At the end of this lecture, students

should be able to:

 Genetic basis of prenatal and pre-
implantation diagnosis.
 Indications of prenatal and pre-implantation
diagnosis.
 Different modalities used for prenatal and
pre-implantation diagnosis
 Introduction
 Congenital abnormalities account for 20-25% of
perinatal deaths.
 Now, many genetic and other disorders can be
diagnosed early in pregnancy.

Aim of Prenatal Diagnosis

 To provide an accurate diagnosis that will allow
the choice to those at increased risk of having
children with genetic disorders or with congenital
abnormalities.
 Indications of Prenatal Diagnosis
 The pregnant woman is 35 years or older at the
time of pregnancy.
 She or her parents have had a previous child with a
chromosomal abnormality.
 She has a history of recurrent abortions, or her
husband's previous wife suffered from several
miscarriages.
 A strong family history is present.
 The couple is known to be carriers of a
chromosomal translocation.
 The pregnant woman is affected with type 1
diabetes mellitus, epilepsy or myotonic dystrophy.
Indications of Prenatal Diagnosis

 She was exposed to viral infections, such as

rubella or cytomegalovirus.
 The mother is exposed to excessive
medication or to environmental hazards.
 History of Down syndrome or some other
chromosomal abnormalities are present in
her or her family.
 A history of single gene disorder is present
in her or her family.
Indications of Prenatal Diagnosis

 Her male relatives have Duchenne

muscular dystrophy or severe hemophilia.

 She is suspected of having some other

harmful gene on her X chromosomes.

 The fetus is diagnosed in utero to have

some hereditary error of metabolism.

 The fetus is detected to be at increased

risk for a NTD.
 Benefits of Prenatal Diagnosis
 Prenatal diagnosis determines the outcome of
pregnancy.
 It is helpful for couples to decide whether to continue
the pregnancy or not.
 It indicates possible complications that can arise at
birth process.
 Prenatal diagnosis is helpful for the management of
the remaining weeks of pregnancy.
 It prepares the couple for the birth of a child with an
abnormality if it was late.
 Prenatal diagnosis can be helpful for the improvement
of the outcome of pregnancy using fetal treatment.
Types of prenatal tests:
• Prenatal screening tests include:
> Ultrasound
> Early pregnancy (first trimester) screening: nuchal translucency
ultrasound together with testing of the mother’s blood
> Second trimester screening: testing the mother’s blood

• Prenatal screening tests should not be considered routine, but rather

offered as a choice to women.

• Prenatal diagnostic tests include:

> Ultrasound
> Chorionic villus sampling (CVS)
> Amniocentesis
• Prenatal diagnostic tests should not be considered routine, but rather
offered as a choice to women.
Diseases that can be screened by
prenatal diagnosis include:

• Neural tube defects (NTDs).

• Trisomy 21 (Down syndrome).
• Cystic fibrosis.
• Sickle cell anemia
• Thalassemia.
• Duchenne muscular dystrophy.
• Achondroplasia.
Techniques
 Noninvasive techniques:
 Fetal visualization
◦ Ultrasound
◦ Fetal echocardiography
◦ MRI
◦ Radiography
 Screening for neural tube defects (NTDs) – by
measuring maternal serum alpha-fetoprotein (MSAFP)
 Screening for fetal Down syndrome
◦ Measuring MSAFP
◦ Measuring maternal unconjugated estriol uE3.
◦ Measuring maternal serum β-human chorionic gonadotropin
(βhCG).
 Separation of fetal cells from the mother's blood
& examination
Invasive techniques
 Fetal visualization
◦ Embryoscopy
◦ Fetoscopy
 Fetal tissue sampling
◦ Amniocentesis.
◦ Chorionic villus sampling (CVS).
◦ Percutaneous umbilical blood sampling (PUBS).
◦ Percutaneous skin biopsy.
◦ Other organ biopsies, including muscle and
liver biopsy.
Cytogenetic investigations

◦ Detection of chromosomal abnormalities.

◦ Fluorescent in situ hybridization (FISH).

Molecular genetic techniques:
◦ Linkage analysis using microsatellite markers.

◦ Restriction fragment length polymorphisms

(RFLPs).

◦ Single nucleotide polymorphisms (SNPs):

 DNA chip.
 Dynamic allele-specific hybridization (DASH).
 Screening for Neural Tube
Defects (NTDs):
It is recommended if the following are
present:

 Ultrasound findings indicated NTDs.

 A child with NTDs is already present in the
family.
 A family history of NTDs exists.
 The mother has type 1 DM during pregnancy.
 Maternal exposure to drugs that are associated
with NTDs, such as valproic acid.
 Elevated level of MSAFP is present.
Measuring maternal serum
alpha-fetoprotein (MSAFP):
 The foetal liver begins to produce AFP
from the 6th week of gestation.
 The highest concentration of AFP in foetal
serum occurs in the mid-trimester, then
it falls progressively until term.
 AFP enters the amniotic fluid and then
the maternal serum via fetal urine.
 Amniotic fluid AFP increases steadily
during early pregnancy reaching
maximum levels at 13-14 weeks & then
decline.
  In an open NTD (eg, anencephaly, spina
bifida), AFP diffuses rapidly from exposed
fetal tissues into amniotic fluid and MSAFP
continues to rise and peaks in the third
trimester.
 So, abnormal amounts of AFP are present
in both amniotic fluid & maternal
serum.
 MSAFP is measured as a screening test for
NTDs and suspected cases are further
investigated by US.
 If NTDs is confirmed, termination of
pregnancy is offered.
 However, the MSAFP levels also increase with
twins, gestational diabetes, abdominal wall
defect, in association with intrauterine growth
retardation and renal anomalies.

The MSAFP test has the greatest sensitivity

between 16-18 weeks of gestation when 80%
of NTD-affected pregnancies can be identified.

 False negative results may be obtained with

closed NTDs where the lesion is covered with a
membrane.
 A combination of the MSAFP test and
ultrasonography detects almost all cases of
anencephaly and most cases of spina bifida.

 Also, a NTDs can be distinguished from other

fetal defects, such as abdominal wall defects,
by the use of an acetyl-cholinesterase test
carried out on amniotic fluid obtained by
amniocentesis.

 If the level of acetyl-cholinesterase rises along

with amniotic fluid AFP, it is suspected as a
condition of a NTD.
Screening for fetal trisomy 21;
Down syndrome:
 Incidence of trisomy 21 is 1 in 800 pregnancies.
 It can be identified by chromosome analysis of
cells obtained by amniocentesis in the mid-
trimester.
 Its incidence varies greatly with maternal age.
 So, Down syndrome can be detected by
amniocentesis in pregnant 35 years or more.
Screening for fetal trisomy 21
Down syndrome
Abnormalities in maternal serum analytes
with Down syndome:

 Decreased levels of MSAFP, pregnancy-

associated plasma protein A (PAPP-A) and
unconjugated estriol (uE3).

 Increased serum levels of total hCG, free

βhCG and inhibin A.
Graph for gestational age and hCGT
Screening for fetal Down syndrome:
 Measuring maternal serum AFP:
Low MSAFP level indicates the condition of Down
syndrome or other chromosomal aneuploidy and failing
pregnancies.
 Measuring maternal unconjugated estriol (uE3):
The amount of estriol in maternal serum depends upon
viable fetus, a properly functioning placenta, and on
maternal well-being. Fetal adrenal glands produce dehydro-
epiandrosterone (DHEA) that gets metabolized to estriol in
the placenta.
 Estriol crosses to the maternal circulation and is
excreted either by maternal kidney in urine or by
maternal liver in the bile.In the third trimester, the
level of estriol gives an indication for the well-being of
the fetus. A low level of estriol is an indication of Down
syndrome
 Screening for fetal Down
syndrome:
 Measuring maternal serum free βhCG:
◦ Following conception and implantation of the
embryo into the uterus, the trophoblasts produce
enough β-hCG, which is an indicator of pregnancy.

◦ In the middle to late second trimester, the level

of free β-hCG can be also used with MSAFP to
screen for chromosomal abnormalities.

◦ An increased free β-hCG level coupled with a

decreased MSAFP level suggests Down syndrome.
 Screening for fetal Down
syndrome:
 Protocols vary between centres but generally
involve the measurement of MSAFP and
either total hCG or free β-hCG.

 Programs achieve detection rates of about

60% for a false - positive rate of about 5%.

 Addition of measuring serum uE3 and inhibin

A improves the sensitivity and reduce the No. of
false positives for a given detection rate.
First trimester screening for Down
Syndrome:

 Screening may also be performed in the first trimester.

 Risks are calculated using a combination of maternal age,

biochemical measurements (as maternal serum free β–
hCG & PAPP-A) & US for measuring foetal nuchal
translucency thickness which is increased in trisomy
21 pregnancies.

• It can yield detection rates of better than 80% for a

false-positive rate of about 5%.

• Diagnosis is confirmed by amniocentesis and

chromosomal analysis of cells found in the amniotic fluid.
 Cystic fibrosis
 It is a serious disorder which has a poor prognosis.

 It is a genetically heterogeneous disease.

 Several hundred mutations of cystic fibrosis gene is

detected in families with the condition, but about 70% of
mutations involve the deletion of a single codon (so called
ΔF508).
Indications of screening for cystic fibrosis:

 When a couple has already had one affected child.

 On prospective parents from families in which the condition

occurs.
Screening can be performed on foetal tissue
obtained by chorionic biopsy:

•Such is capable of detecting up to 90% of

carriers.
•Positive results indicate that an individual was a
carrier.
•Negative results would not exclude the
possibility of carriage of a mutation that had not
been screened for.
Separation of fetal cells from the
mother's blood:

 Fetal blood cells make access to maternal

circulation through the placental villi.
 These cells can be collected safely from
approximately 18 weeks of gestation
onward, although by the successful
procedures, these cells can be collected at
12 weeks of gestation.
 The fetal cells can be sorted out and
analyzed by different techniques.
PCR
 Fetal blood cells can be analyzed for the
diagnosis of genetic disorders using molecular
genetic techniques by isolating DNA and
amplifying it by polymerase chain reaction
(PCR)
 Fetal cells separated from a mother's blood
have been successfully used in the diagnosis
of cystic fibrosis, sickle cell anemia and
thalassemia in a fetus.
 Fetal tissue sampling
Amniocentesis:
It is an invasive, well-established, safe, reliable and
accurate procedure performed between 14 - 20 weeks of
pregnancy.
Itis advised for pregnant women at 35 years or older for
detection of chromosomal abnormalities in the fetus.

Chorionic villus sampling at an early stage:

 The major advantage of CVS over amniocentesis is getting quick
results and its use in early pregnancy.
 Abnormalities can be identified at an early stage & more
acceptable decisions about termination of pregnancy can be
taken.
 Abortion is also much safer at this early stage.
 Percutaneous Umbilical Blood
Sampling:
 PUBS is also known as Cordocentesis.
 It is a method for fetal blood sampling and is
performed after 16 weeks' gestation.
 A needle is inserted into the umbilical cord under
US guidance and fetal blood is collected from the
umbilical vein for chromosome analysis and
genetic diagnosis.
 An advantage of PUBS is the rapid rate at which
lymphocytes grow, allowing genetic diagnosis.
 This technique is also useful for evaluating fetal
metabolism and hematologic abnormalities.
Percutaneous skin biopsy
 To prenatally diagnose a number of serious
skin disorders.

 Fetal skin biopsies are taken under

ultrasonic guidance between 17-20 weeks'
gestation.
 Other organ biopsies, including
liver and muscle biopsy
 Fetal liver biopsy is needed to diagnose an inborn
error of metabolism, as ornithine
transcarbamylase deficiency, glucose-6-
phosphatase deficiency, glycogen storage disease
type IA, non-ketotic hyperglycemia
andcarbamoyl-phosphate synthetase deficiency.
 Fetal liver biopsy also is best performed between
17-20 weeks' gestation under ultrasound
guidance.
 Fetal muscle biopsy is carried out under ultrasound
guidance at about 18 weeks' gestation to analyze
the muscle fibers histochemically for prenatal
diagnosis of Becker-Duchenne muscular dystrophy.
 Cytogenetic Investigations
 Detection of chromosomal aberrations
Chromosomal aberrations, such as
deletions, duplications, translocations and
inversions diagnosed in affected parents or
siblings, can be detected prenatally in a
fetus by chromosomal analysis.

 This analysis can be undertaken on fetal

cells obtained through such techniques as
amniocentesis and CVS.
 Fluorescent in situ hybridization (FISH)

 It is one technique that can be used to diagnose

aneuploid conditions, such as trisomies and
monosomy X.
 In the condition of fetal infection with such viruses
as rubella, cytomegalovirus and toxoplasmosis, the
viral immunoglobulin M (IgM) or DNA can also be
identified in fetal blood.
• FISH uses different fluorescent-labeled probes,
which are single-stranded DNA conjugated with
fluorescent dyes and are specific to regions of
individual chromosomes.
• These probes hybridize with complementary target
DNA sequences in the genome and can detect
chromosomal abnormalities, such as trisomies ,
monosomies and duplications.
FISH
 In 4% of retinoblastoma cases, deletion of
chromosome band 13q14 has been
reported.

 Prenatal diagnosis of retinoblastoma cases

with deletion of this band on chromosome
13 is feasible using fluorescent-labeled
probes for this region.

 Hybridization of fluorescent DNA probes to

interphase nuclei is under investigation as
a screening method for aneuploidy.
 Molecular genetic
techniques
◦ Linkage analysis using microsatellite markers
◦ Restriction fragment length polymorphisms
(RFLPs)
◦ Single nucleotide polymorphisms (SNPs)
 DNA chip
 Dynamic allele-specific hybridization (DASH)