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
↑β-HCGh (β-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 syndrome- trisomy 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- 1 st
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
• ↑β-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 • Stepwise sequential---95%

• NT----64-70% – 1st T +VE dxtic test offered
(CVS????)
• NT, βhCG, PAPP- A- 82-87%
– 1st T –ve 2nd T test offered-
2. 2nd Trimester – Final Risk assessment
• Triple Test- incorporating 1st and 2nd T results
– βhCG, MSAFP, Estriol---69% • Contingent sequential 88- 94
• Quad Test- – 1st T +ve offer Dxtic test
– Triple + Inhibin—81% – 1st T –veno further test
– 1st T intermidate offer 2nd T
3. 1st and 2nd T
test
• Integrated – Final- Risk Assessment
– NT, PAPP A, Quad test-94-96 incorporates 1st and 2nd results
• Serum integrated
– PAPP A+ Quad--- 85-88%
 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 age-
associated 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 cervical-
thoracic 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 obesity–BMI ≥ 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 withdrawn—approximately 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 15–20 weeksa

MSAFP MoM adjusted for Age, Wt, Ethnicity, GA, Type I DM

MSAFP < 2.0MoM MSAFP >2.0MoM

If not already done, standard sonographic

Normal screening result
evaluation is performed to verify GA and
to exclude twinsb or fetal demise, with
recalculation of AFP MoM as neededb

AFP value < 2.50 MoM AFP value ≥ 2.50 MoMb

Normal screening
Abnormal result.
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
rate→blunting 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)