2. Embryo aneuploidy
• Platteau P., Staessen C.et all, 2004 – 40,5-60%
(depends on pathology)
• NADIYA (total index)
– aCGH – 49,7%
– FISH – 59%
2
3. 3
The main causes of chromosomal anomalies
• The inheritance of the parental pathology
- true inheritance
(e.g.: parental translocation)
- Chromosomal nondisjunction
during gametogenesis
(80-85% of causes relate to oocytes
10-15% - relate to spermatozoa)
• Mitotic errors in the zygote
5. • PDG – is the only one method of detection of embryos without
chromosomal / genetic pathology.
• The transfer of the embryos without chromosomal imbalances allows us
to increase the performance of ART cycles and to prevent the
chromosomal pathology of the embryo.
5
Preimplantation genetic testing
Indications to PGS / PGD
•Matermal age > 38-42 y.o.
•Multiple IVF failure
•RPL
•High level of sperm aneuploidy
•The carriage of the gene defects / chromosomal rearrangements
7. PGD TimeLine
FISH – sex selection
Implementation of
the FISH into the
cytogenetics
PCR -PGD for Fresh ET
aCGH- PGD
CGH- PGD
aCGH
First aCGH-delivery
1970 1980 1990 2000 2010
First PCR - PGD
Implementation of the CGH
into the cytogenetics
arrayCGH reported for clinical genetics
SNP-array
PGD
First delivery
after aCGH-PGS
8. (M Shinawi, S. W. Cheung. 2008 )
8
What is aCGH?
Array comparative genomic hybridization (aCGH) is a technique enabling high-resolution,
genomewide screening of segmental genomic copy number variations
M Shinawi, S. W. Cheung. Drug Discovery Today. 13 (17/18). 2008
11. Disadvantages and limitations
• Balanced rearrangements
– Reciprocal translocation
– Inversions
– Robertson translocations
– Reciprocal insertions
• Imbalanced rearrangements below the diagnostic
resolution
– Point mutations
– Three nucleotide expansions
– Deletions / Duplications in not covered regions
11
•Limited ability to detect polyploidy
•Limited ability to detect mosaicism
•The method needs a great quantity of DNA
12. Whole genome amplification (WGA)
• Methods
– Primer extension preamplification (PEP)
– Degenerative oligonucleotide primed-PCR (DOP-PCR)
– Ligation type PCR
– Tagget PCR (T-PCR)
– Multiple displasement amplification (MDA)
– GenpmePlex
• Disadvantages
– Sensitivity to quality and purity of the input material
– Formation of the 100-1000 bp fragments (mean – 400 bp).
– Amplification of the 60-80% of the genome only
– Effect of the preferential amplification
– Secondary DNA structures cause nonspecific amplification
– Presence of the active polymerase after the end of the reraction
causes subsequent degeneration of the products
– Allele drop-out phenomenon not excluded if works with single cell
12
14. What? Where? and When?
14
Исследуемый материал:
• PB 1 & 2
• Blastomere (cleavage stage -day 3)
• Trophectoderm (blastocyst – day 5)
Features
PB biopsy Blastomere biopsy TE biopsy
•Indirect data about the
oocyte genotype
•Male factor is not taken
into account
•Mosaicism is not excluded
•Decreasing the embryo
viability
•Subsequent self-correction of
trisomic embryos is not
excluded
•More cells = more DNA = more
accurate diagnostics
•Less mosaicism
•Reduced impact of embryo biopsy
•Economic factor: less embryos to
be analized
•Facilitates the selective embryo
transfer
•Allows to modify endometrium if
needed
•Ability to blastocyst cultivation
and vitrification are needed
20. aCGH-PGS for different groups of patients
20
Prognosis for ET and pregnancy
after aCGH-PGS
Good
Poor
Egg donors
Young women with good ovarian response
Couples with male factor
Women with age factor
Poor ovarian response
Carriers of the chromosomal
rearrangements
22. Future directions
• arrayCGH will compete with SNP-arrays and next-gen.
sequencing-based methods in the range of the
same indications to testing
• FISH-method will not die out (at least in close and
middle future) due to different indication to testing
22
• Further investigations will be
directed to:
•comparison of the arrayCGH results
with embryo morphokinetics and
‘–omics’ characteristics
•development of the accurate criterions
for the selective ET
23. Conclusions
• PGS/PGD testing allows us to increase the performance
of ART technologies and to reduce the amount of
unsuccessful ET.
• Among all similar PGS-methods, array-Comparative
Gnomic Hybridization seems to be the most suitable
approach to detect the embryo aneuploidy.
• Performing of aCGH-PGS with the number of embryos
less than 3 is inappropriate.
• aCGH should not be the cause of ungrounded rejection
of the normal embryo!
23
24. It’s hard to being a good embryo
24
But even more difficult to detect it…
d.mykytenko@genetics.kiev.ua
