Lecture on prenatal genetic diagnostic techniques and their value in detection of prenatal genetic anomalies. This lecture details techniques employed in the common diagnostic interventions used in prenatal period and their usefulness.

1. Prenatal Genetic diagnosis
Dr. Yogesh D
Department of Anatomy
Basaveshwara Medical College,
Chitradurga, Karnataka

2. Introduction
The problem:
 Approximately 3% of all infants are born with
genetic disorders or congenital anomalies that
lead to mental or physical handicaps or early
death.

3. Introduction
• What is Prenatal genetic diagnosis?
 Prenatal genetic diagnosis involves maternal
serum screening for biochemical markers of
genetic abnormalities in the foetus,
Ultrasonographic imaging and invasive
techniques like amniocentesis for cytogenetic
analysis for a confirmatory genetic diagnosis.

4. Introduction
• What are the available tests?
Genetic screening tests:
• Biochemical markers in maternal serum and
• Non-invasive imaging studies.
Confirmatory genetic tests:
• Chorionic villus sampling,
• Amniocentesis and
• Cordocentesis.

5. Introduction
• What is the purpose of screening tests?
 Screening tests guide us in deciding who needs
an invasive procedure to obtain the tissue
sample so that a cytogenetic test can be
performed for a definitive genetic diagnosis.

6. Introduction
Impact of Prenatal genetic diagnosis:
 Prenatal diagnostic tests have made it possible
for the neonatologists to know many details
about their patients before they have even laid
their hands on them.
 Prenatal detection has allowed for termination
of pregnancies with severe genetic disorders.
 This however is not true of all the neonatal
caregivers particularly in India.

7. Biochemical markers in maternal
serum
What are they?
 Alpha-fetoprotein (AFP),
 Human chorionic gonadotropin (hCG),
 Unconjugated estriol (uE3) and
 Inhibin-A .

8.  This screening (Quadruple screening) is
routinely employed in much of Europe, USA
and Canada and other developed countries.
 India has much to catch up to….
Biochemical markers in maternal
serum

9. Alpha Fetoprotein (AFP):
 Is one of the major proteins in foetal serum.
 It can be detected as early as 4 weeks of
gestation when it is synthesized by yolk sac
initially.
 AFP in maternal serum is exclusively foetal in
origin and.
Biochemical markers in maternal
serum

10. • An increase in AFP is seen in
 Anencephaly
 An open spina bifida
 Turner’s syndrome
 Congenital nephrosis
 Anterior abdominal wall defects
 Cystic hygroma
 Renal agenesis
 Acardia
Biochemical markers in maternal
serum

11. • A decrease in AFP is seen in
 Trisomy 21
 Trisomy 18
 Intrauterine growth restriction
 A normal AFP value does not rule out
trisomy 21 as low AFP only picks up about
one third of foetuses with Down syndrome.
Biochemical markers in maternal
serum

12. • Unconjugated estriol (uE3).
 It is synthesised in foetal adrenal gland
and liver after which it undergoes
modification in the placenta before
crossing over to maternal circulation.
 Lower than normal values of uE3 suggest
trisomy 21, 13 or 18.
 uE3 has a detection rate of 25%,
Biochemical markers in maternal
serum

13.  hCG and inhibin- A.
 hCG
o Increases the sensitivity as well as specificity.
o hCG is produced by the placenta and is readily
testable in maternal serum.
o hCG levels have the best detection rates of 40%.
o hCG is elevated in Down Syndrome and
decreased in trisomy 18.
Biochemical markers in maternal
serum

14.  Inhibin-A
o Inhibin is produced by mainly by placenta and
belongs to transforming growth factor-β
superfamily.
o Addition of inhibin to the panel increases the
combined detection rates by about another 7-8%.
o Inhibin-A is elevated in trisomy 18 where as all the
other markers are reduced greatly.
o Inhibin-A is also elevated in trisomy 21 along with
hCG.
Biochemical markers in maternal
serum

15.  These serum markers have minimal
correlation between them hence do add to
the sensitivity as well as specificity of the
test.
 Pregnancies affected by trisomy 13 and 18
have will show a decrease in both uE3 as
well as βhCG, whereas trisomy 21 will show
an elevated βhCG with decreased AFP and
uE3.
Biochemical markers in maternal
serum

16. Ultrasonographic examination of the
foetus.
 Ultrasonographic evaluation is the mainstay of
detecting congenital anomalies in India.
 It is estimated that, up to 2-3% of live births
have some congenital anomaly.
Non invasive imaging techniques

17.  Ultrasonography is particularly useful in
anencephaly, microcephaly, encephalocele, and
hydrocephalus.
 By 20 weeks facial features can be evaluated for
cyclopia, cleft lip, micrognathia etc.,
Non invasive imaging techniques

18.  Routine ultrasound scanning has a detection
rate of 35% for malformations.
 Nuchal membrane thickening is suggestive of
Down syndrome.
 Ultrasound is also very useful for guidance
while performing procedures such as
amniocentesis, cordocentesis and chorionic
villus sampling to obtain samples for
cytogenetic evaluation.
Non invasive imaging techniques

19. Non invasive imaging techniques
Nuchal Translucency Measurement
 Ultrasound visualisation and quantification of
fluid filled space in the back of the neck is called
Nuchal translucency measurement.
 An increased nuchal translucency (NT)
thickness is associated with a higher risk of
foetal trisomy 21.

20. Non invasive imaging techniques
 Absence of nasal bone:
This is an independent finding associated with
Down Syndrome and is seen in up to 73% of
trisomy 21 cases.
Combining Nuchal translucency measurement
along with nasal bone will increase sensitivity
and specificity for detection of Down syndrome.

21. Invasive (confirmatory) tests
Amniocentesis:
What is it?
 Amniocentesis is removal of up to 20 ml of the
amniotic fluid from the pregnant uterus using a
22G spinal needle.

22. Invasive (confirmatory) tests
 The cells present in the amniotic fluid may not
yield enough substrate for cytogenetic assays
hence requiring cell cultures and thus
increasing the time taken for completion of the
cytogenetic tests.
 Consequently, the amniotic fluid itself can be
utilised to perform biochemical tests like AFP,
acetylcholinesterase, bilirubin, lecithin,
sphingomyelin or phosphatidylcholine.

23. Invasive (confirmatory) tests
 Indications for amniocentesis.
 Maternal age of 35 years or more.
 Previous pregnancy with chromosomal anomalies.
 Parent with balanced chromosomal translocations.
 Abnormal maternal serum screen results.
 Family history of neural tube defects.
 Maternal history of X-linked disorder.
 Family history of genetic disorder for which DNA
diagnosis is available.

24. Invasive (confirmatory) tests
 Advantages and disadvantages:
 Diagnostic accuracy rate exceeds 99%.
 Early (between 12 and 15 weeks)
amniocentesis is not recommended due to
increased risk of club foot and a slightly higher
foetal loss.

25. Invasive (confirmatory) tests
Chorionic Villus Sampling (CVS):
What is CVS?
 This is a procedure where a sample of the
chorion frondosum is aspirated between 10-11
weeks of gestation.
 It offers the advantage of early diagnosis-
before the mother has felt the quickening of the
baby- thus helping in decision making easier.

26. Invasive (confirmatory) tests
 Since the mesenchymal core cells are actively
dividing, the time required for cell culture is
reduced which means the results of the
cytogenetic tests will be available within 1
week’s time.
 Due to mosaicism a separate cytogenetic
analysis of both cultured preparations as well as
direct specimen is recommended.
 Up to 0.8% to 1.7% of the cases show
chromosomal anomalies in villus that are not
present in the foetus

27. Invasive (confirmatory) tests
 How is it performed?
 In transcervical method a flexible catheter is
passed through the endocervix, under
ultrasound guidance and a sample of chorion
frondosum is aspirated into a sterile tissue
culture medium and the catheter is withdrawn.

28. Invasive (confirmatory) tests

29. Invasive (confirmatory) tests
 In transabdominal method, a sterile spinal
needle is inserted under ultrasound guidance
and villi are aspirated into the tissue culture
medium.
 The villi are dissected from maternal decidua
and processed for tissue culture or DNA
extraction.

30. Invasive (confirmatory) tests
 What are the complications of CVS?
 Increased risk of limb anomalies.
 Transcervical method increases the risk of
serious maternal and foetal infections.
 Both methods can slightly increase the risk of
abortion.

31. Invasive (confirmatory) tests

32. Invasive (confirmatory) tests
 Cordocentesis:
 What is it?
 It is a procedure in which a sample of foetal
blood is aspirated from the umbilical cord.

33. Invasive (confirmatory) tests
 How is it done?
 Under continuous ultrasonographic imaging, the
insertion site of the umbilical cord into the placenta
is identified.
 The umbilical vein is punctured with a 20-gauge
needle, the sample is withdrawn, and the umbilical
cord is observed for signs of haemorrhage.
 The lymphocytes are a source of cells for a rapid
karyotype.

34. Invasive (confirmatory) tests
 This is helpful in two situations:
when anomalies have been noted on
Ultrasonographic examination but it is too
late in gestation to perform an amniocentesis
(antenatal diagnosis of trisomy 13 or 18
influences delivery room management).
for confirmation of a foetal karyotype when
amniocentesis or CVS has shown mosaicism

35. Invasive (confirmatory) tests
 What are the complications?
 Foetal bradycardia.
 Foeto-maternal haemorrhage.

36. Preimplantation genetic diagnosis
• What is it?
– The DNA analysis of the cells during or after in-
vitro fertilization and or before blastomere
transfer is known as pre implantation genetic
diagnosis.
• In a few among the centres that offer
assisted/artificial reproductive technologies it has
been possible to analyse the chromosomal
constitution and DNA from the first polar body,
blastomere or a blastocyst.

37. Preimplantation genetic diagnosis
• The rational for doing so is to avoid implanting
embryos that harbour identifiable genetic
disorders.
• It is mostly performed to look for age-related
aneuploidy.

38. Preimplantation genetic diagnosis
• Other indications for PGD are,
– Couples who carry balanced translocations.
– Couples who are at risk for single gene
disorders such as cystic fibrosis.
• This is a particularly challenging technique
because DNA from a single cell needs to be
analysed.

39. Future in Prenatal genetic
diagnosis
 What can be studied in maternal serum?
 Pregnancy Associated Plasma Protein A,
 Free β subunit of hCG.
 Both these are abnormal in foetal trisomy 21.
 Initial studies indicate the specificity and
sensitivity of these are comparable in both 1st
and 2nd trimesters.

40. Future in Prenatal genetic
diagnosis
 Foetal cells in maternal blood.
 Foetal cells like trophoblasts and nucleated
erythrocytes very infrequently cross the
placenta.
 In a limited number of clinical trials, trisomy 21,
18, triploidy, Klinefelter’s syndrome (47,XXY),
Rehsus D genotype have been successfully
detected using the rare foetal cells circulating in
the maternal blood.

41. Future in Prenatal genetic
diagnosis
 Results of one multicentric study show up to
78% detection rates for aneuploidy.
 The major limiting factor seems to be the very
low numbers of foetal cells circulating in
maternal blood.

42. Future in Prenatal genetic
diagnosis
o Cell free foetal DNA in maternal blood:
o In recent years studies have shown that large
amounts of foetal cell free DNA circulating in
the maternal blood and serum.
o Qualitative detection of uniquely foetal DNA
has made it possible to detect Rhesus –D
genotype, myotonic dystrophy and
achondroplasia.
o It should be possible to detect Rh-D genotype
of foetus exclusively by this in near future.

43. References
• Bianchi DW: Chapter 18-Prenatal Genetic Diagnosis, Avery’s Diseases of the
Newborn, 8th ed.
• Stuart Schwartz : Chapter 7 – Genetic Aspects of Perinatal Disease and Prenatal
Diagnosis, Fanaroff and Martin's Neonatal-Perinatal Medicine, 8th ed.
• O'Connor, C. (2008) Prenatal screen detects fetal abnormalities. Nature
Education 1(1):106.
• O'Connor, C. (2008) Karyotyping for chromosomal abnormalities. Nature
Education 1(1):27
• Verdin et al, Ultrasonographic markers for chromosomal abnormalities in
women with a negative nuchal translucency and second trimester maternal
serum biochemistry. Ultrasound Obstet gynecol 2000; 16: 402-406.
• Rose NC, Mennuti MT: Maternal serum screening for neural tube defects and
fetal chromosome abnormalities, In Fetal Medicine [Special Issue]. West J Med
1993; 159:312-31 7)