Dr. Laxmi Shrikhande is a leading fertility expert in India. She has held numerous leadership positions in national obstetrics and gynecology societies. Some of her accomplishments include receiving awards for women's health, publishing over 30 papers nationally and internationally, and conducting health programs that have reached over 25,000 women and girls. She is the Medical Director of Shrikhande Fertility Clinic in Nagpur, India.

1. Dr. Laxmi Shrikhande MD; FICOG; FICMU;FICMCH
• Medical Director-Shrikhande Fertility Clinic, Nagpur
• National Corresponding Editor-The Journal of Obstetrics & Gynecology of India
• Senior Vice President FOGSI 2012
• Chairperson Designate ICOG 2020 , Vice Chairperson ICOG 2019
• National Governing Council member ICOG 2012-2017
• National Governing Council Member ISAR 2014-2019
• National Governing Council Member IAGE for 3 terms
• Patron & President -Vidarbha Chapter ISOPARB
• Chairperson-HIV/AIDS Committee, FOGSI (2007-09)
• Received Best Committee Award of FOGSI
• Received Bharat excellence Award for women’s health
• President Nagpur OB/GY Society 2005-06
• Immediate Past President Menopause Society, Nagpur
• Associate member of RCOG
• Member of European Society of Human Reproduction
• Visited 96 FOGSI Societies as invited faculty
• Delivered 11 orations and 450 guest lectures
• Publications-Twenty National & eleven International
• Received Nagpur Ratan Award at the hands of Union Minister Shri Nitinji Gadkari
• Presented Papers in FIGO, AICOG, SAFOG, AICC-RCOG conferences
• Conducted adolescent health programme for more than 15,000 adolescent girls
• Conducted health awareness programme for more than 10,000 women

2. Preimplantation
Genetic Diagnosis
DR LAXMI SHRIKHANDE
NAGPUR

3. 1st Embryo biopsy on rabbit embryos
in 1968
PGD was developed in UK in the mid
1980s
In 1989, London, Handyside and
collegues reported the 1st unaffected
child born
As of 2010, more than 14,272 PGD
cycles have been reported.
HISTORY OF PGD
PGS pioneers Verlinsky and Kuliev
published a first report in 1996
As of 2010, more than 23,276 PGS
cycles have been reported.
HISTORY OF PGS

4. DEFINITION OF PGD AND PGS
 Preimplantation Genetic Diagnosis (PGD) applies when one or
both genetic parents carry a gene mutation or a chromosomal
rearrangement and testing is performed to determine whether that
specific mutation or an unbalanced chromosomal complement has
been transmitted to the oocyte or embryo.
 Preimplantation genetic screening (PGS) applies when the
genetic parents are known or presumed to be chromosomally
normal and their embryos are screened for aneuploidies.
The Practice Committee of the SART and the Practice Committee of the ASRM (2007)

5. 4
No preliminary work up. The
genetic testing method is the
same for every couple.
Embryological
aspects
DEFINITION OF PGD AND PGS

6. What is pre-implantation genetic screening (PGS)?
PGS (also known as aneuploidy screening) involves checking the chromosomes
of embryos conceived by in vitro fertilisation (IVF) or intra-cytoplasmic sperm
injection (ICSI) for common abnormalities. This avoids having abnormal embryos
transferred to the womb during IVF or ICSI.
In vitro fertilisation
Intra-cytoplasmic sperm injection (ICSI)
Chromosomal abnormalities are a major cause of the failure of embryos to
implant, and of miscarriages. They can also cause conditions such as Down’s
syndrome.

7.  Young female patients who had experience of Recurrent Pregnancy
Loss, usually defined as 2 or more consecutive miscarriages.
 Young female patients who had experience of repeated IVF failures,
usually defined as 2 or more IVF embryo transfers involving good
quality embryos.
 Advanced female age, usually defined as > 35 years
Indication for PGS
ESHRE PGD Consortium, Goossens et al., 2009

8. Moving toward eSET for all IVF patient population
1 ≥ 2

9. Increase the cost-
effectiveness
Decrease time
to pregnancy
Decrease in
abortion rate
Single ET
Increase
implantation rate
Less abnormal
pregnancies
Expected advantages in IVF
PGS application in ART

10. Rationale for PGS
 more aneuploidy in human
embryos (7- 10% aneuploid)
 low implantation potential
 high miscarriage rate (70%
aneuploid)
B
R
U
S
S
E
L
S
P
G
D

11. Rationale for PGS
aneuploidy in oocytes
 factors
1. age
2. intrinsic gamete
abnormalities
 prevalence
1. 3% at 22 years
2. >50% over 40 years
Pellestor et al., 2003; Fragouli et al.,
2009
B
R
U
S
S
E
L
S
P
G
D

12. Rationale for PGS
aneuploidy in embryos
 prevalence
 2/3 human cleavage stage embryos
aneuploid
 58% under 30 years
 77% above 39 years
Delhanty et al., 1997; Magli et
al., 2000; Bielanska et al.,
2005; Mantzouratou et al.,
2007
B
R
U
S
S
E
L
S
P
G
D

13. Rationale for PGS
aneuploidy in embryos
 factors
 chaotic patterns
 abnormal ploidy
 chromosome loss
- not age-related
 mitotic non- disjunction
- age-related
B
R
U
S
S
E
L
S
P
G
D

14. INDICATIONS FOR PGD
• Other indications:
Mitochondrial pathologies
Late onset disorders (as Huntington Disease)
Inherited Predisposition to Cancer
HLA Genotyping

15. DISEASES SCREENABLE BY PGD
•Achondroplasia
•ADPKD1
•ADPKD2
•Adrenoleukodystroph
•Age-related aneuploidies
•Alpha-thalassemia
•Alpha-1-antitrypsin
•Alport disease
•Amyloid precursor protein (APP) mutation
•ARPKD
•Becker muscular dystrophy
•Beta-thalassemia
•Charcot Marie Tooth disease
•Chromosomal translocations
•Congenital adrenal hyperplasia
•Cystic fibrosis
•Down syndrome
•Duchenne muscular dystophy
•Dystonia
•Epidermolysis bullosa
•Familial dysautonomia
•Fanconi anemia
•FAP
•Fragile X syndrome
•Gaucher disease
•Hemophilia A and B
•HLA genotyping
•HSNF5 mutation
•Huntington disease
•Hypophosphatasia
•Incontinentia pigmenti
•Kell disease
•Klinefelter syndrome
•LCHAD
•Lesch Nyhan syndrome
•Marfan syndrome
•Multiple epiphysial dysplasia
•Myotonic dystophy
•Myotubular myopathy
•NF1 and NF2
•Norrie disease
•Osteogenesis imperfecta
•OTC deficiency
•P53 mutations
•PKU
•Retinitis pigmentosa
•SCA6
•Sickle cell anemia
•Sonic hedgehog mutations
•Spinal muscular atrophy (SMA)
•Tay-Sachs disease
•Tuberous sclerosis
•Turner syndrome
•Von Hippel Lindau
•X-linked hydrocephaly
•X-linked hyper IgM syndrome

16. Monogenic disorders
PGD is available for a large number of monogenic disorders
—that is, disorders due to a single gene only (autosomal
recessive, autosomal dominant or X-linked)— or of
chromosomal structural aberrations (such as a balanced
translocation).
 PGD helps these couples identify embryos carrying a
genetic disease or a chromosome abnormality, thus
avoiding diseased offspring.

17. Monogenic disorders
The most frequently diagnosed autosomal recessive disorders are cystic fibrosis, Beta-
thalassemia, sickle cell disease and spinal muscular atrophy type 1.
The most common dominant diseases are myotonic dystrophy, Huntington's disease
and Charcot-Marie-Tooth disease; and in the case of the X-linked diseases, most of the
cycles are performed for fragile X syndrome, haemophilia A and Duchenne muscular
dystrophy.
Though it is quite infrequent, some centers report PGD for mitochondrial disorders or
two indications simultaneously.

18. X-linked diseases
In the case of families at risk of X-linked diseases, patients are provided with a
single PGD assay of gender identification.
Gender selection offers a solution to individuals with X-linked diseases who are
in the process of getting pregnant.
The selection of a female embryo offspring is used in order to prevent the
transmission of X-linked Mendelian recessive diseases. Such X-linked Mendelian
diseases include Duchenne muscular dystrophy(DMD), and hemophilia A and B,
which are rarely seen in females because the offspring is unlikely to inherit two
copies of the recessive allele.
Since two copies of the mutant X allele are required for the disease to be passed
on to the female offspring, females will at worst be carriers for the disease but
may not necessarily have a dominant gene for the disease.

19. X-linked diseases,
Males on the other hand only require one copy of the mutant X allele for the disease to occur in one's
phenotype and therefore, the male offspring of a carrier mother has a 50% chance of having the
disease.
Reasons may include the rarity of the condition or because affected males are reproductively
disadvantaged.
Therefore, medical uses of PGD for selection of a female offspring to prevent the transmission of X-
linked Mendelian recessive disorders are often applied.
Preimplantation genetic diagnosis applied for gender selection can be used for non-Mendelian
disorders that are significantly more prevalent in one sex.
Three assessments are made prior to the initiation of the PGD process for the prevention of these
inherited disorders.
In order to validate the use of PGD, gender selection is based on the seriousness of the inherited
condition, the risk ratio in either sex, or the options for disease treatment
Eduardo C. L, et al. (2012). In vitro fertilization - innovative clinical and laboratory aspects. Preimplantation Genetic testing: Current status and future prospects

20. Minor disabilities
PGD has occasionally been used to select an embryo for the
presence of a particular disease or disability, such as
deafness, in order that the child would share that
characteristic with the parents.
Simoncelli,Tania."Pre-implantation Genetic Diagnosis: Ethical Guidelines for Responsible Regulation."
CTA International Center for Technology Assessment. Retrieved on Nov. 19 2013

21. How does PGS work? Procedure-
Step 1. IVF treatment to collect and fertilise eggs.
Step 2. The embryo is grown in the laboratory for two - three days until the cells have divided
and the embryo consists of about eight cells.
Step 3. A trained embryologist removes one or two of the cells (blastomeres) from the embryo.
Step 4. The chromosomes are examined to see how many are there and whether they are
normal.
Step 5. One, two or three of the embryos without abnormal numbers of chromosomes are
transferred to the womb so that they can develop. Any remaining unaffected embryos can be
frozen for later use.
However, embryos that have been biopsied may not be suitable for cryopreservation and use in
subsequent treatment cycles.
Step 6. Those embryos that had abnormal chromosomes are allowed to perish or may be used
for research

22. Possible variations to this procedure
Testing at five – six days It is possible that instead of removing and
testing one or two cells from a two – three day old embryo, some
centres may allow the embryo to develop to five - six days, when
there are 100-150 cells.
More cells can be removed at this stage without compromising the
viability of the embryo, possibly leading to a more accurate test.
Alternatively some centres may test eggs for chromosomal
abnormalities before they are used to create embryos. Polar bodies
(small cells extruded by eggs as they mature) can be extracted and
tested.

24. We need to perform a biopsy.....when
and how?

25. Timing of Biopsy
Polar Body Biopsy
Cleavage Stage Biopsy
Blastocyst Biopsy

26. Timing of PGS
B
R
U
S
S
E
L
S
P
G
D1. polar body biopsy
 advantages
 less invasive
 both classical non-disjunction and premature
division of chromatids
 most aneuploidies maternal in origin
 disavantages
 accuracy limited to 94%
 maternal information only
 technically more complex

27. Timing of PGS
B
R
U
S
S
E
L
S
P
G
D
2. cleavage stage biopsy
 advantage
 technically more straightforward
 good concordance rate despite more andmore
specific probes
Mir et al., 2013
 disadvantages
 chromosome instability/mosaicism
 no definite conclusion on the basis of 1cell

28. Timing of PGS
B
R
U
S
S
E
L
S
P
G
D
3. Trophectoderm biopsy
 advantage
 less invasive
 less embryos for biopsy
 mosaicism diluted
 disadvantage
 labour intensive (variable stages of blastocyst)
 effect on children insufficiently demonstrated
 need for cryopreservation of embryos

29. Technique
Analysis of genetic material (DNA) from a single cell is
performed either using a technique called
 FISH ( fluorescent in situ hybridisation)
PCR ( polymerase chain reaction) .
Newer techniques such as array CGH ( comparative
genomic hybridization), SNP (Single Nucleotide
Polymorphism) provide even more accuracy .
New Generation Sequencing (NGS )

30. Technique
FISH utilises fluorescent probes, which are specific for a given chromosome, and
therefore allows one to screen embryos for chromosomal normality.
PCR allows one to amplify (mutiply ) a selected DNA sequence of interest, so
that it can be analysed.
After the analysis on the single cell, the embryos are kept in culture and allowed
to further divide.
Once the appropriate molecular diagnosis is made, unaffected embryos can be
transferred back into the uterus in the IVF cycle.

31. Technical aspects
Generally, PCR-based methods are used for monogenic disorders
and FISH for chromosomal abnormalities and for sexing those cases
in which no PCR protocol is available for an X-linked disease.
 These techniques need to be adapted to be performed on
blastomeres and need to be thoroughly tested on single-cell models
prior to clinical use.
Finally, after embryo replacement, surplus good quality unaffected
embryos can be cryopreserved, to be thawed and transferred back in
a next cycle.

32. FISH- Fluorescent in situ hybridization
FISH is the most commonly applied method to determine
the chromosomal constitution of an embryo.
The cells are fixated on glass microscope slides and
hybridised with DNA probes.
Each of these probes are specific for part of a chromosome,
and are labelled with a fluorochrome.
Currently, a large panel of probes are available for different
segments of all chromosomes,

33. FISH- Fluorescent in situ hybridization
The main problem of the use of FISH to study the chromosomal
constitution of embryos is the elevated mosaicism rate observed at
the human preimplantation stage.
A meta-analysis of more than 800 embryos came to the result that
approximately 75% of preimplantation embryos are mosaic, of which
approximately 60% are diploid–aneuploid mosaic and approximately
15% aneuploid mosaic.
Van Echten-Arends, J.; Mastenbroek, S.; et el (2011). "Chromosomal mosaicism in human
preimplantation embryos: A systematic review". Human Reproduction Update 17 (5): 620–627

34. FISH- Fluorescent in situ hybridization
As a consequence, it has been questioned whether the one or two
cells studied from an embryo are actually representative of the
complete embryo, and whether viable embryos are not being
discarded due to the limitations of the technique.
The FISH technique is considered to have an error rate between 5
and 10%.

35. Fluorescent in situ hybridisation
• Fluorescent in situ hybridisation (FISH) uses fluorescently tagged DNA probes that bind
to their complementary sequence and can be visualised under a fluorescent
microscope. Advantages Disadvantages
It does not require DNA
amplification
It is not able to easily test
for all 23 chromosomes.
It can be completed within
4-10 hours
The risk of potential
misdiagnosis is too high
It does not require cell
synchronisation and
division to produce
metaphase chromosomes
for analysis
-
Despite of its promise of improved clinical outcomes, RCT’s performed over the past 10 years have
found that FISH-based PGS does not improve Live birth rate.

36. PCR
It is a highly sensitive and specific technology, which makes it suitable for all
kinds of genetic diagnosis, including PGD.
As PGD is performed on single cells, PCR has to be adapted and pushed to its
physical limits, and use the minimum amount of template possible: one strand.
This implies a long process of fine-tuning of the PCR conditions and a
susceptibility to all the problems of conventional PCR, but several degrees
intensified.
The high number of needed PCR cycles and the limited amount of template
makes single-cell PCR very sensitive to contamination.

37. PCR
Another problem specific to single-cell PCR is the allele drop out (ADO)
phenomenon.
It consists of the random non-amplification of one of the alleles present in a
heterozygous sample.
ADO seriously compromises the reliability of PGD as a heterozygous embryo
could be diagnosed as affected or unaffected depending on which allele would
fail to amplify.
This is particularly concerning in PGD for autosomal dominant disorders, where
ADO of the affected allele could lead to the transfer of an affected embryo

38. Polymerase Chain Reaction
• qPCR is very effective in the assessment of known single gene defects, and has
traditionally been used for PGS.
• The highlighting advantage of qPCR is that it can be completed in just 4 hour.

39. Microarray-Based Comprehensive Genomic Hybridisation
Advantages Disadvantages
CGH arrays platforms are
able to complete the entire
analysis in 12-15 hour
It may not detect some
clinically significant
deletions and duplications
It eliminates the necessity
of cryopreserving the
biopsied embryos prior to
obtain results
It is only specific to
metaphase CGH
It cannot detect
polyploidies
Chromosomal
microdeletions can go
undetected because of its
resolution

40. Single nucleotide polymorphism
• Single nucleotide polymorphisms are a single nucleotide (A, T, C, or G) change in
genomic DNA.
• SNP microarrays for PGS typically evaluate approximately 300,000 SNPs throughout all
23 chromosomes. Advantage Disadvantage
It has the ability to
evaluate all 23 pairs of
chromosomes with highly
dense platform capable
of detecting clinically
significant deletions and
duplications.
It is a costly technology.
It can identify triploidy
and uniparental disomy.
The preparation of SNP
array analysis is
laborious.
It can also detect single
gene defects in
conjunction with PGS
2-3 days of time is
required to analyse the
sample

41. Next Generation Sequencing
• NGS is a high-throughput method used to determine a portion of the nucleotide
sequence of an individual’s genome.
• This technique utilizes DNA sequencing technologies that are capable of processing
multiple DNA sequences in parallel.
The potential role for NGS in PGS was
assessed by Yin et al.
• 38 blastocyst biopsy samples were
analysed with both SNP arrays and
NGS.
• NGS detected 6 embryos with
unbalanced chromosomal
translocations, 1 of which was not
identified by SNP array.

42. NGS + PGS

43. Side Effects to Embryo
PGD/PGS is an invasive procedure
One of the risks of PGD includes damage to the embryo during the biopsy
procedure (which in turn destroys the embryo as a whole)
Another risk is cryopreservation where the embryo is stored in a frozen state
and thawed later for the procedure.
About 20% of the thawed embryos do not survive.
 There has been a study indicating a biopsied embryo has a less rate of surviving
cryopreservation.
"Reduced survival after human embryo biopsy and subsequent cryopreservation".
Human Reproduction vol.14 no.11 pp.2833-2837.

44. Ethical issues
By relying on the result of one cell from the multi-cell embryo, PGD
operates under the assumption that this cell is representative of the
remainder of the embryo.
This may not be the case as the incidence of mosaicism is often
relatively high.
 On occasion, PGD may result in a false negative result leading to the
acceptance of an abnormal embryo, or in a false positive result
leading to the de selection of a normal embryo.

45. PGD can potentially be used
to select embryos to be without a genetic disorder,
to have increased chances of successful pregnancy,
to match a sibling in HLA type in order to be a donor,
to have less cancer predisposition, and
 for sex selection.

46. Policy and legality
In India, Ministry of Family Health and Welfare, regulates the
concept under - "The Pre-Conception and Prenatal Diagnostic
Techniques (Prohibition of Sex Selection) Act, 1994".
The Act was further been revised after 1994 and necessary
amendment were made are updated timely on the official website of
the Indian Government dedicated for the cause.
http://www.pndt.gov.in/

47. Indications
PGD PGS
Couples with a family history of X-linked disorders Women of advanced maternal age
(>35 years)
Couples with chromosome translocations Couples with history of recurrent pregnancy loss
(2 miscarriages)
Carriers of autosomal recessive diseases Couples with repeated IVF failure
(2 failed IVF)
Carriers of autosomal dominant disease Male partner with severe male factor infertility
(Low sperm count)
Patients undergoing Chemotherapy

48. Difference between PGD and PGS
PGD PGS
Aims Identify genetically normal embryos Achieve a
genetically normal pregnancy and birth
Achieve a pregnancy Birth
Identification Monogenetic disorder, X-linked diseases,
unknown chromosomal abnormalities
Advanced maternal age, Repeated implantation
failure, Repeated miscarriages and Severe male
factor
Fertility Often Fertile Infertility or Subfertility
Biopsy Usually day 3 Usually day 3, Recently polar body and
Blastocysts Biopsy are been used
Number of cells for
Analysis
1-2 cells 1 cell
Diagnosis Interphase FISH for chromosome
abnormalities and sexing PCR for monogenetic
disorders,Recently arrays been used.
FISH with as many Probes as possible
Recently Array being used
Undiagnosed or
inconclusive results
Never transfer these Embryos Can transfer these Embryos
Parental diagnosis Indicated Indicated for the same risk factors as natural
conceptions.

49. Conclusion
• The potential capabilities and efficiency of preimplantation genetic testing
have dramatically increased in recent years due largely to improvements in
the ability to accurately test embryos.
• Defining the exact benefit conferred by PGS and determining exactly which
patient populations could be best served by PGS, however, is currently
controversial.
• There are various pitfalls like False positive and false positive results,
Developmental potential of the biopsied embryos and mosaicism which
may affect the Pregnancy outcome.
• Regardless of improvements, patients must be carefully counselled about
error rates inherent in all the PGS technologies, as well as outcomes
reported to date.

50. Conclusion
• Patients must be given accurate information with which to make their
treatment decisions, taking into account their age, ovarian reserve, indication
for PGS and method of PGS to be employed.
• Once the patient is pregnant antenatal testing with chorionic villus sampling
and/ amniocentesis must be discussed, and a strong recommendation for
antenatal testing should be included in all PGT consent forms.
• Alternative treatment strategies, such as using donor gametes, should also be
discussed.

51. Questions

52. Dr. Laxmi Shrikhande
Shrikhande Fertility Clinic
Ph-8805577600 /8805677600
shrikhandedrlaxmi@gmail.com

53. The Art of Living
Anything that
helps you to
become
unconditionally
happy and loving
is what is called
spirituality.
H. H. Sri Sri Ravishakar