 40- 45 .
BUT 70 percent of Down
syndrome pregnancies are in
women younger than 35 years.

Candidates for prenatal diagnosis

Dizygotic twins and maternal age older than

31 years at delivery
Previous autosomal trisomy , triploidy or
other chromosome abnormality birth
Patient or partner is a carrier of
chromosome translocation or inversion
Some cases of repetitive early pregnancy
loss
Major structural defect by sonography

First trimester Screening

Most common used protocol

GA 11-14wks
Nuchal translucency alone
Combined test involoves
Nuchal translucency and gestational age
combines with serum marker PAPP-A and fr
ee or total BhCG
< double test>

Nuchal translucency

This is the maximum thickness of the

subcutaneous translucent area between the
skin and soft tissue overlying the fetal spine
at the back of the neck
It is measured in the sagittal plane, when
the crown-rump length measures between
38 and 84 mm.

Nuchal translucency

 The NT measurement is expressed as a

multiple of the gestational age specific me
dian.
NT 3.5 mm , offered targeted
sonography, in addition to fetal karyotypin
g
One third of fetuses with increased nuchal
translucency thickness will have a chromo
some abnormality

 isolated marker, NT detects 70 percent of

fetuses with Down syndrome at a false-positi
ve rate of 5 percent
maximal sensitivity at 11 weeks
NT is used as an isolated marker in
multifetal gestations as serum screening is n
ot as accurate or not available.

Serum analytes in first trimester

free -hCG and PAPP-A <pregnancyassociated plasma protein A >
fetal Down syndrome :
free -hCG level 2.0 MoM
PAPP-A level 0.5 MoM.

With trisomy 18 and trisomy 13, levels of

both analytes are lower

Combined first trimester testing

NT measurement with serum hCG and PAPPA.

Down syndrome detection rates is 79 to 87
percent, a false-positive rate of 5 percent
trisomies 18 and 13 detection rates is 90
percent, 2-percent false-positive rate

Full Integrated test

Ultrasound measurement of nuchal

translucency and serum analytes at the age
of 10 -13 then
At GA of 15-18 wks AFP,uE3, hCG, and inhibin
A
Detection rate 85-95 percents
Result withheld until quad test completed
Lowest false positive test in down syndrome

Sequential and contingent testing

The disadvantages of integrated test is

patients have to wait until the second trimes
ter to perform the screening test thereby th
e sequential and contingent testing develop
s.

Sequential testing

Performing at first trimester

first trimester screening test and then quad
test :
1 % offer diagnostic test after screening
99% procede to quad test ,result withheld
until quad test completed
Using first trimester screening and then
offering CVS only to the high risk one.
If not a very high risk ,go on to the quad test

Contingent testing

First trimester screen and quad test

Three risk cut-offs
1. women at a very high risk< 1%>, > 1 in 50 ,
after 1 st trimester testing , CVS should be perfor
med.
2. women at a very low risk< 84%> ,< 1 in 2000 ,
no additional testing after the first trimester
screening
3. women at intermediate risk < 15%>, > 1 in
2000, but < 1 in 50, receiving second trimester te
st.

Quadruplet test

Best available screening test for women who

present for prenatal care in second trimester
GA 15-18 wks
Maternal serum for AFP uE3 hCG and inhibin
A

Abnormal Serum analyte in Down syndromes

Serum analyte

Result

1.Total or Free BhCG

2. PAPPA
3. AFP
4. Unconjugated estriol
5. Inhibin alpha

2
0.5
0.74
0.8
1.7

Cell free fetal DNA in maternal blood

Next generation genomic sequencing

99.5% detection of trisomy 13 18 and 21
and possibly sex aneuploid
False positive rate of 0.5% or less
Perform 12-20 wks of gestation.

Free cell fetal DNA in maternal serum

Candidates for using cell free fetal DNA

testing as a primary screening in singleton
maternal age of 35 years and older
Presence of sonographic finding
associated with fetal aneuploidy
History of previous pregnancy with fetal
trisomy
Screening positive test
Patients carry a balanced robertsonian
translocation

Free cell fetal DNA in maternal serum

Limitation : uninformative test, expensive
High sensitivity and specificity : selecting
the one who will be beneficial for the
invasive prenatal diagnosis

Soft sign

Soft signs: Normal variants

Used in 15-22 weeks of gestation
Risk increase steeply with the number of
markers identified.

Sonographic markers

Likelihood ratio of markers in down syndrome

 10 percents will have this soft signs in

unaffected pregnancy
ACOG recommends the risk adjustment
based on second trimester sonographic mar
kers is concerned because more fetal losses
would result than case of of down syndrome
identified.

Bernaceraf scoring index

Minor sonographic finding that associated with Down syndrome

1. nuchal skinfold thickening

2. echogenic intracardiac focus
3. mild renal pelvis dilatation< pyelectasis>
4.echogenic bowel
5.clinodactyly
6. sandal gap

Minor sonographic finding that associated with Down syndrome

 Nuchal skinfold
Transcerebellar view of fetal head from
the outer edge of skull to the outer skin
A measurement of 6 and greater is
considered abnormal.
More than 10 fold risk of down syndrome
Amniocentesis should be performed even
found as an isolated finding.

Nuchal skinfold

Echogenic intracardiac focus

Echogenic intracardiac focus < EIF> is focal

intracardiac papillary calcification
Not a cardiac abnormality
Usually left sided
Double the risk of down syndrome if found
isolated
Common in trisomy -13

Echogenic intracardiac focus

Pyelectasis

Transient or physiological finding in normal

fetus
Measured on transverse image of the pelvis
4mm or greater is found about 2 percents of
of fetuses and double the risk of down
syndrome
Likelyhood of having renal abnormality and
further additional testing should perform at
GA34wks

Pyelectasis

Echogenic fetal bowel

Six fold increased risk of down syndrome

Appears as bright as bones
Represents :
Small amounts of swallowed blood
Fetal CMV infection
Cystic fibrosis in fetus

Echogenic fetal bowel

Femur and Humeral length

Short femur and humerus

Short femur: 90 percents of expected
which correlated with the BPD measurement
Short humerus: 89 percents of expected
which correlated with the BPD measurement

Clinodactyly, Hypoplasia of the fifth

finger middle phalanx

Sandal Gap

First trimester sonographic finding in Down syndrome

Absent of fetal nasal bone

Nuchal Translucency
Wider frontomaxillary facial angle : flat
facial profile
Tricuspid Regurgitation
Abnormal ductus venosus flow
also associated with trisomy 18 and 13
Not routinely used in the US

Fetal nasal bone

Two thirds of fetuses with down syndrome,

the nasal bone is not visible in GA of 11-14
wks
adequate assessment : 45-degree angle of
insonation with the fetal profile; in the
midsagittal plane, with the tip of the nose an
d the third and fourth ventricles visible; and
that the nasal bone brightness be greater th
an or equal to that of the overlying skin

Fetal nasal bone

Prenatal and preimplantation

diagnosis

Prenatal and preimplantation diagnosis

Invasive procedure
Chorionic villi sampling
Amniocentesis
Fetal blood sampling
Preimplantation diagnostic

Amniocentesis

Transabdominal withdrawal of amniotic fluid

Most common prodedure
Used to diagnose the fetal genetic
conditions
GA 15-20 wks to be performed
Assess fetal karyotype
Takes 7 to 10 days to culture the amniocyte
and the assessment of the karyotype

Technique

Sonographic guidance
Using 20 to 22 gauze spinal needle
Initially 1 -2 ml of fluid is contaminated
20 ml of AF was collected
After removing of the needle , puncture site
is observed to check bleeding and fetal
cardiac motion is documented
If maternal Rh-D negative ,RhoGram is given
after the procudure

Typical volume required for selected test.

 AF should be clear
If blood tinged fluid is detected ,
transplacental passage is suspected but not
associated with pregnancy loss
In Multifetal pregnancy : indigo carmine dye
is often injected before removing the first ne
edle as the second sac entrance with clear fl
uid would verify the needle in the second sa
c.

complication

Pregnancy loss : 1 in 300-500

Double the pregnancy loss if BMI 40 kg/m2
Twins : 1.8 percents
Amniocentesis leakage < usually within 48
hrs after the procedure>
Chorioamnionitis

Early amniocentesis

Performing at the age of 11 -14 wks

Less fluid is withdrawn : I ml per each GA
Higher rate of complication : AF leakage,
talipes equinovarus ****and fetal loss
ACOG not recommend

CVS

Between 10 -13 wks

Karyotype and specialized genetic test
Results are earlier ,allowing safer pregnancy
termination
Karyotype available in 7 -10 days

CVS Technique

Transabdominal
Transcervical, both are equally safe
Transcervical villus sampling is performed
using a specifically designed catheter made
from flexible polyethylene that contains a
blunt-tipped, malleable stylet.

CVS Technique

Transabdominal sampling is performed

using an 18- or 20-gauge spinal needle.
With either technique, transabdominal
sonography is used to guide the catheter or
needle into the early placenta chorion fron
dosum, followed by aspiration of villi into a s
yringe conta

CVS Technique

 Relative contraindications:
vaginal bleeding or spotting
active genital tract infection,
extreme uterine ante- or retroflexion
. If the patient is Rh D-negative and
unsensitized, anti-D immune globulin is ad
ministered following the procedure

 fetal loss rate following CVS was 2 percent

compared with less than 1 percent
following amniocentesis.
An early CVS was its association with limbreduction**** defects and oromandibular
limb hypogenesis performed at 7 weeks g
estation
When performed at 10 weeks gestation,
as is commonly done today, the incidence
of limb defects does not exceed the backg
round rate of 1 per 1000

 Vaginal spotting is common following

transcervical sampling, but it is self-limited
and not associated with pregnancy loss.
The incidence of infection is less than 0.5
percent

limitation of CVS

chromosomal mosaicism is identified in up to 2

percent of specimens.
In most cases, the mosaicism reflects confined
placental mosaicism rather than a true second
cell line within the fetus.
Amniocentesis should be offered, and if the
result is normal, the mosaicism is presumed to
be confined to the placenta.
Confined placental mosaicism has been
associated with fetal-growth impairment and
stillbirth.

Fetal Blood Sampling

cordocentesis or percutaneous umbilical

blood sampling (PUBS).
It was initially described for fetal transfusion
of red blood cells in the setting of anemia
from alloimmunization
fetal anemia assessment remains the most
common indication.

Fetal Blood Sampling

Fetal blood sampling is also performed for

fetal karyotype determination, particularly in
cases of mosaicism identified following
amniocentesis or CVS.
Fetal blood karyotyping can be
accomplished within 24 to 48 hours.
Although fetal blood can be analyzed for
virtually any test performed on neonatal
blood

Fetal Blood Sampling Technique

Under direct sonographic guidance,

using a 22- or 23-gauge spinal needle into
the umbilical vein, and blood is slowly
withdrawn into a heparinized syringe.
Adequate visualization of the needle is
essential.
Fetal blood sampling is often performed
near the placental

Fetal Blood Sampling Technique

cord insertion site, where it may be easier to

enter the cord if the placenta is anterior
(Fig. 14-9). Alternatively, a free loop of cord
may be punctured.
a local anesthetic may be administered.
Prophylactic antibiotics are used at some
centers, although there are no trials to supp
ort this policy.

Fetal Blood Sampling Technique

Arterial puncture is avoided, because it may

result in vasospasm and fetal bradycardia.
After the needle is removed, fetal cardiac
motion is documented, and the site is obser
ved for bleeding.

Fetal Blood Sampling Technique

 fetal loss rate is approximately 1.4 percent

Other complications
cord vessel bleeding in 20 to 30 percent
of cases,
fetal-maternal bleeding in 40 percent of
cases in which the placenta is traversed
fetal bradycardia in 5 to 10 percent
Most complications are transitory, with
complete recovery, but some result in fetal

Preimplantation Genetic Testing

Genetic testing performed on oocytes or

embryos before implantation ,in vitro
fertilization (IVF), may provide valuable infor
mation regarding the chromosomal comple
ment and single-gene disorders

Preimplantation Genetic Testing

Preimplantation genetic diagnosis
Preimplantation genetic screening
1. polar body analysis
2. blastomere biopsy
3. trophectoderm biopsy

Polar body analysis

Maternally inherited genetic disorder.

The first and second polar bodies are
extruded from the developing oocyte.
Sampling should not affect fetal
development
Disadvantages : paternal genetic
contribution is not evaluated.

Blastomere biopsy

Done at the 6- to 8-cell (cleavage) 3 days

old, then one cell is removed.
limitation : mosaicism of the blastomeres
may not reflect the chromosomal complemen
t of the developing embryo
The technique is associated with a 10percent reduction in the pregnancy rate

Blastomere biopsy

Trophectoderm biopsy
5 to 7 cells from a 5- to 6-day blastocyst
Advantage :no embryal cells are removed
as trophectoderm cells give rise to the tro
phoblast .
Disadvantageously :performed later in
development

Trophectoderm biopsy

Preimplantation Genetic diagnosis

PCR amplified genome region
FISH : chromosome rearrangement
CVS or AC should be done to confirm the
PGD