1) Comparative genomic hybridization (aCGH) allows for high-resolution screening of chromosomal copy number variations in embryos, detecting aneuploidies and structural rearrangements. 2) Array CGH analysis of embryos from patients with multiple IVF failures found a higher rate of euploid embryos and pregnancies compared to FISH or no PGS. 3) Future directions include comparing aCGH results to embryo development and 'omics' characteristics to improve embryo selection for transfer.

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

9. 9

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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