<ul><ul><li>USA: </li></ul></ul><ul><li>Livingston, NJ </li></ul><ul><li>Europe: </li></ul><ul><li>Barcelona, Spain </li><...
<ul><li>Conversion </li></ul><ul><li>Sequential FISH with 24 probes </li></ul><ul><li>Comparative Genome Hybridization (CG...
Kallioniemi et al. (1992), applied to single cells by Wells et al. (1999) Normal DNA Test DNA Comparative Genome  Hybridiz...
<ul><li>Disadvantage :  time-consuming </li></ul><ul><li>requiring  embryo freezing. </li></ul><ul><li>Indication : blasto...
<ul><li>Cycles Mat. Prev. embryos  implantation </li></ul><ul><li>age failed replaced   (+ sac)   cycles </li></ul><ul><li...
Validation of aCGH
CGH-based DNA  Microarray (aCGH) Test  DNA Normal  DNA 2700 probes Same band resolution as karyotype
aCGH advantages <ul><li>Results in  24  hours; allows for PB or day 3 biopsy </li></ul><ul><li>Parental DNA  not  required...
46,XX
47,XX+2
44,XX-9-17
46,XY-10 +16   aCGH detected  50%  more abnormalities than FISH-12 and  20%  more abnormal embryos (Colls et al. 2009) Det...
aCGH validation: no results <ul><li>Old 1  array: </li></ul><ul><li>Embryos undiagnosed (biopsy day 5):  50% (n=30) </li><...
aCGH validation: error rate <ul><li>Validation method: Reanalysis of the rest of the embryo by FISH with 12 chromosomes pl...
aCGH results on day 3: validation data Cells from the same embryo: X O , + 2,  - 4p, + 11, - 13,  - 18,  - 22 XX,   - 2,  ...
Comparison of platforms FISH  CGH array SNP   12 CGH arrays probes   quant./qual. Day 3 biopsy/ day 5 results yes no yes y...
<ul><li>Blastocyst biopsy + CGH + vitrification shows very high implantation rates (72%, av. Age 38). </li></ul><ul><li>Ar...
Santiago Munn é , PhD, Director Jacques Cohen, PhD, Director munne@reprogenetics.com  www.reprogenetics.com  Pere Colls, P...
aCGH, CGH cannot detect polyploidy 91,070 embryos analyzed by FISH with 9-12 probes: Polyploid or haploid: 7.7% - plus ane...
SNP arrays may not detect mitotic trisomies 91,070 embryos analyzed by FISH with 9-12 probes: Complex abnormal mosaics: 26...
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Munne et al ASRM 2009 Abstract O6

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presentation on the validation studies of array CGH validation for its use in PGD for the detection of ALL chromosome aneuploidies.

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Munne et al ASRM 2009 Abstract O6

  1. 1. <ul><ul><li>USA: </li></ul></ul><ul><li>Livingston, NJ </li></ul><ul><li>Europe: </li></ul><ul><li>Barcelona, Spain </li></ul><ul><li>Oxford, UK </li></ul><ul><li>Hamburg, Germany </li></ul><ul><li>Asia: </li></ul><ul><li>Kobe, Japan </li></ul><ul><li>South America: </li></ul><ul><li>Lima, Peru </li></ul>VALIDATION OF MICROARRAY CGH FOR PGD BY FISH REANALYSIS Santiago Munné, Cristina Gutierrez-Mateo, Jorge Sanchez-Garcia, Kelly Ketterson, Renata Prates, Daniel Kenigsberg Reprogenetics, Livingston, NJ; Long Island IVF, Melville, NY
  2. 2. <ul><li>Conversion </li></ul><ul><li>Sequential FISH with 24 probes </li></ul><ul><li>Comparative Genome Hybridization (CGH) </li></ul><ul><li>Single Nucleotide Polymorphism (SNP) array </li></ul><ul><li>Array CGH (aCGH) </li></ul>Different strategies for Analyzing all chromosomes
  3. 3. Kallioniemi et al. (1992), applied to single cells by Wells et al. (1999) Normal DNA Test DNA Comparative Genome Hybridization (CGH) Normal Trisomy Monosomy
  4. 4. <ul><li>Disadvantage : time-consuming </li></ul><ul><li>requiring embryo freezing. </li></ul><ul><li>Indication : blastocyst biopsy </li></ul><ul><li>>300 cases done </li></ul><ul><li>blastocysts analyzable: 94% </li></ul><ul><li>Normal blastocysts: 52% </li></ul><ul><li>100% survival after freeze/thaw </li></ul>CGH on blastocyst biopsies: Results Schoolcraft et al. (in press)
  5. 5. <ul><li>Cycles Mat. Prev. embryos implantation </li></ul><ul><li>age failed replaced (+ sac) cycles </li></ul><ul><li>CGH : 45 37.7 2.4 2.0 72% </li></ul><ul><li>control : 113 37.1 1.2 2.7 46% p<0.0001 </li></ul><ul><li>Schoolcraft et al. (in press) </li></ul>CGH on blastocyst biopsies: Preliminary clinical results
  6. 6. Validation of aCGH
  7. 7. CGH-based DNA Microarray (aCGH) Test DNA Normal DNA 2700 probes Same band resolution as karyotype
  8. 8. aCGH advantages <ul><li>Results in 24 hours; allows for PB or day 3 biopsy </li></ul><ul><li>Parental DNA not required: ad hoc decisions possible </li></ul><ul><li>Detects trisomy originating from mitotic errors or MII meiotic errors without crossing-over (SNP array may not). </li></ul>
  9. 9. 46,XX
  10. 10. 47,XX+2
  11. 11. 44,XX-9-17
  12. 12. 46,XY-10 +16 aCGH detected 50% more abnormalities than FISH-12 and 20% more abnormal embryos (Colls et al. 2009) Detection of abnormalities: aCGH vs FISH-12 Detectable by FISH
  13. 13. aCGH validation: no results <ul><li>Old 1 array: </li></ul><ul><li>Embryos undiagnosed (biopsy day 5): 50% (n=30) </li></ul><ul><li>New 2 array, old 3 amplification: </li></ul><ul><li>Embryos undiagnosed (biopsy day 3): 12% (n=163) </li></ul><ul><li>New 2 array, New 4 amplification: </li></ul><ul><li>Embryos undiagnosed (biopsy day 3): 0% (n=73) </li></ul><ul><li>Embryos undiagnosed (biopsy day 5): 0% (n=16) </li></ul>1: Undisclosed; 2: Bluegnome; 3: Genomplex, Sigma; 4: Sureplex, Bluegnome
  14. 14. aCGH validation: error rate <ul><li>Validation method: Reanalysis of the rest of the embryo by FISH with 12 chromosomes plus those found abnormal by aCGH </li></ul><ul><li>Error rate (biopsy day 3): 6% (same as expected by mosaicism), biopsy d5 still n/a </li></ul>
  15. 15. aCGH results on day 3: validation data Cells from the same embryo: X O , + 2, - 4p, + 11, - 13, - 18, - 22 XX, - 2, - 4p, + 4q31, - 13, - 18, - 22 XX X , + 2, - 4p, - 4q31, + 11, - 13, - 18, - 22 X O , + 2, + 4p, - 4q31, - 11, - 13, - 18, - 22 Who said mosaicism was a FISH artifact?
  16. 16. Comparison of platforms FISH CGH array SNP 12 CGH arrays probes quant./qual. Day 3 biopsy/ day 5 results yes no yes yes Parental DNA needed no no no yes Detect gene defects no no no yes Detect polyploidy (errors) yes 0.2% 0.2% yes Detect MII trisomies w/o crossover yes yes yes yes / no Detect mitotic trisomies (errors) yes yes yes yes / 3.4% Error rate 7% 8% 6% unk No Results Rate 3% 6% 0% unk Increased implantation rate + + + + unk unk Reprogenetics data.
  17. 17. <ul><li>Blastocyst biopsy + CGH + vitrification shows very high implantation rates (72%, av. Age 38). </li></ul><ul><li>Array CGH most likely will produce similar benefits in that combination </li></ul><ul><li>Array CGH and day 3 biopsy (day 5 results) will detect 20% more abnormal embryos than FISH-12 probes </li></ul><ul><li>Additional vitirifcation step may still be advantageous </li></ul>Conclusions: comprehensive chromosome analysis
  18. 18. Santiago Munn é , PhD, Director Jacques Cohen, PhD, Director munne@reprogenetics.com www.reprogenetics.com Pere Colls, PhD Dagan Wells, PhD Tomas Escudero, MS Kelly Ketterson, MS Jill Fischer, MS John Zheng, MD George Pieczenik, PhD Cristina Gutierrez, PhD Piedad Garzon Jorge Sanchez, MS Tim Schimmel, BS Sasha Sadowy, BS Sophia Tormasi, BS Jessica Vega, MS N-neka Esprit-Ngachou, BS Laurie Ferrara Renata Prates, BS Bekka Sellon-Wright USA Spain Mireia Sandalinas, MS Carles Giménez, PhD César Arjona, MS Ana Jiménez, PhD Elena Garcia, MS Japan Tetsuo Otani, MD Muriel Roche Miho Mizuike UK Dagan Wells, PhD Elpida Fragouli, PhD Samer Alfarawati, MS South America Paul Lopez, BS Luis Alberto Guzman, BS Francisco Parera, PhD Germany Karsten Held, MD
  19. 19. aCGH, CGH cannot detect polyploidy 91,070 embryos analyzed by FISH with 9-12 probes: Polyploid or haploid: 7.7% - plus aneuploidy: 5.9% (detectable by aCGH) - no other abnormalities: 1.8% (not detectable by aCGH) - Arrested or dysmorphic: 1.6% (unlikely replaced) - Good morphology: 0.2% (risk of misdiagnosis)
  20. 20. SNP arrays may not detect mitotic trisomies 91,070 embryos analyzed by FISH with 9-12 probes: Complex abnormal mosaics: 26,624 (29%) - with only trisomies: 4,029 - of mitotic origin (76.2%)*: 3,070 (3.4% potential error) * Munne et al. (2002)
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