Development and validation of an accurate quantitative real time

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Development and validation of an accurate quantitative real time

  1. 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 1
  2. 2. Down 2
  3. 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 3
  4. 4. 4
  5. 5. Preimplantation Genetic Diagnosis Sperm Ovum PGD Program Evil 6
  6. 6. Current Methods 1. Comparative genomic hybridization (CGH) 7
  7. 7. Current Methods 1. Comparative genomic hybridization (CGH) 2. SiNP microarray technologies 8
  8. 8. Current Methods 1. Comparative genomic hybridization (CGH) 2. SiNP microarray technologies 3. Fluorescence in situ hybridization (FISH) 9
  9. 9. Current Methods 1. Comparative genomic hybridization (CGH) Waste Time 2. SiNP microarray technologies 3. Fluorescence in situ hybridization (FISH) 10
  10. 10. Current Methods 1. Comparative genomic hybridization (CGH) Chip is not cheap 2. SiNP microarray technologies 3. Fluorescence in situ hybridization (FISH) 11
  11. 11. Solution 咻 咻 咻 咻 12
  12. 12. A Brief Introduction to Quantitative PCR 13
  13. 13. 14
  14. 14. MATERIALS AND METHODS Experimental Design 16
  15. 15. Two-phase design Phase I Using the cell-lines to evaluate the system and establish baseline database. 17
  16. 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. 18
  17. 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 19
  18. 18. Phase II : Embryos 20
  19. 19. qPCR Statistics Alkaline lysis 18 cycles of multiplex amplification Real-time PCR 21
  20. 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 22
  21. 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 23
  22. 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 24
  23. 23. Establish baseline database GM00323 25
  24. 24. RESULT To evaluate the utility 26
  25. 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. 27
  26. 26. FIGURE 1 28
  27. 27. FIGURE 2 29
  28. 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 30
  29. 29. FIGURE 2 Chromosome-specific consistency of 99.94% (1,703/1,704) and an overall 24-chromosome diagnosis consistencyof 98.6% (70/71) 31
  30. 30. FIGURE 3-1 Examples of (gray) singlenucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies. 32
  31. 31. FIGURE 3-2 Examples of (gray) singlenucleotide polymorphism microarray– and (white) qPCR-based 24-chromosome copy number results from blastocyst-stage embryo biopsies. 33
  32. 32. DISCUSSION qPCR-based methodology provides the first opportunity for same-day trophectoderm biopsy 24-chromosome aneuploidy screening and fresh blastocyst transfer. 34
  33. 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 35
  34. 34. NO question ! No comment ! 36

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