development and validation of an accurate quantitative real time
TRANSCRIPT
![Page 1: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/1.jpg)
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
![Page 2: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/2.jpg)
2
Down
![Page 3: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/3.jpg)
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
![Page 4: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/4.jpg)
4
![Page 5: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/5.jpg)
6
Sperm Ovum
PGD Program
Preimplantation Genetic Diagnosis
Evil
![Page 6: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/6.jpg)
7
Current Methods1. Comparative genomic
hybridization (CGH)
![Page 7: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/7.jpg)
8
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies
![Page 8: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/8.jpg)
9
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies3. Fluorescence in situ
hybridization (FISH)
![Page 9: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/9.jpg)
10
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies3. Fluorescence in situ
hybridization (FISH)
Waste Time
![Page 10: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/10.jpg)
11
Current Methods1. Comparative genomic
hybridization (CGH)
2. SiNP microarray technologies3. Fluorescence in situ
hybridization (FISH)
Chip is not cheap
![Page 11: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/11.jpg)
12
Solution 咻咻
咻咻
![Page 12: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/12.jpg)
13
A Brief Introduction to Quantitative PCR
![Page 13: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/13.jpg)
14
![Page 14: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/14.jpg)
16
MATERIALS AND METHODS
Experimental Design
![Page 15: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/15.jpg)
17
Two-phase design
Phase IUsing the cell-lines to evaluate the system and establish baseline database.
![Page 16: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/16.jpg)
18
Two-phase design
Phase IUsing the cell-lines to evaluate the system and establish baseline database.
Phase II
Use 71 of blastocysts to evaluate the system.
![Page 17: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/17.jpg)
19
Catalog ID Cell Type Karyotypes Numbers GM00323 Fibroblast 46,XY 10+7
GM04610 Fibroblast 47,XX,t8 5
GM09286 Fibroblast 47,XY,t9 4
GM02948 Fibroblast 47,XY,t13 5
GM04435 Fibroblast 48,XY,t16,t21 2
AG16778 B-Lymphocyte 46,XX 3
AG16782 B-Lymphocyte 46,XY 3
GM01454 B-Lymphocyte 47,XY,t12 5
AG16777 B-Lymphocyte 47,XX,t21 5
Phase I : Cell Lines
![Page 18: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/18.jpg)
20
Phase II : Embryos
![Page 19: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/19.jpg)
21
qPCRStatistics
Alkaline lysis 18 cycles of multiplex amplification
Real-time PCR
![Page 20: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/20.jpg)
22
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
![Page 21: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/21.jpg)
23
StatisticsΔCt 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
Specific Chromosome
4 16
![Page 22: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/22.jpg)
24
StatisticsΔCt 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
ΔCt 4 16 4 21 336
![Page 23: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/23.jpg)
25
Establish baseline database
GM00323
![Page 24: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/24.jpg)
26
RESULTTo evaluate the utility
![Page 25: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/25.jpg)
27
FIGURE 1Examples of qPCR-based 24-chromosome copy number results from 5-cell samples derived from nine cell lines with previously well characterized karyotypes.
![Page 26: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/26.jpg)
28
FIGURE 1
![Page 27: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/27.jpg)
29
FIGURE 2
![Page 28: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/28.jpg)
30
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
![Page 29: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/29.jpg)
31
FIGURE 2
Chromosome-specific consistency of 99.94% (1,703/1,704) and an overall 24-chromosome diagnosis consistencyof 98.6% (70/71)
![Page 30: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/30.jpg)
32
FIGURE 3-1Examples of (gray) single-nucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies.
![Page 31: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/31.jpg)
33
FIGURE 3-2Examples of (gray) single-nucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies.
![Page 32: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/32.jpg)
34
DISCUSSIONqPCR-based methodology provides the first opportunity for same-day trophectoderm biopsy 24-chromosome aneuploidy screening and fresh blastocyst transfer.
![Page 33: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/33.jpg)
35
Back to the start …
1. Enhance embryo selection
2. Increase implantation rates
3. Reduce the incidence of miscarriage
4. Reduce the time of implantation
5. Cost-Down
PGD
qPCR
![Page 34: Development and validation of an accurate quantitative real time](https://reader038.vdocuments.us/reader038/viewer/2022110118/55504589b4c9058f768b4d41/html5/thumbnails/34.jpg)
36
NO question !No comment !