USA: Livingston, NJ Europe: Barcelona, Spain Oxford, UK Hamburg, Germany Asia: Kobe, Japan South America: Lima, Peru 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
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USA: Livingston, NJ Europe: Barcelona, Spain Oxford, UK Hamburg, Germany
Asia: Kobe, Japan
South America: Lima, Peru
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
• ConversionConversion
• Sequential FISH with 24 probesSequential FISH with 24 probes
control : control : 113113 37.137.1 1.2 1.2 2.7 2.7 46%46% p<0.0001 p<0.0001
Schoolcraft et al. (in press)Schoolcraft et al. (in press)
CGH on blastocyst biopsies:CGH on blastocyst biopsies:Preliminary clinical resultsPreliminary clinical results
Validation of aCGHValidation of aCGH
CGH-based DNA CGH-based DNA Microarray (aCGH)Microarray (aCGH)
Test DNA
Normal DNA
2700 probesSame band resolution as karyotype
aCGH advantagesaCGH advantages
• Results in Results in 2424 hours; allows for PB or day 3 biopsy hours; allows for PB or day 3 biopsy
• Parental DNA Parental DNA notnot required: ad hoc decisions possible required: ad hoc decisions possible
• Detects trisomy originating from mitotic errors or MII Detects trisomy originating from mitotic errors or MII meiotic errors without crossing-over (SNP array may not).meiotic errors without crossing-over (SNP array may not).
46,XX
47,XX+2
44,XX-9-17
46,XY-10 +16
aCGH detected aCGH detected 50%50% more abnormalities than FISH-12 and more abnormalities than FISH-12 and 20%20% more abnormal embryos (Colls et al. 2009)more abnormal embryos (Colls et al. 2009)
Detection of abnormalities: Detection of abnormalities: aCGH vs FISH-12aCGH vs FISH-12
Detectable by FISH
aCGH validation: no resultsaCGH validation: no results
• OldOld11 array: array:Embryos undiagnosed (biopsy day 5): Embryos undiagnosed (biopsy day 5): 50% (n=30)50% (n=30)
• NewNew22 array, old array, old33 amplification: amplification:Embryos undiagnosed (biopsy day 3): Embryos undiagnosed (biopsy day 3): 12% (n=163)12% (n=163)
• NewNew22 array, New array, New44 amplification: amplification:Embryos undiagnosed (biopsy day 3): Embryos undiagnosed (biopsy day 3): 0% (n=73)0% (n=73)Embryos undiagnosed (biopsy day 5): Embryos undiagnosed (biopsy day 5): 0% (n=16)0% (n=16)
• Validation method: Reanalysis of the rest of the embryo Validation method: Reanalysis of the rest of the embryo by FISH with 12 chromosomes plus those found abnormal by FISH with 12 chromosomes plus those found abnormal by aCGHby aCGH
• Error rate (biopsy day 3): Error rate (biopsy day 3): 6% 6% (same as expected by (same as expected by mosaicism), biopsy d5 still n/amosaicism), biopsy d5 still n/a
aCGH results on day 3: validation dataaCGH results on day 3: validation data
Cells from the same embryo:Cells from the same embryo:
- Blastocyst biopsy + CGH + vitrification shows very Blastocyst biopsy + CGH + vitrification shows very high implantation rates (72%, av. Age 38).high implantation rates (72%, av. Age 38).
- Array CGH most likely will produce similar benefits in Array CGH most likely will produce similar benefits in that combinationthat combination
- Array CGH and day 3 biopsy (day 5 results) will detect Array CGH and day 3 biopsy (day 5 results) will detect 20% more abnormal embryos than FISH-12 probes 20% more abnormal embryos than FISH-12 probes
- Additional vitirifcation step may still be advantageousAdditional vitirifcation step may still be advantageous
91,070 embryos analyzed by FISH with 9-12 probes:91,070 embryos analyzed by FISH with 9-12 probes:
Polyploid or haploid:Polyploid or haploid: 7.7%7.7%- plus aneuploidy:- plus aneuploidy: 5.9% (detectable by aCGH)5.9% (detectable by aCGH)
- no other abnormalities:- no other abnormalities: 1.8% (not detectable by aCGH)1.8% (not detectable by aCGH)- Arrested or dysmorphic:- Arrested or dysmorphic: 1.6% (unlikely replaced)1.6% (unlikely replaced)- Good morphology:- Good morphology: 0.2%0.2% (risk of misdiagnosis) (risk of misdiagnosis)
SNP arrays may not detect mitotic trisomiesSNP arrays may not detect mitotic trisomies
91,070 embryos analyzed by FISH with 9-12 probes:91,070 embryos analyzed by FISH with 9-12 probes:
Complex abnormal mosaics:Complex abnormal mosaics: 26,624 (29%)26,624 (29%)- with only trisomies:- with only trisomies: 4,029 4,029
- of mitotic origin (76.2%)*:- of mitotic origin (76.2%)*: 3,070 3,070 (3.4% potential error)(3.4% potential error)