1. PRE-IMPLANTATION GENETIC
DIAGNOSIS (PGD)
Dr. Iyer

2. THE UNDERSTANDING
 The background
 The test procedures
 The current status and the potential

3. THE BACKGROUND

4. PRE-IMPLANTATION GENETIC
DIAGNOSIS (PGD)
 What is PGD?
 First reported in 1990, PGD combines advances in
molecular genetics and in assisted reproductive
technology and is conducted before the embryo is
placed in the womb of the woman .
 PGD is a procedure used in conjunction with In Vitro
Fertilization (IVF) in which genetic analysis is done of
a single cell from an eight-cell embryo.
 PGD is conducted with an intention to improve the
chances of a “normal” pregnancy.
 WHY?

5. PRE-IMPLANTATION GENETIC
DIAGNOSIS (PGD)
 Why PGD?
 Reproductive failure (i.e. failed implantation, miscarriages
and major birth anomalies) are far more likely to be due to
embryo incompetence (70-75%) than to a lack of uterine
receptivity (25-30%).
 The number of chromosomes in a cell is referred to as its
ploidy.
 A cell with a normal number of chromosomes is
referred to, as euploid, while one with an irregular
chromosome number is aneuploid.
 Itappears that it is the ploidy of the mature egg (rather
than the sperm) that determines the post-fertilization
chromosome configuration of the embryo.
 The embryo's ploidy, in turn, determines its competence.

6. ANEUPLOIDY
 Aneuploidy = abnormal number of chromosomes,
and is a type of chromosome abnormality.
 An extra or missing chromosome is a common cause
of genetic disorders (birth defects).
 Aneuploidy occurs during cell division when the
chromosomes do not separate properly between the
2 cells.
 Aneuploidy =most frequent cause of spontaneous
abortions.
 Aneuploidy outcome- termination of developing fetus,
 Most common extra chromosomes among live births are
21, 18 and 13.

7. WHY CONSIDER PGD IN ADDITION TO
IVF?
 Chromosome abnormalities (aneuploidies) are
associated with failed implantation, pregnancy loss,
and the birth of children with multiple congenital
anomalies.
 Preimplantation genetic diagnosis (PGD) provides a
means of testing for these chromosome abnormalities
and selecting the best embryos for transfer.
 So, what is aneuploidy?

8. WHEN TO CONSIDER PGD IN ADDITION
TO IVF?
 Recurrent miscarriages
 One child already affected with a genetic disease
 Family history of inherited disease
 Maternal age older than 38
 Couples with >3 IVF failures
 Epididymal or Testicular sperm aspiration with >1 IVF
failures
 Family “balancing” for sex

9. INDICATIONS FOR PGD
 When there is suspicion of Chromosomal Disorders
 Chromosomal rearrangements
 Inversions
 Translocations
 Chromosome Deletions
 Severe monogenic diseases
 cystic fibrosis,
 ß thalassaemia,
 sickle cell anemia,
 fragile X syndrome,
 myopathies

10. STAGES IN PGD
1. Monitor egg maturation in
the ovary by the use of
Ultrasound & Hormone
levels
2. Collect eggs (mother’s own
or from donor) in 2 steps -
A. Injection of human chorionic
gonadotropin (hCG) and
follicle stimulating hormone
(FSH) to time egg ripening
B. Transvaginal aspiration
using hollow needle

11. STAGES IN PGD
3. Obtain sperm from father/donor) & assess quality
4. Combine eggs and sperm in vitro, using
intracytoplasmic sperm injection (ICSI), if sperm is
low quality
5. Nurture embryo growth by incubating in medium
containing various nutrients and hormones
6. For doing PGD, remove one cell called blastomere
from the 6-8-cell embryo after 2-3 days (6-8 cell
stage) for testing.
7. If fine on PDG, incubate until embryo is 5-6 days old
(blastocyst) and transfer embryos (usually 3-6) to
uterus, artifically removing zona pellucida.

12. METHOD OF PGD
 In skilled hands, this generally does not harm
the developing embryo and this is done using a
fine glass needle to puncture the zona pellucida
and aspirate the cell.
Blastomere Biopsy on Day 3
Genetic
Analysis

13. PROBLEMS WITH PGD
 The single blastomere cell cells contains too little DNA
to do extensive testing.
 So “modified multiple displacement amplification” is
done to allow researchers to amplify or make carbon
enough copies of the DNA they obtain from an embryo
obtained by in vitro fertilization, to do multiple tests.
 The amplified DNA from the couple’s embryos are then
sent for testing for aneuploidy using a test called 23-
chromosome microarray.

14. BENEFITS OF PGD
 Increases implantation success rate
 Reduction in the chance of having a child with
aneuploidy
 Reduces the possibility of having to choose to
terminate the pregnancy following a diagnosis of
a probable genetic disorder.
 Reduction in pregnancy losses

15. THE TESTS

16. PGD PROCESS
 2 types of PGD assessment techniques are common:
1. Genetic testing for specific disease loci by Polymerase
Chain Reaction (PCR) or gene chips) amplification of DNA
specific to a gene of interest (family history guides choice
of genes).
2. Chromosome “painting” or Fluorescence In Situ
Hybridization (FISH)

17. PCR
 Molecular genetic disorders, i.e single gene
disorders - diagnosed by PCR
 Appears to be available in India in some clinics
 Needs to be adapted to single-cell PCR due to
scarcity of embryonic material available

18. PCR - GENETIC DISORDERS
DETECTABLE
 Tay Sachs (autosomal recessive; ~98% accuracy)
 Cystic fibrosis (autosomal recessive; ~85% for
common allele mutation)
 Huntington’s disease (autosomal dominant)
 Thalassemias (autosomal recessive blood disorder)
 Duchenne muscular dystrophy (X-linked recessive)
 Spinal muscular atrophy
 As more genetic tests are developed, more will be
available for predictive purposes in PDG.

19. FISH
 Appears to be the most widely performed PGD test in
India & involves chromosome “painting” or
karyotyping, using fluorescent probes specific for each
chromosome.
 This procedure destroys the tested cell but permits
number and size of each chromosome to be checked.
 Useful for identifying aneuploidies (incorrect
chromosome numbers) and translocations.
 Conventional FISH cannot fully access all the
chromosomes - in fact, only about 12 of them.

20. FISH
 Thus, even when FISH reveals that all the accessed
chromosomes are normal, there still remains more
than a 40% chance of chromosomal aneuploidy
involving those chromosomes not targeted by the test.
 FISH testing is able to detect the most common
chromosome abnormalities in order to reduce the risk
of having an affected pregnancy or child., including
 Down syndrome,
 Trisomy 18,
 Trisomy 13, and
 sex chromosome anomalies.

21. CGH / CMA
 Comparative genomic hybridization (CGH) is a
molecular-cytogenetic method for the analysis of copy
number changes (gains/losses) in the DNA content of a
given subject's DNA
 This technique compares the amount of DNA present
for each chromosome in a single cell, and compares it
to that of a normal standard.
 Chromosomal Microarray Analysis (CMA) allows for
evaluation of all 23 chromosome pairs in a single cell.
 Research shows, however, that abnormalities
involving any chromosome can increase the risk of
miscarriage and reduce the effectiveness of IVF.

22. CGH / CMA
 This test is valuable at looking for previously unknown
mutations that can lead to children with dysmorphic
features, developmental delays, mental retardation,
and autism.
 Microarray can be performed on three different sample
types:
 polar bodies (from fertilized eggs),
 blastomeres (from Day 3 embryos), or
 blastocyst/trophectoderm (from Day 5 embryos).
 23-chromosome microarray will replace FISH for this
reason .

23. CGH / CMA
 Relatively new technique
 Does not appear to be
available in India.
 By the CMA chromosome
testing, one is able to
analyze the entire
chromosome complement
of a single cell, reducing
the risk of failed
implantation and
miscarriage, and
increasing the chance of
having a healthy baby

24. TRANSLOCATION TESTING
 Accuracy of the PGD for translocation is 90%
and may be of 2 types:
1. “Balanced” if chromosome material merely
switches locations with no net loss or gain; or
2. “Unbalanced” if switch is accompanied by a net
loss or net gain of genetic material

25. THE CURRENT STATUS AND
THE POTENTIAL

26. PGD - ADVANTAGES
 PGD can help eliminate some genetic diseases cures for
which are not likely to be found soon (eg,
 Tay-Sachs disease,
 cystic fibrosis,
 Huntington disease,
 X-linked dystrophies.
 genetic disease testing is currently performed through
amniocentesis or chorionic villus testing (CVS) with
fetus aged 10-16 weeks.
 If positive for genetic defect, the option is to have a child
with a genetic disease or to undergo MTP.
 Difficult and often traumatic decision that PGD can
eliminate.

27. PGD - LIMITATIONS
 PGD is expensive, time and labor intensive to develop
and work with single-cell diagnostic techniques.
 PGD can only detect a specific genetic disease in an
embryo.
 Itcannot detect many genetic disorders at a time and
cannot guarantee that the fetus will not have an unrelated
birth defect.
 Removal of a single cell without breaking it or causing
serious damage is technically difficult and requires
skill and experience. Damage to the embryo (projected
to be 0.1%) may accidentally occur during removal of
the cell.

28. PGD - LIMITATIONS
 For aneuploidy screening, not all chromosomal or
genetic abnormalities can be diagnosed with PGD
because only a restricted number of chromosomes can
be examined at one time during the course of a single
procedure.
 Currently, FISH offers evaluation of less than half of
the 23 chromosomes; usually 9-11 are analyzed.
 Studies using comparative genetic hybridization (CGH)
and FISH demonstrate that as many as 25% of
aneuploid embryos are characterized as normal
because the abnormal chromosomes were not analyzed.

29. PGD - LIMITATIONS
 Current recommendations from the Society for Assisted
Reproductive Technology (SART) and American Society
for Reproductive Medicine (ASRM) state that
 Available evidence does not support the use of PGS to
improve live-birth rates for advanced maternal age,
recurrent pregnancy loss, or implantation failure
 Also recommends that patients be counseled about the
limitations of the technique and should not make future
treatment decisions based solely on PGD result.

30. PGD - POTENTIAL
 In the future, genetic links to common diseases (eg,
diabetes, hypertension, cardiovascular diseases,
endometriosis, cancers) may be identified, and PGD
will become available to control the transmission of
these diseases to future generations.

31. PGD –
EQUIPMENT/CONSUMABLES/MANPOWE
R
 Equipment cost is negligible and consists of:
 Thermal cyclers, fluorescence microscopes
 The problem is with:
 High consumable cost – for PCR, FISH and CGH
 Manpower cost – also very high. Need highly trained
personnel

32. PGD - LOCATION
 A PGD test center would presumably need to be
attached to an IVF clinic – time between testing and
implantation is 1-2 days.
 Transport of genetic material/embryos does not seem
feasible.
 Hospitals – some offer PGD (*), others just have IVF
clinics
 Dozens of IVF clinics across India
 Many claim to offer PGD (primarily FISH)
 The Centre for DNA Fingerprinting and Diagnostics
(CDFD) – currently not offering PGD but a VERY
wide range of pre-natal tests offered.
 http://www.cdfd.org.in/index.html

33. THAN
K
YOU!