The techniques used for pre-implantation genetic testing were briefly explained.

1. RECENT ADVANCES IN PRE-
IMPLANTATION GENETIC TESTING
M. Vharshini
M.Sc. 2nd year

2. Pre-Implantation Genetic Testing
• Preimplantation genetic (PG) testing is the practice of
obtaining a cellular biopsy sample from a developing human
oocyte or embryo, obtained via a cycle of in vitro fertilization
(IVF); evaluating the sample’s genetic composition; and using
this information to determine which embryos will be optimal
for subsequent uterine transfer.
• PG testing was 1st described in 1990 for X chromosome
linked diseases by Handyside et al.
• PGT is of two broad categories:
Pre-Implantation Genetic
Testing
Pre-Implantation Genetic Diagnosis
Pre-Implantation Genetic Screening

3. Pre-Implantation Genetic Screening
• The term ‘‘preimplantation genetic screening’’ (PGS) applies when the
genetic parents are known or presumed to be chromosomally normal
and their embryos are screened for aneuploidy.
• PGS is now referred as PGT-A (PGT-“Aneuploidy”).
• The screening helps to reduce the chance of having a child with extra
or missing chromosomes.
• E.g.: Down syndrome, Patau’s syndrome, Edward’s syndrome, Turners
syndrome, klinefelter syndrome, abnormal sperm parameters etc.
• INDICATIONS:
– Advanced maternal age
– History of recurrent early pregnancy loss
– Severe male infertility
– Sex selection (controversial)
– Repeated IVF failure

4. Pre-Implantation Genetic Diagnosis
• The term PGD applies when one or both genetic parents carry a gene
mutation or a balanced chromosomal rearrangement.
• Testing is performed to determine whether that specific mutation or an
unbalanced chromosomal complement has been transmitted to the
oocyte or embryo.
• PGD is classified as PGT-M and PGT-SR
PGT-M (PGT- Monogenic/Single Gene Disorders):
 PGT-M is a sophisticated reproductive technology used in conjunction with
IVF treatment to test early embryos for specific or single gene disorders.
 E.g.: sickle cell anaemia, cystic fibrosis, beta-thalassemia etc
 HLA matching for the purposes of creating a tissue donor for an existing
diseased sibling.
PGT- SR (PGT-“Structural Rearrangements”):
 PGD for chromosomal translocations (balanced or unbalanced) or
inversions is used to test embryos for chromosomal abnormalities as well as
abnormal chromosomal positions and rearrangements.

5. Sampling Approaches
Invasive - BIOPSY TECHNIQUES

6. Techniques for PGT
Fluorescence in situ Hybridization (FISH):
– FISH is the most frequently used method for analysis of the
chromosomal complement of the cell.
– Collected cells are spread on a slide after which DNA probes
labeled with flurochromes specific for chromosomes of interest are
applied.
– The type and number of probes used depends on the indication.
– The most commonly tested chromosomes are 13,18,21,X & Y.
– The main limitation of the FISH technology was that only around 5–
12 pairs of chromosomes could be tested for aneuploidy from a
total of 23 pairs of human chromosomes.

7. Techniques for PGT
• Polymerase Chain Reaction (PCR):
– PCR, sometimes called DNA amplification, is a technique in which a
particular DNA sequence is copied many times in order to facilitate its
analysis.
– The specific chromosomes evaluated are chosen according to the
patient’s prior history and include those involved in the most common
aneuploidies identified in spontaneous miscarriages.
- Amplification of DNA specific to a gene of interest (family history guides
choice of genes)
- Second round PCR used for specific exonic sequencing and/or linkage
analysis (Fragment analysis)
LIMITATIONS:
– Incapability of detecting de-novo genetic mutations,
– contamination,
– Allele dropouts and sensitivity issues that lead to the false positive or
negative.

8. Recent Advances in PGT
• To overcome the limitations of FISH and PCR advance diagnostic
techniques were developed. They are:
 array-Comparative genomic hybridization (aCGH),
 Single-nucleotide polymorphism (SNP) microarray,
 Multiplex quantitative PCR (qPCR), and
 Next generation screening (NGS)

9. Multiplex qPCR
• PCR has been adapted for chromosome copy number
analysis.
• This technique requires a high-order multiplex reaction after
pre-amplification is conducted to amplify at least two
sequences on each arm of each chromosome for rapid
quantification and comparison of each product across the
genome within 4–6h.
• It is reliable in determining aneuploidy, but not ideal for
detecting structural chromosomal aberrations or uniparental
disomy.

10. Microarray Based Techniques
• The two modalities that are most commonly used today utilize
microarray technology, either by using
– single nucleotide polymorphism (SNP) or
– comparative genomic hybridization (CGH) technology.
• Both of these technologies rely on obtaining embryonic DNA,
fragmenting and then amplifying this DNA, and evaluating this
amplified product using microarrays.
• This amplification process is a potential source of error, as
failure to amplify the entire embryonic DNA product could
produce a false result.

11. Microarray Based Techniques
 Array -Comparative Genome Hybridization:
– Microarray-based CGH has higher resolution, throughput, and
speed than conventional CGH.
– First techniques used to provide comprehensive PGT of all 23
pairs of chromosomes
– aCGH can assess chromosomal abnormalities such as copy
number and unbalanced translocations.
– CGH arrays, in contrast to SNP, evaluate far fewer genetic
markers and compare this result to a known normal DNA
sample.
– An advantage of CGH arrays is that they may be performed in
12–16 hours as opposed to several days for most SNP arrays.

12. Array -Comparative Genome
Hybridization:

13. Microarray Based Techniques
SNP Arrays:
• SNP array was initially designed for genome-wide association
studies (GWAS) before its first application in PGD/PGS in 2011.
• SNP arrays directly evaluate ploidy status using a dense array of
approximately 300,000 genetic markers.
– Capable of identifying all parental translocation imbalances in
embryos.
• An advantage of SNP arrays is their ability to detect relatively small
genetic duplications or deletion.
• For Single gene disorders, the SNP genotypes of the parents and a
reference (an affected child or another affected relative) can be
analyzed at a gene of interest, and linkage information can be used
to select unaffected embryos during PGD.

14. Next Generation Sequencing
• NGS is based on ultra-high throughput parallel DNA sequencing that
achieves genome-scale sequencing within days, or even within 24
h.
• It can detect genetic mutations at single nucleotide level, with the
capacity of detecting aneuploidy such as triploidy and uniparental
disomy.
• NGS was performed on trophectoderm cells and on blastomeres for
the detection of aneuploidies and of unbalanced chromosomal
rearrangements; the results of these experiments were successful.
• The sensitivity and specificity of this method depends on the
sequencing depth and the coverage of the regions of interest.

16. Recent perspectives on sampling
approaches
 Invasive – BF sampling:
– Palini et al. showed, for the first time,
• the presence of genomic DNA in the
BF by whole-genome amplification
(WGA), quantitative PCR (qPCR),
and
• analyzed multicopy genes such as
TSPY1 (Y chromosome ) and
TBC1D3 (chromosome 17)
– Gianaroli et al. used aCGH to evaluate
BF biopsies for ploidy prediction in
comparison with the conventional biopsy
methods.
– concluded that BF biopsy was
comparable to conventional biopsy
materials for chromosomal analysis.

17. Non Invasive Techniques
 Cell-free nucleic-acid collection in culture medium:
• Inspired by BF sampling, Assou et al. collected cell-free
nucleic acids released from embryos in the culture medium
and successfully determined the embryo sex by PCR.
 Time lapse Imaging:
• Time-lapse imaging, was developed to select the best
embryos for single-embryo transfer (SET) by correlating
cellular morphology and morphokinetic parameters.

18. Time lapse Imaging

19. Recent - Microarray Based
Techniques
KARYOMAPPING:
• It is a PGT – M test, which is advanced than Next generation
sequencing.
• By knowing the genotyping of the parents and a close relative of
known disease status, generally a previously affected child, this
technology eliminates the need for customized test development.
• Karyomap identifies
– the offspring’s SNP genotypes from four possible inherited
haplotypes across each chromosome
– maps the inheritance of these haplotypes and
– the position of any crossovers in the proband as well as in the
preimplantation embryos.
• Thus, it identifies the embryo-carrying normal chromosome copies
and also reveals potential chromosomal abnormalities and single-
gene mutations.

20. Recent - MICROARRAY
BASED TECHNIQUES
KARYOMAPPING
• ADVANTAGES:
– Targeted approach, karyomap is applicable to any inherited SGD within
the informative SNP loci without the development of costly, time-
consuming, and laborious patient- or disease-specific designs.
– In addition, SNP data enable the detection of chromosomal
abnormalities (meiotic trisomies, monosomies, and deletions)
• DISADVANTAGES:
• Karyomap does not include a mutation detection method; therefore,
a reference is always needed to establish linkage information, which
is not always available for every couple.

21. References
• Brezina PR, Ke RW, Kutteh WH. Preimplantation genetic
screening: a practical guide. Clin Med Insights Reprod Health.
2013;7:37–42. Published 2013 Feb 27.
doi:10.4137/CMRH.S10852
• Schoolcraft WB et al. Clinical application of comprehensive
chromosomal screening at the blastocyst stage. Fertil Steril.
2010;94(5):1700–6.
• Sermon K, Van Steirteghem A, Liebaers I. Preimplantation
genetic diagnosis. Lancet 004;363:1633-41
• Wilton L, Voullaire L, Sargeant P, Williamson R, McBain J.
Preimplantation aneuploidy screening using comparative
genomic hybridization or fluorescence in situ hybridization of
embryos from patients with recurrent implantation failure. Fertil
Steril 2003;80:860–8.

22. THANK YOU