development and validation of an accurate quantitative real time

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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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Down 

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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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Sperm Ovum

PGD Program

Preimplantation Genetic Diagnosis

Evil

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Current Methods1. Comparative genomic

hybridization (CGH)

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Current Methods1. Comparative genomic

hybridization (CGH)

2. SiNP microarray technologies

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Current Methods1. Comparative genomic

hybridization (CGH)

2. SiNP microarray technologies3. Fluorescence in situ 

hybridization (FISH)

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Current Methods1. Comparative genomic

hybridization (CGH)

2. SiNP microarray technologies3. Fluorescence in situ 

hybridization (FISH)

Waste Time

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Current Methods1. Comparative genomic

hybridization (CGH)

2. SiNP microarray technologies3. Fluorescence in situ 

hybridization (FISH)

Chip is not cheap

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Solution 咻咻

咻咻

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A Brief Introduction to Quantitative PCR

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MATERIALS AND METHODS

Experimental Design

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Two-phase design

Phase IUsing the cell-lines to evaluate the system and establish baseline database.

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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.

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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

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Phase II : Embryos

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qPCRStatistics

Alkaline lysis 18 cycles of multiplex amplification

Real-time PCR

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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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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

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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

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Establish baseline database

GM00323

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RESULTTo evaluate the utility

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FIGURE 1Examples of qPCR-based 24-chromosome copy number results from 5-cell samples derived from nine cell lines with previously well characterized karyotypes.

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FIGURE 1

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FIGURE 2

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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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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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FIGURE 3-1Examples of (gray) single-nucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies.

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FIGURE 3-2Examples of (gray) single-nucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies.

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DISCUSSIONqPCR-based methodology provides the first opportunity for same-day trophectoderm biopsy 24-chromosome aneuploidy screening and fresh blastocyst transfer.

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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

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NO question !No comment !