Prenatal Diagnosis

Introduction
Prenatal diagnosis
is the science of identifying Malformations,
Disruptions, Chromosomal Abnormalities, and
other genetic syndromes in the fetus.
Its goal is to provide accurate information
regarding short- and long-term prognosis,
recurrence risk, and potential therapy and to
thereby improve counseling and optimize
outcomes
It encompasses routine screening tests for
aneuploidy and neural-tube defects,
invasive diagnostic tests such as chorionic villus
sampling and amniocentesis,

Prenatal Dx-ANEUPLOIDY
There are three main approaches:
(1) first-trimester screening, and
(2) second-trimester screening,
(3) sequential screening in both the first and second
trimester

Screening for Down syndrome and other

aneuploidies is more complex than screening for
NTDs in that it involves more than one analyte,
and because the risk of Down syndrome is age
specific.
Sensitivity for noninvasive detection of Down
syndrome is age dependent

Screening for Downs Syndrome (Trisomy 21) - 1st

Trimester

1st Trimester screening - b/n 10 and 13 weeks

1. Nuchal translucency (NT) measurement

the width of the translucent space at the back of the

fetal neck determined by ultrasound.

2. Combined test- NT and maternal serum

measurements:
NT
-HCGh (-hCG: free or total), and
pregnancy-associated plasma protein-A (PAPP-A),
together with maternal age.

The nuchal translucency (NT) measurement 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. Calipers are placed on the inner borders of the nuchal space, at its widest portion,
perpendicular to the long axis of the fetus. In this normal fetus at 12 weeks' gestation, the
measurement is 2.0 mm.

NT
-NT measured between 10 and 13weeks is a useful marker for
increased risk of chromosome abnormalities(Down syndrometrisomy 18 -Turner syndrome in descending order)
-Other Abnormalities are associated with large NT, even in fetuses
with normal karyotype
Eg. -Cardiac abnormalities
-Skeletal
-CNS malformations
-Diaphramatic Hernia
-Fetal demise
NT> 3.5mm-- increased risk of structural anomalies even in the
absence of aneuploidy. Such pts require targeted Us and Fetal
Echo)
2nd T NTD screening (MSAFP, US) should be offered to those who had
only 1st T aneuploidy screening who had need (ACOG, AAP-2012)

Dx of Aneuploidy- 1st Trimester

Chorionic Villous Sampling (CVS)
Invasive diagnostic test for abnormal serum marker results
Biopsy of chorionic villi is generally performed between 10 and
13 weeks gestation.
Purpose- Fetal karyotype , specialized genetic tests
The primary advantage of villus biopsy is that results are
available earlier in pregnancy, allowing safer pregnancy
termination, if desired.
A full karyotype is available in 7 to 10 days, and some
laboratories provide preliminary results within 48 hours.
Transcervical or transabdominal approaches are
consideredequally safe and effective (ACOG, 2012a; Jackson, 1992)

CVS- Complications
The procedure-related fetal loss rate is
comparable to that with amniocentesis ACOG, 2012a).
overall loss rate following CVS is approximately 2
% compared with <1% following amniocentesis
CVS has association with limb reduction defects
and oromandibular limb hypogenesis (Burton, 1992; Firth,
1991, 1994; Hsieh, 1995).

These were commonly seen with procedures

performed at 7 weeks gestation (Holmes, 1993)
. When performed at 10 weeks gestation, as is
commonly done today, the incidence of limb defects
does not exceed the background rate of 1 per 1000
(Evans, 2005; Kuliev, 1996).

1st T. screening Vs. multiple gestation

For MG, NT measurements alone may be the only

screening option for fetal aneuploidy.
Maternal serum marker screening is not as
accurate for twins and is unavailable for triplets.
The distribution of each individual NT
measurement in multiple gestations is similar to
that in a singleton pregnancy.
Thus, in dichorionic or trichorionic pregnancies, it
is possible to obtain an independent risk for each
fetus.
This makes NT the marker of choice for screening
in multiple pregnancies.

2nd Trimester screening for T21

Offered b/n 15-20 weeks

Triple test- based on measurement of
maternal serum level of
MSAFP,
unconjugated estriol (uE3), and
-hCG (free or total), together with maternal age.

Quadruple test (Quad test)

Triple Test + inhibin-A, together with maternal
age.

Downs Syndrome screening Tests and detection

rates- (ACOG, AAP perinatal Guidelines, 2012)
1. 1st Trimester
NT----64-70%
NT, hCG, PAPP- A- 82-87%
2. 2nd Trimester
Triple Test

hCG, MSAFP, Estriol---69%

Quad Test

Triple + Inhibin81%

3. 1st and 2nd T

Integrated

NT, PAPP A, Quad test-94-96

Serum integrated

PAPP A+ Quad--- 85-88%

Stepwise sequential---95%

1st T +VE dxtic test offered

(CVS????)
1st T ve 2nd T test offeredFinal Risk assessment
incorporating 1st and 2nd T
results

Contingent sequential 88- 94

1st T +ve offer Dxtic test

1st T veno further test
1st T intermidate offer 2nd T
test
Final- Risk Assessment
incorporates 1st and 2nd
results

2nd Trimester Screening for Trisomy 18

Trisomy 18 can be readily detected by maternal

serum analyte screening.
Decreased levels occur for AFP, uE3 and hCG.
NB This pattern differs from trisomy 21,in which
hCG
offer invasive prenatal diagnosis
(amniocentesis)whenever serum screening for
each of these three markers falls below certain
thresholds (MSAFP 0.6 MoM; hCG 0.55 MoM; uE3
0.5 MoM).
This detects 60 to 80 percent of trisomy 18
fetuses, with a 0.4 percent amniocentesis rate

Confounding Factors in 2nd T Screening

CF influence serum screening, and adjustments may be
necessary.
Adjustments for maternal weight and ethnic group are
routinely employed.
With increased maternal weight, decreased levels of
AFP, uE3, and hCG levels all occur.
Type 1DM is associated with decreased uE3 and hCG.
Maternal smoking increases MSAFP by 3 percent and
decreases maternal serum uE3, and hCG levels by 3
percent and 23 percent, respectively.
Maternal serum hCG is higher and MSAFP lower in
pregnancies conceived in vitro, compared with
pregnancies conceived spontaneously

MG Vs Aneuploidy
Since DZ twins carry independent risks of aneuploidy, the
chance of having at least one affected live-born twin at
term is twice the maternal age associated risk.
At a maternal age of 32, the age-associated risk of
aneuploidy is
1 in 481 singleton births.
1 in 240 DZ twins , which is equivalent to a 35-year-old
woman carrying a singleton.

For a woman carrying MZ twins, her risk of having at least

one affected live-born twin at term is the same as her ageassociated risk (i.e., 1 in 240 at 35 years of age).
Unfortunately, since her twins are MZ, this risk actually is
the risk of both fetuses being affected

Neural tube defects (NTD)

NTDs include anencephaly, spina bifida,
cephalocele, and other rare spinal fusion (schisis)
abnormalities.
Are the 2nd most common class of birth defect
after cardiac anomalies
Reported frequency of NTDs is app 0.9/1000
births (Cragan, 2009; Dolk, 2010).
NTDs are classic examples of multifactorial
inheritance.
Their development may be influenced by
hyperthermia, hyperglycemia, teratogen
exposure, ethnicity, family history, fetal gender,
and various genes.

NTDs
Selected risk factors are more strongly
associated with specific NTD location.
Hyperthermia has been associated with
anencephaly risk;
pregestational diabetes with cranial and cervicalthoracic defects; and
valproic acid exposure with lumbosacral defects
(Becerra, 1990; Hunter, 1984; Lindhout, 1992).

Screening for NTDs

Neural Tube Defect screening can include
Second-trimester MSAFP and/or
Ultrasound.

Both have sensitivity (detection rate) of 80% to 90% for

open NTDs (However PPV of AFP 2-6%)
MSAFP can be performed from 15 to 22 weeks GA
AFP value (MoM) adjusted for maternal age, weight,
ethnicity, gestational age, and insulin-dependent diabetes
An elevated MSAFP is indicative of an increased risk for a
NTD and other disorders .
A low MSAFP is indicative of an increased risk for Down
syndrome.
(MSAFP is on average 27% lower in women carrying fetuses
with Down syndrome-)
Risk of NTD in elevated MSFP ~ 1 in 50

Conditions Associated with AbnormalMSAFP Concentrations

Screening for NTDs

US~100% sensitivity in detecting NTD
Abnormalities of intra cranial anatomy in open
NTDs:
Ventriculomegally
The Lemon Sign Scalloped frontal bones
Banana Sign
Cisterna Magna Obliteration/effacement in the
anterior curvature of/with herniation of cerebral
Hemisphere

NTD Dxtic Algorithm

MSAFP Amniocentesis* ( to determine AF

AFP level)-AF AFP Acetylcholinestrase
assay- if this is positive NTD(98% sensitivity)

*Usually after US to confirm GA & fetal viablity, No of

fetuses + anatomic defects
although amniocentesis was once considered the "gold
standard" for diagnosis of open NTDs, in many centers it
has been replaced by, or used in conjunction with,
specialized sonography

contd.
Other fetal abnormalities may be associated
with AF AFP and + ve Achsterase assay Eg. ventral wall defects,
esophageal atresia,
fetal teratoma,
cloacal exstrophy,
skin abnormalities such as epidermolysis bullosa.

Amniocentesis
Transabdominal withdrawal of AF
the most common procedure for Dx of fetal aneuploidy
and other genetic conditions.
generally performed between 15 -20 weeks gestation
but may be performed later as well.
anterior placenta (half of all placenta locations)-60% of the
time traversed by the needle-not associated with preg loss

The indication is usually to assess fetal karyotype

use of FISH and array-based comparative genomic
hybridization studies have increased considerably
Because the amniocytes must be cultured ,the time
needed for karyotyping is 7 to 10 days

Amniocentesis- Complications
The procedure-related loss rate following midtrimester
amniocentesis is considered to be 1 per 300 to 500 (ACOG, 2012a).
The loss rate may be doubled in women with class 3 obesityBMI
40 kg/m2(Harper, 2012).
In twin pregnancies, Cahill and coworkers (2009) reported an
increased loss rate attributable to amniocentesis of 1.8 %.
Some losses are unrelated to the procedure and instead are due to
abnormal placental implantation or abruption, uterine
abnormalities,
fetal anomalies, or infection. Wenstrom and colleagues (1990)
analyzed 66 fetal deaths following nearly 12,000 2nd trimester
amniocenteses and found that 12 percent were caused by
preexisting intrauterine infection.
Other complications of amniocentesis include amnionic fluid
leakage in 1 to 2% and chorioamnionitis in < 0.1 % (ACOG, 2012a)
Fetal survival folloing leakage in 48 hrs > 90%(Borgida, 2000).

Selected Tests Performed on Amnionic

Fluid and Typical Volume of Fluid Required

aThe volume of fluid needed for each test may vary according to individual laboratory
specifications.
bFISH: Fluorescence in situ hybridization is typically performed
for chromosomes 21, 18, 13, X, and Y, or in the
case of a fetal conotruncal cardiac abnormality, for the
22q.1.1 microdeletion.
PCR = polymerase chain reaction.

Early Amniocentesis
Amniocentesis performed between 11 -14 weeks,
The technique is the same as for traditional amniocentesis,
Sac puncture may be more challenging due to lack of membrane
fusion to the uterine wall.
Also, less fluid is typically withdrawnapproximately 1 mL for
each gestational week (Shulman, 1994; Sundberg, 1997)
significantly higher rates of procedure-related complications than
other fetal procedures.
significantly Higher rates of amnionic fluid leakage, fetal loss, and
talipes equinovarus (clubfoot) than with traditional amniocentesis.
Compared with CVS Fourfold increased rate of talipes equinovarus
(Philip, 2004).
Anotherproblem of EA is that the higher cell culture failure rate
necessitating a second procedure.
Forall these reasons, ACOG (2012a) recommends against the use
of early amniocentesis.

MSAFP determined as part of multiple marker

screening at 1520 weeksa
MSAFP MoM adjusted for Age, Wt, Ethnicity, GA, Type I DM
MSAFP < 2.0MoM
Normal screening result

AFP value < 2.50 MoM

Normal screening
result

MSAFP >2.0MoM
If not already done, standard sonographic
evaluation is performed to verify GA and
to exclude twinsb or fetal demise, with
recalculation of AFP MoM as neededb
AFP value 2.50 MoMb

Abnormal result.
Patient counseled,
offered specialized
US ,consideration for
amniocentesis

Second-Trimester Screening in Multiple Gestations

Down syndrome occurs 20 % more often in twin

pregnancies than in singletons, given the known
+ve correlation between twinning and maternal
age
Down syndrome screening using multiple serum
markers is less sensitive in twin pregnancies than
in singleton
Using singleton cutoffs, 73 % of MZ twin
pregnancies but only 43% of DZ twin pregnancies
with Down syndrome are detected, given a 5%
false-positive rateblunting effect of one normal
and one aneuploid fetuses in DZ twin It may be
preferable to perform invasive procedures on
both fetuses/NT in 1st T

Effect of vanishing Twin

NB A vanishing twin may cause:
An elevated AFP level (MSAFP and amniotic fluid )
a positive AF acetylcholinesterase assay result .
A discrepancy between the karyotype established by
CVS and the karyotype of a surviving twin when tissue
from a vanished twin is inadvertently sampled
Therefore, the diagnosis of vanishing twin should be
excluded to avoid confusion during maternal serum
screening for Down syndrome or neural-tube defects .
For these reasons, amniocentesis for karyotype may
be preferable if a vanishing twin is suspected .

Components of Standard Ultrasound Examination (WILLIAMS

First Trimester
Gestational sac location=> intrauterine vs Ectopic
Embryo and/or yolk sac identification
CRL
Cardiac activity
Fetal number, including amnionicity and
chorionicity of multiples when possible
Assessment of embryonic/fetal anatomy
appropriate for the first trimester
Evaluation of the uterus, adnexa, and cul-de-sac
Assessment of the fetal nuchal region if possible

Components of Standard Ultrasound Examination by

(WILLIAMS)

Second and Third Trimester

Fetal number; multifetal gestations: amnionicity,
chorionicity, fetal sizes, amnionic fluid volume, and fetal
genitalia, if visualized
Presentation
Fetal cardiac activity
Placental location and its relationship to the internal
cervical os
Amnionic fluid volume
Gestational age
Fetal weight
Evaluation of the uterus, adnexa, and cervix
Fetal anatomical survey, including documentation of
technical limitations

Prenatal Dx of CAH- for those with risk

Amniotic fluid anlysis for elevated level of
17alpha hydroxyprogesterone or
11 B deoxycortisol in amniotic fluid.

Genetic diagnosis using specific probes and

cells obtained by CVS or amniocentesis also is
possible

Fetal lung maturity tests by amniocentecis

Lecithin/sphyngomyelin ratio > 2

Phosphotidylglycerol presence
Florescence polarization(S:A)
Surfactant: albumin> 55( 55mg S to 1mg A)

Optical Density(OD) 0.15

Lamellar body count 30-40,000

Fetal lung maturity

NB:
a mature FLM test result before 39 weeks of
gestation, in the absence of appropriate
clinical circumstances, is not an indication for
delivery.
RDS , IVH, NEC and other complications have
been reported in premature newborns
delivered with mature lecithin
(phosphatidylcholine)/sphingomyelin ratios or
the presence ofphosphatidylglycerol
(ACOG_2008)