Comparative genomic hybridization in PGD

1. Comparative genomic
hybridization in PGD
Dmytro Mykytenko, M.D.,Ph.D.
2012

2. Embryo aneuploidy
• Platteau P., Staessen C.et all, 2004 – 40,5-60%
(depends on pathology)
• NADIYA (total index)
– aCGH – 49,7%
– FISH – 59%
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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
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4. Abnormal oocytes
Gianaroli, L, Magli, C, et al, Glob. libr. women's med., 2008 4

5. Preimplantation genetic testing
• 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.
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
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6. Methods of PGD/PGS
• PCR-based
– PCR, real-time-PCR
– QF-PCR
• Sequencing, mini-Sequencing, next-gen
sequencing
• F.I.S.H.
• array-CGH
• SNP-array
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7. PGD TimeLine
PCR -PGD for Fresh ET
FISH – sex selection
Implementation of aCGH- PGD
the FISH into the CGH- PGD aCGH
cytogenetics
First aCGH-delivery
1970 1980 1990 2000 2010
SNP-
array
PGD
First PCR - PGD
Implementation of the CGH
into the cytogenetics First delivery
after aCGH-PGS
arrayCGH reported for clinical genetics

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. 2008 )
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M Shinawi, S. W. Cheung. Drug Discovery Today. 13 (17/18). 2008

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10. Advantages of the array-CGH
• Microscopic chromosomal rearrangements
– Aneuploidy (with limited mosaicism detection)
– imbalanced rearrangements
– Marker chromosomes
• Submisroscopic alterations
– Subtelomeric imbalanced rearrangements
– Micro-deletion/duplication syndromes
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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
•Limited ability to detect polyploidy
•Limited ability to detect mosaicism
•The method needs a great quantity of DNA
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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
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13. aCGH platforms
Technologies:
•BAC (1-2 Mbp)
•Oligo (10-140kpb)
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14. What? Where? and When?
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 •Mosaicism is not excluded •More cells = more DNA = more
oocyte genotype •Decreasing the embryo accurate diagnostics
•Male factor is not taken viability •Less mosaicism
into account •Subsequent self-correction of •Reduced impact of embryo biopsy
trisomic embryos is not •Economic factor: less embryos to
excluded be analized
•Facilitates the selective embryo
transfer
•Allows to modify endometrium if
needed
•Ability to blastocyst cultivation
and vitrification are needed
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15. 12h BlueGnome
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16. PGD-aCGH (own results)
Euploid embryo
46, XY Aneuploid embryo
47, XY, +7
Aneuploid embryo
Euploid embryo
45, XY, -16
46, XX
arr 20q13.32-q.ter x 1
arr CGH 22q11.1-q.ter x 1
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17. The comparative analysis of aCGH and FISH PGS (own data)
for patients with multiple IVF failure
CGH-PGS * FISH-PGS No PGS
Cycles total 21 41 134
Main Age 33,9±5,4 33,45±5,1 34,3±4,3
Oocytes retrieved 16,7±8,2 13,6±5,1 12,8±4,2
Previously failed cycles 3,9 ±0,7 3,8±0,6 3,2±0,4
Embryos per ET 1,7±0,5 2,1±1,1 2,6±1,3
ET total 12 (+6 expected) 32 132
Completed cycles 84.4% 78,0% 98,5%
Pregnancies total 6 9 18
Pregnancies / OR 40% 22,0% 13,4%
Pregnancies / ET 50% 28,1% 13,6%
* - frequency of aCGH-PGS cancelation – 34,4%
Mykytenko DO, Zukin VD. 1st BRM meeting, 2012
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18. Low Y signal
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19. Low Y signal
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20. aCGH-PGS for different groups of patients
Good Egg donors
Young women with good ovarian response
Prognosis for ET and pregnancy
after aCGH-PGS
Couples with male factor
Women with age factor
Poor ovarian response
Carriers of the chromosomal
rearrangements
Poor
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21. PGD/PGS: aCGH vs other technologies
Criterion aCGH SNP array Next-Gen QF-PCR/ FISH
Sequencing PCR PGS
Comprehensive + + + - -
chrom. screening
Balanced - - ? - Limited
aberrations
Nonbalanced + + + + +
abberations
Microdeletion Limited + + + +
syndromes
UPD - + + - -
Single Gene - +/- + + -
disorders
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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
• 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
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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!
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24. It’s hard to being a good embryo
But even more difficult to detect it…
d.mykytenko@genetics.kiev.ua
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