USA: Livingston, NJ Europe: Barcelona, Spain Oxford, UK Hamburg, Germany

Asia: Kobe, Japan

South America: Lima, Peru

Santiago MunnéSantiago Munné

Preimplantation Genetic Diagnosis

Reprogenetics servicesReprogenetics services

• CLIA and NYDH approved laboratory. CLIA and NYDH approved laboratory. • Approximately 1/3 (150 centers) of US PGD casesApproximately 1/3 (150 centers) of US PGD cases• >20,000 PGD cycles performed>20,000 PGD cycles performed• PGD Services: PGD Services:

- 24-chromosome analysis by array CGH- 24-chromosome analysis by array CGH- translocations by FISH or array CGH- translocations by FISH or array CGH- gene defects by PCR or SNP array- gene defects by PCR or SNP array- Blasotcyst biopsy and vitrification teaching- Blasotcyst biopsy and vitrification teaching

• PGD portal for the electronic medical records of PGD portal for the electronic medical records of eeIVFIVF

High rates of High rates of Chromosome abnormalitiesChromosome abnormalities

embryos analyzed: 6054. Morphologically normal embryos: 3751. Source: Munnembryos analyzed: 6054. Morphologically normal embryos: 3751. Source: Munnéé et al. 2007. et al. 2007.Similar results also found by Munne et al 1995, Marquez et al. 2000, Magli et al. 2007.Similar results also found by Munne et al 1995, Marquez et al. 2000, Magli et al. 2007.

% chromosomally abnormal embryos

56%

Maternal age

Morphology:

The majority of embryos with ‘good’ The majority of embryos with ‘good’ morphology are chromosomally abnormalmorphology are chromosomally abnormal

PGDPGDHypothesisHypothesis

PGD may improve ART outcome in women of advanced maternal agePGD may improve ART outcome in women of advanced maternal ageMunné et al. (1993)

Despite large studies indicating the Despite large studies indicating the

advantages of aneuploidy screening, the advantages of aneuploidy screening, the

notion that PGS for infertility is beneficial is notion that PGS for infertility is beneficial is

not yet shared uniformly.not yet shared uniformly.

Contradicting PGD results Contradicting PGD results using day 3 biopsy and FISHusing day 3 biopsy and FISH

Positive effectGianaroli et al. 1999Munne et al 1999Gianaroli et al 2001aGianaroli et al. 2001bMunne et al. 2003Gianaroli et al. 2004Munne et al. 2005Munne et al 2006Verlinsky et al. 2005Colls et al. 2007Garrisi et al. 2009Rubio et al. 2009

No effect (small) Werlin et al. 2003Jansen et al. 2008Mersereau et al. 2008Scholcraft et al. 2009

No effect (Large)Staessen et al. 2004Platteau et al. 2005

Negative effect Mastenbroek et al. 2007Hardarson et al. 2008

Contradicting PGD results Contradicting PGD results using day 3 biopsy and FISHusing day 3 biopsy and FISH

Proposed explanations:

1)Mosaicism

2)Self-correction of abnormal embryos

3)Sub-optimal PGD and biopsy methods

Mosaicism Mosaicism

592 embryos found abnormal by PGD were reanalyzed and found to be: 592 embryos found abnormal by PGD were reanalyzed and found to be: normal normal 1313 mosaic <49% abnormalmosaic <49% abnormal 2727 mosaic 50-99% abnormal mosaic 50-99% abnormal 124124 mosaic 100% abnormal mosaic 100% abnormal 297297 homogeneously abnormal homogeneously abnormal 131131

Colls et al. (2007)Colls et al. (2007)

Mosaicism produces <7% misdiagnosisMosaicism produces <7% misdiagnosis

1[13]1[16]2[18]2[21]1[22]

2[13]1[16]2[18]2[21]2[22]

1[13]1[16]2[18]2[21]1[22]

1[16] 2[13]2[16]2[18]2[21]2[22]

2[13]1[16]2[18]1[21]1[22]

2[13]3[18]1[21]1[22]

3[13]1[16]2[18]1[21]3[22]

1[13]1[16]1[18]1[21]1[22]3[13]1[16]2[18]1[21]3[22]

6.8%6.8%

The FISH error of the assay translates into a The FISH error of the assay translates into a

clinical misdiagnosis rate of 0.5%clinical misdiagnosis rate of 0.5%

NEGATIVE PREDICTIVE VALUE: NEGATIVE PREDICTIVE VALUE: 99.5%99.5%- Data from >150 IVF centers referring to ReprogeneticsData from >150 IVF centers referring to Reprogenetics

- 10 aneuploid abortions of 2148 implanted fetuses 10 aneuploid abortions of 2148 implanted fetuses

- Average maternal age = 37 years- Average maternal age = 37 years

Clinical misdiagnosisClinical misdiagnosis

self-correction mythsself-correction myths

UNIPARENTAL DISOMY: UNIPARENTAL DISOMY: a trisomy correction creates a zygote with 2 chromosomes of one parent, a trisomy correction creates a zygote with 2 chromosomes of one parent, none of the other (trisomy rescue). Theoretical chance : 1/3 none of the other (trisomy rescue). Theoretical chance : 1/3

EXAMPLE TRISOMY 15:EXAMPLE TRISOMY 15:Prognosis is ethal because of imprintingPrognosis is ethal because of imprinting

Trisomy 15 in cleavage stage embryos:Trisomy 15 in cleavage stage embryos: 1.874% a1.874% aUPD-15 in newborns: UPD-15 in newborns: 0.001% b0.001% bEstimated correction of trisomy 15 to UDP:Estimated correction of trisomy 15 to UDP: 1/3 c 1/3 c

Trisomy 15 day 3 embryos that self-corrected: Trisomy 15 day 3 embryos that self-corrected: 1.874 x 0.001 / 3 = 1.874 x 0.001 / 3 = 0.56%0.56%

a: Munne et al. (2004), b: From: OMIM, c: 3 X UPDa: Munne et al. (2004), b: From: OMIM, c: 3 X UPD

Trisomy correction is rare: UPD evidenceTrisomy correction is rare: UPD evidence

Fetus seldom self correct: Fetus seldom self correct: it’s the placenta that becomes abnormalit’s the placenta that becomes abnormal

This work questions the assumption that placental confined mosaicism is the result of fetal self-correction. At the contrary, it suggests that normal fetuses may develop abnormal placenta.

Sub-optimizal methodsSub-optimizal methods(FISH studies, day 3 biopsy)(FISH studies, day 3 biopsy)

Optimal PGSOptimal PGS Questionable PGSQuestionable PGS

Biopsy mediaBiopsy media with aminoacidswith aminoacids simple mediasimple media

Biopsy time / embryoBiopsy time / embryo 1 min1 min > 5 min> 5 min

# cells biopsied# cells biopsied oneone twotwo

Fixation methodFixation method Carnoy’sCarnoy’s Tween 20Tween 20

# chromosomes tested# chromosomes tested ≥≥88 66

# analysts / case# analysts / case 22 11

Use of NRR*Use of NRR* yesyes nono

Large experienceLarge experience yesyes nono

Error rateError rate <10%<10% 10-50%10-50%

Number of zygotesNumber of zygotes >5>5 5 5

*NRR: No result rescue, or re-testing of dubious chromosome with different probes.

Optimal PGS methodsOptimal PGS methods

CONTROL 2-CELLS BIOPSIED

Staessen et al (2004)

11.5%

17.1%

Implantation

Staessen et al. (2004):

- No significant differences- But 2 cells biopsied

PGD for AMA: randomized studiesPGD for AMA: randomized studies

“ The data presented here clearly indicates that two cell biopsy significantly impacts clinical outcome. Our previous report providing no arguments in favor of PGS (Staessen et al., 2004) was criticized by others arguing that PGS might have been beneficial if only one cell had been removed (Cohen et al., 2007). In respect to the present findings, this criticism seems justified”.

P < 0.001

Two cell biopsy is detrimentalTwo cell biopsy is detrimental

1)1) 20% of 20% of cyclescycles undiagnosed (literature: 1-3% of undiagnosed (literature: 1-3% of embryos embryos *)*)2) 59% implantation reduction due to biopsy:2) 59% implantation reduction due to biopsy:

3) 3) Average number of embryos analyzed was only 5Average number of embryos analyzed was only 54) Chromosomes 15 and 22 (21% abnormalities) not analyzed4) Chromosomes 15 and 22 (21% abnormalities) not analyzed

59% reduction59% reduction

implantationimplantation Control Control 14.7% 14.7% Biopsied, no PGDBiopsied, no PGD 6.0%6.0% Biopsied and PGDBiopsied and PGD 16.8% 16.8%

* 1% Gianaroli et al. (2004), 3.1% Colls et al. (2007)

PGD for AMA: randomized studiesPGD for AMA: randomized studiesMastenbroek et al. (2007)Mastenbroek et al. (2007)

Analysis of remaining cells of embryos previously analyzed by PGD:Analysis of remaining cells of embryos previously analyzed by PGD:

studystudy techniquetechnique error rateerror rate

Baart et al 2004 Baart et al 2004 FISHFISH 50.0%50.0% Li et al. 2005 Li et al. 2005 FISHFISH 40.0%40.0% Gleicher et al. 2009Gleicher et al. 2009 FISHFISH 15-20%15-20% Munne et al. 2002 Munne et al. 2002 FISH-9FISH-9 7.2% 7.2% Colls et al., 2007 Colls et al., 2007 FISH-9FISH-9 4.7% 4.7% Magli et al. 2007 Magli et al. 2007 FISH-9FISH-9 3.7% 3.7% Munne et al. 2010Munne et al. 2010 array CGHarray CGH 1.8% 1.8%

Error rate should be <10%Error rate should be <10%

New approach to PGD:New approach to PGD:

• 24 chromosome analysis by 24 chromosome analysis by arraysarrays

• Blastocyst biopsy and Blastocyst biopsy and vitrificationvitrification

• 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

CGH and array CGHCGH and array CGH

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)

Array CGHArray CGH

Test DNA

Normal DNA

2700 probesSame band resolution as karyotype

46,XY

47,XY+2

44,XY-9-17

aCGH advantagesaCGH advantages

• All All 24 chromosome 24 chromosome type of aneuploidies detectedtype of aneuploidies detected

• 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)

• Used in Used in >15,000>15,000 patients with mental retardation patients with mental retardation

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

Embryos undiagnosed:Embryos undiagnosed:

biopsy on day biopsy on day 33: : 2% 2% (16/724)(16/724)

biopsy on day biopsy on day 55: : >2%>2% (0/54) (0/54)

Method: Bluegnome array, Sureplex amplification.Method: Bluegnome array, Sureplex amplification.

aCGH validation: error rateaCGH validation: error rate

• Validation method 1: single cells from cell lines analyzed* Validation method 1: single cells from cell lines analyzed*

Error rate in euploid cell lines: 0/9Error rate in euploid cell lines: 0/9Error rate in aneuploid cell lines: 0/42Error rate in aneuploid cell lines: 0/42

• Validation method 2: Reanalysis of the rest of the embryo Validation method 2: Reanalysis of the rest of the embryo

by FISH with 19 chromosomes probes** by FISH with 19 chromosomes probes**

Error rate from day 3 biopsies: Error rate from day 3 biopsies: 1.8% 1.8% (1/56)(1/56)

* Mamas et al (submitted), ** Gutierrez et al. (submitted)* Mamas et al (submitted), ** Gutierrez et al. (submitted)

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?

Day 3 biopsy, day 5 transferDay 3 biopsy, day 5 transferand array CGHand array CGH

Cycles performed: Cycles performed: 151 151Maternal age (av.)Maternal age (av.) 37.2 37.2

Pregnancy RatePregnancy Rate Ongoing Pregnancy RateOngoing Pregnancy RatePer CyclePer Cycle Per ETPer ET Per CyclePer Cycle Per ETPer ET

ControlControl 38%38% 38%38% 31%31% 31%31%PGDPGD 46%46% 59%59% 42%42% 54%54%

NSNS < 0.001< 0.001 NSNS < 0.001< 0.001

* Expected from each center SART data, controlled by age* Expected from each center SART data, controlled by ageData from 24 centers. Munné et al. (2010) PGDIS, and unpublished dataData from 24 centers. Munné et al. (2010) PGDIS, and unpublished data

CGH on blastocyst biopsies:CGH on blastocyst biopsies:Why?Why?

Advantages:1) More DNA: More robust diagnosis2) Eliminates some mosaic embryos3) Reduces error rate4) Reduced impact of embryo biopsy5) Less embryos to process6) Facilitates single embryo transfer7) Uterine environment optimized after thaw

Effect of embryo vitrification andEffect of embryo vitrification andReplacement on next cycleReplacement on next cycle

• Average age 37.7Average age 37.7• Av. Previous failed cycles: 2.4Av. Previous failed cycles: 2.4• Average # blastocysts: 6.4Average # blastocysts: 6.4• Normal blastocysts: 52%Normal blastocysts: 52%• 99.3% survival after freeze/thaw99.3% survival after freeze/thaw

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

CGH on blastocyst biopsies:CGH on blastocyst biopsies:Clinical results (2)Clinical results (2)

CyclesCycles Mat.Mat. Prev.Prev. embryosembryos implant. implant. ongoingongoingageage failedfailed replaced replaced (+ sac) (+ sac) preg.preg.

cyclescycles

CGH : CGH : 45 45 37.737.7 2.4 2.4 2.0 2.0 67%67% 79%79%

control : control : 113113 37.137.1 1.2 1.2 2.7 2.7 28%28% 60%60%

p=0.0003p=0.0003Schoolcraft et al. (in press)Schoolcraft et al. (in press)

CGH on blastocyst biopsies:CGH on blastocyst biopsies:Preliminary clinical resultsPreliminary clinical results

0

10

20

30

40

50

60

70

80

90

30-34 35-38 39-42 43-45

implantation rateper replacedembryo

aneuploidy rate

cycles with allembryos abnormal

CGH on blastocyst biopsies:CGH on blastocyst biopsies:Implantation is independent of ageImplantation is independent of age

Patients <43 who are eligible for blastocyst transfer have a >95% change of having normal embryos available for transfer

• Pregnancy rates were above those for controls for all age groups Pregnancy rates were above those for controls for all age groups

• However, pregnancy rates were lower for older patients due to However, pregnancy rates were lower for older patients due to the increased frequency of cycles with no euploid embryos. the increased frequency of cycles with no euploid embryos.

• Spontaneous abortion rates were reduced for all patients, Spontaneous abortion rates were reduced for all patients, including those with a history of multiple miscarriageincluding those with a history of multiple miscarriage

• Chromosomally normal embryos from older patients have a Chromosomally normal embryos from older patients have a similar chance of producing a child as those derived from similar chance of producing a child as those derived from young patients.young patients.

CGH on blastocyst biopsies:CGH on blastocyst biopsies:Conclusions on pilot studyConclusions on pilot study

• What patient groups will benefit the most from this type of screening?What patient groups will benefit the most from this type of screening?

Young patients may benefit, particularly in countries where single embryo Young patients may benefit, particularly in countries where single embryo transfer is mandatorytransfer is mandatory

• Can the results obtained in the current study be replicated in a randomized Can the results obtained in the current study be replicated in a randomized controlled trial?controlled trial?

• How much of the observed benefit is due to transfer in a subsequent cycle?How much of the observed benefit is due to transfer in a subsequent cycle?

An RCT is currently being contemplated in the UKAn RCT is currently being contemplated in the UK

• Aneuploidy explains ‘some’ of the decline in IVF success with advancing Aneuploidy explains ‘some’ of the decline in IVF success with advancing maternal age. What explains the remainder?maternal age. What explains the remainder?

Uterine receptivity factor may be involvedUterine receptivity factor may be involved

CGH on blastocyst biopsies:CGH on blastocyst biopsies:Remaining questionsRemaining questions

PGD for translocationsPGD for translocationsand 24 chromosome and 24 chromosome

abnormalitiesabnormalities

aCGH for translocations andaCGH for translocations and24 chromosome aneuploidy 24 chromosome aneuploidy

Patient: 46,XY,t(3;11)(q22.2;q23.3)

Comparison ofComparison ofdifferent approachesdifferent approaches

aCGH vs. SNP arrays: Genome coverageaCGH vs. SNP arrays: Genome coverage

# of probe genomeprobes size covered

aCGH 4,000 x 150,000 kb = 600.0 Mb (25%)

SNPs 300,000 x 50 kb = 1.5 Mb (>0.1%)

aCGH vs. SNP arrays: ResolutionaCGH vs. SNP arrays: Resolution

More resolution, more counseling nightmares:

In pre-natal diagnosis the minimum resolution reported is 20 MB: Smaller abnormalities are not well understood and difficult to council

At 1.5 Kb resolution there are >300,000 CNPs described: Difficult to differentiate a new polymorphism from an abnormality

• Embryos analyzed with 9-12 probes:Embryos analyzed with 9-12 probes: 91,47391,473• Polyploid or haploid:Polyploid or haploid: 6,609 (8%) 6,609 (8%)

• Abnormal embryos fully reanalyzed:Abnormal embryos fully reanalyzed: 1,416 1,416• polyploid or haploid by PGD and polyploid or haploid by PGD and polyploid or haploid after reanalysis:polyploid or haploid after reanalysis: 140 140• Of the above, that had only trisomies:Of the above, that had only trisomies: 27 (24%) 27 (24%)• Not diagnosed by aCGH or CGH:Not diagnosed by aCGH or CGH: 1.8%1.8%

aCGH vs SNPs: polyploidyaCGH vs SNPs: polyploidy

aCGH vs SNPs: mitotic abnormalitiesaCGH vs SNPs: mitotic abnormalities

• Embryos analyzed with 9-12 probes:Embryos analyzed with 9-12 probes: 91,47391,473• Complex abnormal:Complex abnormal: 26,609 (29%)26,609 (29%)

• Abnormal embryos fully reanalyzed:Abnormal embryos fully reanalyzed: 1,416 1,416• Complex abnormal by PGD and mosaic andComplex abnormal by PGD and mosaic and not aneuploid after reanalysis:not aneuploid after reanalysis: 330 (52%) 330 (52%)• Of the above, that had only trisomies:Of the above, that had only trisomies: 53 (16%) 53 (16%)• Not diagnosed by qualitative SNP array:Not diagnosed by qualitative SNP array: 3%3%

aCGH vs SNPsaCGH vs SNPs

Other SNP advantages:Other SNP advantages: Usefulness?Usefulness?- Origin of aneuploidy detectedOrigin of aneuploidy detected 95% are maternal anyway95% are maternal anyway- Embryo fingerprintingEmbryo fingerprinting None in SET. aCGH also doableNone in SET. aCGH also doable- Differentiates normal / balancedDifferentiates normal / balanced balanced are fine. balanced are fine. >80% abnormalities>80% abnormalities

Other SNP disadvantages:Other SNP disadvantages: Problem:Problem:-Requires parental testingRequires parental testing Adds costs, cancellation fees,Adds costs, cancellation fees,

planning in advance, prevents planning in advance, prevents add oc prescription, 20% add oc prescription, 20% patients cancelpatients cancel

Comparison of platformsComparison of platforms

FISH-12 FISH-12 CGHCGH aa arrayarray SNPSNP probes probes aa CGHCGH aa arraysarrays

Day 3 biopsy/ day 5 resultsDay 3 biopsy/ day 5 results yesyes nono yesyes yesyesParental DNA neededParental DNA needed nono nono nono yesyesAneuploid embryos detectedAneuploid embryos detected 89%89% 100%100% 100%100% 97%97%Detect gene defectsDetect gene defects nono nono nono yesyesDetect polyploidy (errors)Detect polyploidy (errors) yesyes (1.8%)(1.8%) (1.8%(1.8% ) ) yesyesDetect MII trisomies w/o crossoverDetect MII trisomies w/o crossover yesyes yesyes yesyes nono if qual. if qual.Detect mitotic trisomies (errors)Detect mitotic trisomies (errors) yesyes yesyes yesyes (3%)(3%)Error rate (cell lines analysis)Error rate (cell lines analysis) 2%2% 1%1% 0% 0% dd 4%4% bb

Error rate (embryo reanalysis) Error rate (embryo reanalysis) 7%7% 8%8% 2%2% unkunkEmbryos with no resultsEmbryos with no results 3%3% 6%6% 2%2% 8%8% bb

Increased implantation rate (day 3) Increased implantation rate (day 3) ++ n/an/a ++++ unkunk Increased implantation rate (day 5)Increased implantation rate (day 5) unkunk +++ +++ ++++++ +++ +++ cc

aaReprogenetics data,Reprogenetics data, b b Jonhson et al. (2010), Jonhson et al. (2010), c Scott et al. (2010, PCRS), d Mamas et al (submitted)

PGD for gene defects PGD for gene defects and 24 chromosome and 24 chromosome

abnormalitiesabnormalities

PGD for gene disordersPGD for gene disorders

Disease tested: Acetil Co Oxidase type I defficiency, Adrenoleucodistrophy, Alpha-thalassemia, Alport syndrome, Autosomal Dominant Polycystic Kidney Disease (ADPKD), Autosomal Recesive Polycystic Kidney Disease (ARPKD), Beta-thalassemia, Branchio-Oto-Renal syndrome (BOR), BRCA1 breast cancer predisposition, BRCA2 breast cancer predisposition, CanavanCharcot-Marie-Tooth type IA (CMT1a), Choroideremia, Congenital adrenal hyperplasia (CAH), Congenital neutropenia, Connexin 26 hearing loss, Cystic fibrosis, Duchenne/Becker Muscular Dystrophy (DMD), Ectrodactyly, Ectodermal dysplasia, and Cleft lip/palate syndrome (EEC1), Fabry Disease, Familial adenomatous poliposis coli (FAP), Familial dysautonomia, Familial intrahepatic cholestasis 2, Fanconi anemia, Fragile site mental retardation , Gangliosidosis type 1 (GM1), Gaucher disease, Glomuvenous malformations (GVM), Glycogen-storage disease type I (GSD1), Glycosylation type 1C, Hemoglobin SC disease, Hemophilia A, Hemophilia B, Hereditary nonpolyposis colon cancer (HNPCC), Hereditary pancreatitis, HLA matching Huntington disease, Hurler syndrome, Hypophosphatasia, Incontinential pigmenti, Krabbe disease (Globoid cell leukodystrophy), Long QT syndrome, Marfan syndrome, Meckle gruber, Metachromatic leukodystrophy (MLD), Methylmalonic aciduria cblC type (MMACHC), Myotonic Dystrophy 1, Myotubular myopathy, Neurofibromatosis 1, Neurofibromatosis 2, Niemann-Pick Disease, Noonan syndrome, Oculocutaneous albinism 1 (OCA1), Ornithine carbamoyltransferase deficiency (OTC), Osteogenesis Imperfecta 1, Rapp Hodgkin ectodermal dysplasia, Retinitis pigmentosa, Retinoblastoma, Sickle Cell Anemia, Smith-Lemli-Opitz syndrome (SLOS), Spinal bulbar muscular atrophy (SBMA), Spinal Muscular Atrophy Type 1 (SMA1), Tay Sachs, Tuberous sclerosis 1 (TSC1), Tuberous sclerosis 2 (TSC2), Von Hippel-Lindau Syndrome (vHL), X-linked dominant Charcot–Marie–Tooth (CMTX), etc…… (see review Gutierrez et al. (2008))

We can do PGD for any disease with known mutation

accurate accurate amplification of amplification of

both lociboth loci

ADO affecting ADO affecting normal allelenormal allele

ADO affecting ADO affecting mutation sitemutation site

MutationMutation sitesite

Polymorphic Polymorphic sitesite

Conventional approach to PGD of gene Conventional approach to PGD of gene defects to avoid Allele Drop Out (ADO)defects to avoid Allele Drop Out (ADO)

Δ508

Father:

Δ508

Mother:

Slide adapted from A.Handyside

Monosomy 11

Trisomy 21

PGD of gene defects using SNP array KaryomappingPGD of gene defects using SNP array Karyomapping

Carrier Δ508

Example:Example: First Clinical Application of Karyomapping for PGD of Gaucher Disease Combined with 24 Chromosome Screening

Handyside A, Grifo, J, Gabriel A, Thornhill A, Griffin D, Ketterson K, Prates R, Tormasi S, Fischer J, Munné S (2010) PGDIS

Embryo # Gene defects Chromosome defects

5 Affected normal7 Affected monosomy 228 Carrier monosomy 10, 21,229 Affected monosomy 222 unaffected Monosomy 2210 Affected Monosomy 9,17,19,22, trisomy 14

Results confirmed by a-CGH and by PCR

ConclusionsConclusions

- Age and morphology are poor indicators of - Age and morphology are poor indicators of aneuploidy aneuploidy

- Less than 50% of good morphology day 3 embryos Less than 50% of good morphology day 3 embryos and less than 60% of blastocysts are normal in and less than 60% of blastocysts are normal in patients >35patients >35

- Selecting for euploid embryos should improve ART Selecting for euploid embryos should improve ART outcome outcome

Conclusions: chromosome abnormalitiesConclusions: chromosome abnormalities

Studies with improved results differ from those that show Studies with improved results differ from those that show no improvement in that:no improvement in that:

1) Reduce biopsy damage (1 cell, experience, blast?)1) Reduce biopsy damage (1 cell, experience, blast?) 2) Low error rate (fixation, NRR, 2 analyzers)2) Low error rate (fixation, NRR, 2 analyzers) 3) Analyze 16,15,21,22 chromosomes + ≥4 more3) Analyze 16,15,21,22 chromosomes + ≥4 more 4) Extensive experience4) Extensive experience 5) Appropriate population (≥5 embryos, 5) Appropriate population (≥5 embryos, ≥≥ 35 y. old)35 y. old)

Conclusions: FISH studiesConclusions: FISH studies

- Blastocyst biopsy + CGH + vitrification shows very high Blastocyst biopsy + CGH + vitrification shows very high implantation rates (72%, av. Age 38).implantation rates (72%, av. Age 38).

- Array CGH and day 5 biopsy will produce same resultsArray CGH and day 5 biopsy will produce same results

- Array CGH and day 3 biopsy improves results when Array CGH and day 3 biopsy improves results when normal embryos are available. normal embryos are available.

- Additional vitirifcation step may still be advantageousAdditional vitirifcation step may still be advantageous

Conclusions: array CGHConclusions: array CGH

- Single gene defects and 24 chromosome analysis can Single gene defects and 24 chromosome analysis can be detected simultaneously for couples carriers of gene be detected simultaneously for couples carriers of gene defects. defects.

- It requires parental testing.It requires parental testing.

Conclusions: gene defects andConclusions: gene defects and24 chromosome analysis24 chromosome analysis

Integration withIntegration with eIVF

- Reduce time of form-filling to a few clicks: Reduce time of form-filling to a few clicks:

patient demographics, cycle progress, PGD results, patient demographics, cycle progress, PGD results, follow up, automatically accessed by IVF center and follow up, automatically accessed by IVF center and ReprogeneticsReprogenetics

Advantages (1)Advantages (1) eIVF

- Minimize the risk of mismanaging patients:Minimize the risk of mismanaging patients:

Translocations and gene defects preparations, billing, … Translocations and gene defects preparations, billing, …

Advantages (2)Advantages (2) eIVF

- Access to the aggregate data of Reprogenetics, de-Access to the aggregate data of Reprogenetics, de-identified by patient and center, with standard queries identified by patient and center, with standard queries and access to the raw data.and access to the raw data.

Example: Out of the 20,000 PGD cycles performed by Example: Out of the 20,000 PGD cycles performed by Reprogenetics, what is the proportion of normal Reprogenetics, what is the proportion of normal embryos in a patient 43 years old? 17% (n = 345)embryos in a patient 43 years old? 17% (n = 345)……with an E2 between 8 and 10…. etcwith an E2 between 8 and 10…. etc

Advantages (3)Advantages (3) eIVF

Fields to choose: Fields to choose:

- Age by 1 year or by SART groups (drop list)Age by 1 year or by SART groups (drop list)- stim protocol (drop list)stim protocol (drop list)- ICSI or IVFICSI or IVF- pick E2 level (chose range)pick E2 level (chose range)- number of eggs (chose range)number of eggs (chose range)- number of zygotes (chose range)number of zygotes (chose range)- zygote pm stage (chose range)zygote pm stage (chose range)- number of blastocysts(chose range)number of blastocysts(chose range)- motphology grade on day 3 (chose range)motphology grade on day 3 (chose range)- motphology grade on day 5 (chose range)motphology grade on day 5 (chose range)- number of day 3 embryos of grade X (chose range)number of day 3 embryos of grade X (chose range)- number of day 5 embryos of grade X (chose range)number of day 5 embryos of grade X (chose range)- ICM grade (chose range)ICM grade (chose range)- trophectoderm grade (chose range)trophectoderm grade (chose range)- # of previous miscarriages (chose range)# of previous miscarriages (chose range)- # of previous failed cycles (chose range)# of previous failed cycles (chose range)- # of previous conceptions that were chromosomally abnormal (0, >0)# of previous conceptions that were chromosomally abnormal (0, >0)- PGD indication (drop list)PGD indication (drop list)- PGD test (drop list)PGD test (drop list)- pregnancy rate of centers included in the query (chose range)pregnancy rate of centers included in the query (chose range)

Outcome:Outcome:

- % of cycles with these characteristics out of the total% of cycles with these characteristics out of the total- % normal embryos% normal embryos- % aneuploid embryos% aneuploid embryos- % other abnormal embryos% other abnormal embryos- implantation rate per pick upimplantation rate per pick up- implantation rate per transferimplantation rate per transfer- pregnancy rate per pick uppregnancy rate per pick up- pregnacy rate per transferpregnacy rate per transfer- miscarriage ratemiscarriage rate- % miscarraiges chromosomally abnormal% miscarraiges chromosomally abnormal- take home baby ratetake home baby rate- center ranking compared to other non identified centerscenter ranking compared to other non identified centers

eIVF

Santiago Munné, PhD, DirectorSantiago Munné, PhD, DirectorJacques Cohen, PhD, DirectorJacques Cohen, PhD, Director

munne@reprogenetics.com munne@reprogenetics.com www.reprogenetics.comwww.reprogenetics.com

Pere Colls, Ph.DPere Colls, Ph.DDagan Wells, Ph.DDagan Wells, Ph.DGeorge Pieczenik, Ph.DGeorge Pieczenik, Ph.D Cristina Gutiérrez, Ph.DCristina Gutiérrez, Ph.D Jorge Sanchez, PhDJorge Sanchez, PhD John Zheng, MDJohn Zheng, MD Tomas Escudero, MSTomas Escudero, MSKelly Ketterson, MSKelly Ketterson, MSJill Fischer, MSJill Fischer, MS

Jessica Vega, MSJessica Vega, MS Tim SchimmelTim SchimmelSasha SadowySasha SadowySophia TormasiSophia TormasiN-neka GoodallN-neka GoodallRenata PratesRenata Prates Piedad GarzonPiedad Garzon Laurie FerraraLaurie FerraraBekka Sellon-WrightBekka Sellon-WrightMaria FeldhausMaria Feldhaus

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

Michalis KonstantinidisMichalis Konstantinidis

South AmericaSouth AmericaPaul LopezPaul Lopez

Luis Alberto GuzmanLuis Alberto Guzman

GermanyGermanyKarsten Held, MDKarsten Held, MD