1. Development and validation of an accurate
quantitative real-time polymerase chain reaction–
based assay for human blastocyst comprehensive
chromosomal aneuploidy screening
Author : Nathan R. Treff (Ph.D), et al.
Fertility and Sterility (IF: 4.174) VOL. 97 NO. 4 / APRIL 2012
Speaker : Hung,
Mau-Ren
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2. Down
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3. The Reason We Start
1. Enhance embryo selection
2. Increase implantation rates
3. Reduce the incidence of miscarriage
4. Reduce the time of implantation
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4. 4

5. Preimplantation Genetic Diagnosis
Sperm Ovum
PGD Program
Evil
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6. Current Methods
1. Comparative genomic
hybridization (CGH)
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7. Current Methods
1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies
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8. Current Methods
1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies
3. Fluorescence in situ
hybridization (FISH)
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9. Current Methods
1. Comparative genomic
hybridization (CGH)
Waste Time
2. SiNP microarray technologies
3. Fluorescence in situ
hybridization (FISH)
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10. Current Methods
1. Comparative genomic
hybridization (CGH)
Chip is not cheap
2. SiNP microarray technologies
3. Fluorescence in situ
hybridization (FISH)
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11. Solution
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12. A Brief Introduction to Quantitative PCR
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13. 14

14. MATERIALS
AND METHODS
Experimental Design
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15. Two-phase design
Phase I
Using the cell-lines to evaluate
the system and establish
baseline database.
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16. Two-phase design
Phase I
Using the cell-lines to evaluate
the system and establish
baseline database.
Phase II
Use 71 of blastocysts to
evaluate the system.
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17. Phase I : Cell Lines
Catalog ID
GM00323
GM04610
GM09286
GM02948
GM04435
AG16778
AG16782
GM01454
AG16777
Cell Type
Karyotypes
Numbers
Fibroblast
46,XY
10+7
Fibroblast
Fibroblast
47,XX,t8
47,XY,t9
5
4
Fibroblast
Fibroblast
47,XY,t13
48,XY,t16,t21
5
2
B-Lymphocyte
46,XX
3
B-Lymphocyte
B-Lymphocyte
46,XY
47,XY,t12
3
5
B-Lymphocyte
47,XX,t21
5
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18. Phase II : Embryos
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19. qPCR
Statistics
Alkaline lysis
18 cycles of
multiplex amplification
Real-time PCR
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20. Statistics
ΔCt was calculated from the average ∆Ct of the
16 reactions targeting a specific Chromosome
minus the average ∆ Ct of all of the 336 reactions
targeting all of the remaining autosomes
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21. Statistics
ΔCt was calculated from
the average ∆Ct of the 16
reactions targeting a
specific Chromosome
minus the average ∆ Ct of
all of the 336 reactions
targeting all of the
remaining autosomes
4
16
Specific Chromosome
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22. Statistics
ΔCt was calculated from
the average ∆Ct of the 16
ΔCt a 4
reactions targeting
specific Chromosome
minus the average ∆ Ct of
all of the 336 reactions
targeting all of the
remaining autosomes
16
4
21
336
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23. Establish baseline database
GM00323
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24. RESULT
To evaluate the utility
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25. FIGURE 1
Examples of qPCR-based
24-chromosome copy
number results from 5cell samples derived from
nine cell lines with
previously well
characterized karyotypes.
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26. FIGURE 1
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27. FIGURE 2
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28. FIGURE 2
97.6% reliability of obtaining a
diagnosis and a100% level of
consistency of chromosome-specific
(n =984) and 24-chromosome copy
number (n =41) assignments
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29. FIGURE 2
Chromosome-specific consistency
of 99.94% (1,703/1,704) and an
overall 24-chromosome diagnosis
consistencyof 98.6% (70/71)
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30. FIGURE 3-1
Examples of (gray) singlenucleotide polymorphism
microarray–
and (white) qPCR-based
24-chromosome copy
number results from
blastocyst-stage embryo
biopsies.
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31. FIGURE 3-2
Examples of (gray) singlenucleotide polymorphism
microarray–
and (white) qPCR-based
24-chromosome copy
number results from
blastocyst-stage embryo
biopsies.
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32. DISCUSSION
qPCR-based methodology
provides the first opportunity
for same-day trophectoderm
biopsy 24-chromosome
aneuploidy screening and
fresh blastocyst transfer.
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33. Back to the start …
1. Enhance embryo selection
2. Increase implantation rates
PGD
3. Reduce the incidence of miscarriage
4. Reduce the time of implantation
5. Cost-Down
qPCR
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34. NO question !
No comment !
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