Munne et al ASRM 2009 Abstract O6

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

• Comparative Genome Hybridization (CGH)Comparative Genome Hybridization (CGH)

• Single Nucleotide Polymorphism (SNP) arraySingle Nucleotide Polymorphism (SNP) array

• Array CGH (aCGH)Array CGH (aCGH)

Different strategies forDifferent strategies forAnalyzing all chromosomesAnalyzing all chromosomes

Kallioniemi et al. (1992), applied to single cells by Wells et al. (1999)

NormalNormal TrisomyTrisomy MonosomyMonosomy

Normal DNANormal DNATest DNATest DNA

Comparative Genome Comparative Genome Hybridization (CGH)Hybridization (CGH)

DisadvantageDisadvantage: time-consuming: time-consumingrequiring embryo freezing.requiring embryo freezing.

IndicationIndication: blastocyst biopsy: blastocyst biopsy• >300 cases done>300 cases done• blastocysts analyzable: 94%blastocysts analyzable: 94%• Normal blastocysts: 52%Normal blastocysts: 52%• 100%100% survival after freeze/thaw survival after freeze/thaw

CGH on blastocyst biopsies:CGH on blastocyst biopsies:ResultsResults

Schoolcraft et al. (in press)Schoolcraft et al. (in press)

CyclesCycles Mat.Mat. Prev.Prev. embryosembryos implantation implantationageage failedfailed replaced replaced (+ sac) (+ sac)

cyclescycles

CGH : CGH : 45 45 37.737.7 2.4 2.4 2.0 2.0 72%72%

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)

1: Undisclosed; 2: Bluegnome; 3: Genomplex, Sigma; 4: Sureplex, Bluegnome

aCGH validation: error rateaCGH validation: error rate

• 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:

XXOO,, ++2, 2, --4p,4p, ++11,11, --13, 13, --18, 18, --2222 XX,XX, --2, 2, --4p,4p, ++4q31,4q31, --13, 13, --18, 18, --2222 XXXXXX,, ++2, 2, --4p,4p, --4q31,4q31, ++11,11, --13, 13, --18, 18, --2222 XXOO,, ++2, 2, ++4p,4p, --4q31,4q31, --11,11, --13, 13, --18, 18, --2222

Who said mosaicism was a FISH artifact?

Comparison of platformsComparison of platforms

FISH FISH CGHCGH arrayarray SNPSNP 1212 CGHCGH arraysarraysprobesprobes quant./qual. quant./qual.

Day 3 biopsy/ day 5 resultsDay 3 biopsy/ day 5 results yesyes nono yesyes yesyesParental DNA neededParental DNA needed nono nono nono yesyesDetect gene defectsDetect gene defects nono nono nono yesyesDetect polyploidy (errors)Detect polyploidy (errors) yesyes 0.2%0.2% 0.2%0.2% yesyesDetect MII trisomies w/o crossoverDetect MII trisomies w/o crossover yesyes yesyes yesyes yes yes / / nonoDetect mitotic trisomies (errors)Detect mitotic trisomies (errors) yesyes yesyes yesyes yes yes / / 3.4%3.4%Error rate Error rate 7%7% 8%8% 6%6% unkunkNo Results RateNo Results Rate 3%3% 6%6% 0%0% unkunk

Increased implantation rate Increased implantation rate ++ + + ++ + + unkunk unkunkReprogenetics data.Reprogenetics data.

- 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

Conclusions: comprehensive Conclusions: comprehensive chromosome analysischromosome analysis

Santiago MunnSantiago Munnéé, PhD, Director, PhD, DirectorJacques Cohen, PhD, DirectorJacques Cohen, PhD, Director

[email protected] [email protected] www.reprogenetics.com www.reprogenetics.com

Pere Colls, PhDPere Colls, PhDDagan Wells, PhDDagan Wells, PhD

Tomas Escudero, MSTomas Escudero, MSKelly Ketterson, MSKelly Ketterson, MS

Jill Fischer, MSJill Fischer, MSJohn Zheng, MD John Zheng, MD

George Pieczenik, PhDGeorge Pieczenik, PhDCristina Gutierrez, PhDCristina Gutierrez, PhD

Piedad GarzonPiedad Garzon

Jorge Sanchez, MSJorge Sanchez, MSTim Schimmel, BSTim Schimmel, BSSasha Sadowy, BSSasha Sadowy, BS

Sophia Tormasi, BSSophia Tormasi, BSJessica Vega, MSJessica Vega, MS

N-neka Esprit-Ngachou, BSN-neka Esprit-Ngachou, BSLaurie FerraraLaurie Ferrara

Renata Prates, BSRenata Prates, BSBekka Sellon-WrightBekka Sellon-Wright

USAUSA

SpainSpainMireia Sandalinas, MSMireia Sandalinas, MSCarles Giménez, PhDCarles Giménez, PhD

César Arjona, MSCésar Arjona, MSAna Jiménez, PhDAna Jiménez, PhDElena Garcia, MS Elena Garcia, MS

JapanJapanTetsuo Otani, MDTetsuo Otani, MD

Muriel RocheMuriel RocheMiho MizuikeMiho Mizuike

UKUKDagan Wells, PhDDagan Wells, PhD

Elpida Fragouli, PhDElpida Fragouli, PhDSamer Alfarawati, MSSamer Alfarawati, MS

South AmericaSouth AmericaPaul Lopez, BSPaul Lopez, BS

Luis Alberto Guzman, BSLuis Alberto Guzman, BSFrancisco Parera, PhDFrancisco Parera, PhD

GermanyGermanyKarsten Held, MDKarsten Held, MD

aCGH, CGH cannot detect polyploidyaCGH, CGH cannot detect polyploidy

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)

* Munne et al. (2002)* Munne et al. (2002)