william kearns, usawilliam kearns, usacontroversies for pgs • does aneuploidy screening work? –...

William Kearns, USAWilliam Kearns, USAShady Grove Center forPreimplantation GeneticsWashington, USA

He declares receipt of grants and contracts from MerckSerono. Declares receipt of honoraria or consultation feesfrom Merck Serono. Declares participation in a companysponsored speaker’s bureau: Merck Serono.

PGD: Genetics or Proteomics?

WG Kearns

Shady Grove Center forShady Grove Center for PreimplantationPreimplantation GeneticsGenetics

The Johns Hopkins University School of MedicineThe Johns Hopkins University School of Medicine

Shady Grove Fertility Reproductive Science CenterShady Grove Fertility Reproductive Science Center

Rockville and Baltimore, MD USARockville and Baltimore, MD USA

Indications for Genetic Assessmentof Embryos

• Aneuploidy screening (PGS)• Structural chromosome aberrations

(PGD)• Single gene disorders (PGD)• Mitochondrial disorders (PGD)

Ethics

• Non-medical sex selection?

• Huntington disease

• Deafness

• HLA typing– “My Sister’s Keeper”

Cell Types - Biopsy

• Polar Bodies– Meiosis I errors– Meiosis II errors– X-linked disorders

• Blastomeres

Current Benefits and Limitations

• Aneuploidy• Structural chromosome aberrations• Single gene disorders• Mitochondrial disorders

Aneuploidy

• Fluorescence in situ hybridization(FISH) on interphase nuclei

• Comparative genomic hybridization(CGH) on metaphase chromosomes

FISH Limitations

• Single “spot-check” to testfor the presence ofa specific chromosome

• Cells are in interphase

• Limited fluorochromes

• Reduced accuracy withadditional probes

• Fixation/nuclear spreading~ 10-12 chromosomes tested

CGH on Metaphase Chromosomes

CGH Benefits and Limitations

• Tests for all 23-pairs of chromosomes

• Limitations – hybridization takes ~ 3 daysand requires an FET

What are the Issues/Risks of PGS?

• Test Limitations– FISH - Limited chromosomes– CGH – requires an FET

• Embryo mosaicism?

• Damaging the embryo during the biopsy?– Reduced implantation?– Biochemical pregnancy?

What are the Issues/Risks of PGS?

• Embryo correction of day-3 embryo?– CVS – Placental aneuploid colonies

Biopsy a Better Cell Type?

• Trophectoderm

Structural Chromosome Imbalances

• Fluorescence in situHybridization (FISH)

• Reciprocal translocations– Telomere clones– Genetic balance

• May have the translocation– Breakpoint clones

• No translocations

• Robertsonian translocations– Locus specific clones– Genetic balance

• May have the translocation

Structural Chromosome Disorders

• Pericentric inversions– Telomere probes– Identify

duplication/deficientchromosomes inembryos

• Paracentric inversions– Centromere probes– Telomere probes– Identify dicentric

chromosomes inembryos

Structural Chromosome Aberrations

• If male carrier– Test sperm for % genetically

unbalanced?

Duplication and Microdeletion Syndromes

• Wolf-Hirchhorn syndrome• Williams syndrome• Prader-Willi/ Angelman syndrome• Smith-Magenis syndrome• Miller-Dieker syndrome• DiGeorge/Velo-Cardio-Facial syndrome• Kallman syndrome• Steriod sulfatase deficiency

Data

• Translocations and Inversions• Aneuploidy• Single Gene

Structural Chromosome Disorders

• n = 343 cycles

• 3270 embryos

• 29% no transfer – poor embryo quality orabnormal

• 8% - no dx due to cell lysis / poor embryo quality

Structural Chromosome Disorders

• Clinical Pregnancy = 38%– 41% – recriprocal translocations– 35% – rob translocations– 33% – pericentric inversions

• 9% – biochemical• 9% – miscarriages

Single Gene and Mitochondrial Disorders

• Single gene disorders– PCR– DNA sequencing– Linkage analysis

• Mitochondrial– Recessive

Segregation Patterns

Single Gene and MitochondrialAnalyses Limitations

• Single gene disorder– Mutation must be known– Allele dropout – misdiagnosis

• Mitochondrial disorders– Few offered

Single Gene PGD

n = 209 cycles

• 2015 embryos

• 8% - No transfer

• 7% - No Dx due to cell lysis

• 51% abnormal

• 7% monosomy or trisomy

• CP = 51%

• Biochemical = 9%

• Miscarriages = 6%

• Mis-Dx

Aneuploidy

Controversies for PGS

• Does aneuploidy screening work?– Mastenbroek S et al. N Engl J Med. July 2007

• 8 chromosomes assessed• Showed no improvement in implantation

– BUT• Maternal age (35-41)

– We wouldn’t offer PGS• Many 4-cell embryos biopsied• 20% no FISH results• Failed to test for chromosomes 22 and 15

(~ 25% of aneuploidy)• Tested for chromosome 1 ???• Biopsied 2 cells sometimes

Mastenbroek S et al. N Engl J Med. 2007;357(1):9-17.

Controversies for PGS

• PGS doesn’t improve clinical pregnancy rates(gestational sac and fetal heart beat) and deliveryrates

– ASRM Practice Statement, October 2007– ESRE Practice Statement – July 2008– ACOG – May 2009

– No prospective, randomized studies to date

Shady Grove Fertility (10-Probe FISH)

< 35CP = 35% / ETMiscarriage = 4%

35-37CP = 32% / ETMiscarriage = 8%

38-40CP = 20% / ETMiscarriage = 17%

>40CP = 14%Miscarriage = 20%

Biopsy a Better Cell Type?

• Trophectoderm

What are the Issues / Risks ofTrophectoderm Biopsy and PGS?

• Mosaicism?– Inner cell mass– Trophectoderm

23-Chromosome PGS - Trophectoderm

• Trophectoderm cells– CGH on metaphase chromosomes

(Wells et al)– Can’t R/O mosaicism

• n = 72• 2-6 unsuccessful IVF cycles• CP = 72%• Delivery rate = 60%

PGS – Trophectoderm

• Patient recruitment – 3+ blasts• Indication for PGS• 24 hr turnaround from thaw to transfer• 114 blasts thawed• 78% blast survival• 67% re-expansion• 83% were biopsied• 100% PGD results• PGS (10 chromosomes) - 41% normal, 59% abnormal• Biopsied cells (mean = 5/patient)

n = 41 casesFISH for Chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y

PGS – Trophectoderm

• n = 41 cases• 3 had no transfer• 70% + FHT with gestational sac

(all > 12 weeks)• 3% Biochemical• 16 normal deliveries• No miscarriages

• Blastocyst biopsy and PGS may be a less-invasive and more beneficial option to day-3blastomere biopsy and PGS

New Technologies- MicroarraysNew Technologies- Microarrays

• Single nucleotide polymorphisms (SNPs)

5’-ACTGGGAATCCCGAAGTGTGC TGATTACA-3’

• Normally occurring genetic variant• ~11,000,000 estimated to be in human genome

• Stable in >1% of the human population

– More dense• Various density arrays

– Up to ~ 1,000,000 genomic hits

• CGH

– Less dense

– Ratio

– Oligonucleotides or BACs• ~ 42,894 genomic hits (~ Exonic (16404), Intronic (19805), Intergenic (6685))

T

FISH vs MicroarraysFISHFISH vsvs Microarrays

FISH: hybridization of a long oligonucleotide toa peri-centromeric or locus specific location- detection via microscopy- analysis and interpretation via microscopy

SNP or CGH: DNA markers throughout thechromosome- detection via microarray and scanner- analysis and interpretation viaalgorithm

SNP and CGH Microarrays

SNP Arrays vs CGH Arrays

Genetic Diagnostics and Screening SNP CGH

23/24 chromosome aneuploidy X X

Copy number variations (CNVs) X X

Structural chromosome imbalances ~1.5kb ~.1-10mb

Genome-wide scans X

What embryo implanted? X

What partner provided the extra chromosome? X

Single gene disorders X

Mitochondrial mutations X

Uni-parental disomy X

Copy neutral event X

Validation -Materials and Methods

•• n = 802 single cells /n = 802 single cells / blastomeresblastomeres (110 day(110 day--3 abnormal embryos) and 34 cell lines3 abnormal embryos) and 34 cell lines

•• Embryo biopsy of a single cellEmbryo biopsy of a single cell –– laserlaser

–– BlastomereBlastomere

•• Modified whole genome amplificationModified whole genome amplification(WGA)(WGA)

Experimental Problem to Overcome

•• ~ 6~ 6 picogramspicogramsDNADNA

•• Need ~ 250,000xNeed ~ 250,000xfor microarrayfor microarrayanalysisanalysis

•• PCRPCR–– ArtifactsArtifacts

–– AmplificationAmplification

–– Allele dropoutAllele dropout

Modified Whole Genome Amplification (WGA)

•• Home BrewHome Brew

•• Parental DNA not required for PGSParental DNA not required for PGS

Materials and Methods

• Invariant DNA genomic loci identified by PCRto ensure the entire genome was amplified

Materials and Methods

•• TaqManTaqMan PCR to ensure heterozygous allelePCR to ensure heterozygous alleleamplificationamplification

•• MicroarraysMicroarrays

•• IlluminaIllumina HumanHap370 ~370,000HumanHap370 ~370,000 SNPsSNPs

•• IlluminaIllumina CytoCyto--12 ~ 320,00012 ~ 320,000 SNPsSNPs

•• IlluminaIllumina 1M ~ 1,000,0001M ~ 1,000,000 SNPsSNPs

Materials and Methods

• Two-channel intensity values – high-resolution copy-numberprofile

– – Identifies copy number variations (CNVs)

• Genome-Wide scans / Genotyping– Identifies SNP Genotypes– Genome-Wide Scan Analysis

• Single Gene Disorders• Complex Genetic Disorders

•• BioinformaticsBioinformatics– deCode genetics Disease Minor Professional, Illumina BeadStudio,

GenomeStudio and KaryoStudio software

Results

•• DNA yields of ~ 8000DNA yields of ~ 8000 ngng / 4 hr reaction/ 4 hr reaction

•• In many cases, a genomic coverage > 98%In many cases, a genomic coverage > 98%(Range 30(Range 30--98%)98%)

•• Correlates with dayCorrelates with day--3 embryo quality3 embryo quality

•• Heterozygous allele detection rate > 90%Heterozygous allele detection rate > 90%

Results

•• Microarray detection rate > 90%Microarray detection rate > 90%(some cases > 99%)(some cases > 99%)

•• Genotype call rate > 90%Genotype call rate > 90%(some cases > 99%)(some cases > 99%)

Two Copies of Chromosome 1

Two Copies of Chromosome 21

Three Copies of Chromosome 21

Three Copies of Chromosome 12One is Deleted from Band q14 ?

Embryo Day-3FISH

1 +13, +17,XY

Embryo Day-4FISH

1-1 +13,+17,XY

1-2 +13,+17,XY2 +14, XX 2-1 +14, XX

2-2 +14, XXX

6 +14,+17, +22,XXX

10 +18,-21,XX

6-1 +14,+17,+22,XXX

6-2 +14,+17,+22,XXX10-1 +18,-21,XX

10-2 +18,-21,XX

11 -13,+16,+22,XY

11-1 -13,+16,+22,XY

11-2 -13,+16,+22,XY

23-ChromosomeMicroarray

+13,+17,XY

+13,+17,XY+14, XX

+8, +14, XXX

+5,-6,+14,+17,+22,XXX

+5,-6,+14,+17,+22,XXX

+7, +9, +18, -21, XX

+7, +9, +18, -21, XX

-5, +6, -13, +16, +22, XY

-5, +6, -13, +16, +22, XY

Correlate FISH and Microarrays ( n = 30)

Results

• Structural chromosome imbalances wereidentified from all 9 cytogenetically abnormal celllines

– del(8q), add(17p), del(17p), add(4q),add(9p), add(14q), dup(18p), dic(5),del(12p) and del(9p)

• Based upon the density of the SNPmicroarray

• CGH array couldn’t identify geneticimbalances

Unbalanced Reciprocal Translocation

der(18)t(13;18)(q33.2;q22.3)der(18)t(13;18)(q33.2;q22.3)

8p Deletion

2p Duplication

8q Trisomy and 8p Deletion?

Copy Number VariationsCopy Number Variations

•• A highA high--resolution copyresolution copy--number mapnumber mapidentifiedidentified CNVsCNVs in all 61 embryos and cellin all 61 embryos and celllineslines

–– InheritableInheritable

•• Segmental deletionsSegmental deletions

•• DuplicationsDuplications

Copy Number VariationsCopy Number Variations

Genome-Wide Scans / Genotyping

•• BeckwithBeckwith--WiedmannWiedmann SyndromeSyndrome

•• Some forms ofSome forms of PraderPrader WilliWilli // AngelmanAngelmanSyndromeSyndrome

•• DiGeorgeDiGeorge SyndromeSyndrome

•• Some forms of Autism (~2%)Some forms of Autism (~2%)

•• UniparentalUniparental disomydisomy

•• Single gene disordersSingle gene disorders

Genome-Wide Scans / Genotyping

NF-1 Prader- Willi

Genome-Wide Scans / Genotyping

• What embryo implanted?

• What partner provided the extrachromosome in aneuploid embryos?– Sperm?– Oocyte?

SNP Microarray Clinical Cases n = 25CP = 64% (9 / 14) Pending hcg = 3Frozen day-5 = 4 No Transfer = 4

< 35 (n = 2)< 35 (n = 2)

CP = 100% / ETCP = 100% / ET

Miscarriage = 0%Miscarriage = 0%

3535--37 (n = 3)37 (n = 3)

7 consecutive7 consecutivemiscarriagesmiscarriages

CP = 33% / ETCP = 33% / ET

Miscarriage = 0%Miscarriage = 0%

3838--40 (n = 5)40 (n = 5)

CP = 80% / ETCP = 80% / ET

Miscarriage = 0%Miscarriage = 0%

>40 (n = 4)>40 (n = 4)

CP = 50%CP = 50%

Miscarriage = 0%Miscarriage = 0%

* One 44 yr old went* One 44 yr old wentthrough twice withthrough twice withnegneg resultsresults

Conclusions

• Complete molecular karyotype for all23-pairs of chromosomes

• Simultaneously determinechromosomal imbalances due totranslocations, inversions, deletionsor duplications

• Simutaneously determine complexgenetic disorders and many singlegene mutations

ConclusionsConclusions

•• Results available for a dayResults available for a day--5 transfer5 transfer

•• BlastomeresBlastomeres,, trophectodermtrophectoderm or polar bodiesor polar bodies

ConclusionsConclusions

•• What embryo implanted?What embryo implanted?

•• Who provided the extra chromosome?Who provided the extra chromosome?

•• Sperm?Sperm?

•• OocytesOocytes??

Metabolomics and Proteomics

Metabolomics / Proteomics vs PGD?

• Single Gene?– NO

• Structural chromosome aberrations?– NO

• Aneuploidy?– ?

Laboratory and Clinical Collaborators

• Shady Grove Center for PreimplantationGenetics and Shady Grove Fertility ReproductiveScience Center

– Rasmei Pen– Andy Benner– Adam Kittai– Andrew Siegel– Chris Chipko– Eric Widra– Richard Leach– William G Kearns

Contact Information

• William G. Kearns– 301-251-2804– William.kearns@integramed.com