Transcriptomics and comprehensive methods ofTranscriptomics and comprehensive methods ofchromosome screeningchromosome screening

Dr. Dagan Wells, FRCPathDr. Dagan Wells, FRCPath

dagan.wells@obsdagan.wells@obs--gyn.ox.ac.ukgyn.ox.ac.uk

Testing chromosomes in embryosTesting chromosomes in embryos

AneuploidyAneuploidy

The incidence of aneuploidyThe incidence of aneuploidy

Chromosome abnormality is extremely common in oocytesChromosome abnormality is extremely common in oocytesProblem increases with advancing maternal ageProblem increases with advancing maternal age

40%40%

10%10%

20%20%

30%30%

2020--3434 3535--3939 4040--4545 Maternal ageMaternal age

50%50%

Also reflected in the increase in Down syndrome with ageAlso reflected in the increase in Down syndrome with age

AneuploidyAneuploidy

Aneuploidy and IVF failureAneuploidy and IVF failure

As aneuploidy increases age, so implantation rate decreasesAs aneuploidy increases age, so implantation rate decreases

40%40%

10%10%

20%20%

30%30%

2020--3434 3535--3939 4040--4545 Maternal ageMaternal age

50%50%

ImplantationImplantation

6565--75% of first trimester miscarriages are aneuploid75% of first trimester miscarriages are aneuploid

regularregular after chromosomeafter chromosomescreeningscreening

Standard embryo evaluations do not reveal embryos withStandard embryo evaluations do not reveal embryos withthe wrong number of chromosomesthe wrong number of chromosomes

Preimplantation genetic screeningPreimplantation genetic screening

oror

Chromosomal indicationsChromosomal indications

Preimplantation genetic screeningPreimplantation genetic screening

22--3 rounds of FISH3 rounds of FISHBiopsyBiopsy

Reduce aneuploid syndromesReduce aneuploid syndromes

Reduce miscarriageReduce miscarriage

Increase embryo implantation/pregnancy rateIncrease embryo implantation/pregnancy rate

Reduce incidence of highReduce incidence of high--order multiplesorder multiples rate ?rate ?

Anticipated benefits for IVF patientsAnticipated benefits for IVF patients

Reduction in aneuploidies 13, 18, 21, XY achieved using PGSReduction in aneuploidies 13, 18, 21, XY achieved using PGS

From 2,300 cases with followFrom 2,300 cases with follow--up data available, mean age 37up data available, mean age 37

PGSPGS –– reduction in aneuploid pregnancyreduction in aneuploid pregnancy

Munne et al 2006 and Reprogenetics data to 10/2007Munne et al 2006 and Reprogenetics data to 10/2007

2.60%

0.50%

0.00%

0.50%

1.00%

1.50%

2.00%

2.50%

3.00%

trisomic conceptions

expectedobserved

P<0.001P<0.001

PGSPGS –– reduction in aneuploid pregnancyreduction in aneuploid pregnancy

Are patients interested in PGS for this purpose?Are patients interested in PGS for this purpose?

If PGS was assumed to have no effect on pregnancy rateIf PGS was assumed to have no effect on pregnancy rate

Recent study of subfertile women (Twisk et al., 2007)Recent study of subfertile women (Twisk et al., 2007)

83% of patients would request PGS83% of patients would request PGS

If PGS was assumed to reduce pregnancy rate from 20% to 14%If PGS was assumed to reduce pregnancy rate from 20% to 14%

36% of patients would still request PGS36% of patients would still request PGS

(75% if 80% detection)(75% if 80% detection)

(31% if 80% detection)(31% if 80% detection)

•• Screening clearly reduces incidence of Down syndromeScreening clearly reduces incidence of Down syndrome

•• Miscarriage rate is probably reducedMiscarriage rate is probably reduced

•• Increase in pregnancy/birth rate remains controversialIncrease in pregnancy/birth rate remains controversial

•• New methods may have finally solved controversyNew methods may have finally solved controversy

Increase in implantation/pregnancyIncrease in implantation/pregnancy-- controversycontroversy

Problems with older technologiesProblems with older technologies

Former technologies only permitted analysis of a restrictedFormer technologies only permitted analysis of a restrictedset of chromosomesset of chromosomes

Cells are in interphaseCells are in interphase

Limited range of fluorochromesLimited range of fluorochromes

Errors caused by spreadingErrors caused by spreading

-- use FISHuse FISH

Problems with older technologiesProblems with older technologies

Less than half the chromosomes are currently testedLess than half the chromosomes are currently tested

Former technologies only permitted analysis of a restrictedFormer technologies only permitted analysis of a restrictedset of chromosomesset of chromosomes

Cells are in interphaseCells are in interphase

Errors caused by spreadingErrors caused by spreading

MosaicismMosaicism

Limited range of fluorochromesLimited range of fluorochromes

Poses a significant problemPoses a significant problemfor diagnosis. However,for diagnosis. However,51% of embryos are51% of embryos areaneuploid in every cell.aneuploid in every cell.

•• RapidRapid –– results in 24 hoursresults in 24 hours

•• Allows the copy number of every chromosome to be determinedAllows the copy number of every chromosome to be determined

NormalNormalTrisomyTrisomy MonosomyMonosomy

Normal DNANormal DNAEmbryo DNAEmbryo DNA

Microarray comparative genomic hybridizationMicroarray comparative genomic hybridization

MicroarrayMicroarray--CGH (arrayCGH (array--CGH or aCGH)CGH or aCGH)

--22 --1313 --1818 --2222

XXXX

--4p4p

Clinical application of CGHClinical application of CGH

•• 100% of chromosome losses/gains detected100% of chromosome losses/gains detected

BenefitsBenefits

•• DNADNA--basedbased

•• No spreadingNo spreading

Diagnosis more robust and accurateDiagnosis more robust and accurate•• Biopsy of several cells is possibleBiopsy of several cells is possible

Less risk of misdiagnosis due to mosaicismLess risk of misdiagnosis due to mosaicism

Analysis of blastocyst stageAnalysis of blastocyst stage

•• Can overcoming the principal challenges to accurate screeningCan overcoming the principal challenges to accurate screeningallow PGS to fulfill the potential predicted by theory?allow PGS to fulfill the potential predicted by theory?

Little or no impact of embryo biopsyLittle or no impact of embryo biopsy

•• Blastocyst cryopreservation (vitrification) necessaryBlastocyst cryopreservation (vitrification) necessary

Clinical application of CGHClinical application of CGH

•• Near 98% survival after biopsy, freeze and thawNear 98% survival after biopsy, freeze and thaw

•• 170 patients, mean age 38 years, 1170 patients, mean age 38 years, 1--6 previous failed IVF cycles6 previous failed IVF cycles(mean 2 failed attempts)(mean 2 failed attempts)

•• Birth rate per cycle with transferBirth rate per cycle with transfer 79%79%

•• Implantation rate per embryoImplantation rate per embryo 67%67%

Blastocyst CGHBlastocyst CGH-- clinical resultsclinical results

60%60%

28%28% **

Control group matched for: maternal age, dayControl group matched for: maternal age, day--3 FSH, day of transfer, # oocytes3 FSH, day of transfer, # oocytesretrieved, # of failed cyclesretrieved, # of failed cycles

*p<0.0003*p<0.0003 -- Extremely promising for single embryo transferExtremely promising for single embryo transfer

•• Embryo loss rates are lowEmbryo loss rates are low

•• 91% of embryos that produced a fetal sac resulted in an91% of embryos that produced a fetal sac resulted in anongoing third trimester pregnancy or live birthongoing third trimester pregnancy or live birth

•• 97% of embryos that produced a fetal heart beat resulted in an97% of embryos that produced a fetal heart beat resulted in anongoing third trimester pregnancy or live birthongoing third trimester pregnancy or live birth

•• Expected pregnancy loss rate for IVF patients in this ageExpected pregnancy loss rate for IVF patients in this agerange is ~25%range is ~25%

Blastocyst CGHBlastocyst CGH-- rates of pregnancy lossrates of pregnancy loss

Blastocyst CGHBlastocyst CGH-- clinical resultsclinical results

Implantation rate

Aneuploidy rate

Cycles with all embryosaneuploid

Patients aged 30Patients aged 30--40 who are eligible for blastocyst transfer40 who are eligible for blastocyst transfer(according to day(according to day--3 criteria) have a >95% chance of having3 criteria) have a >95% chance of havingnormal embryos available for transfernormal embryos available for transfer

R. Scott et al (reported at ASRM 2008):R. Scott et al (reported at ASRM 2008):

•• Blinded prospective studyBlinded prospective study

•• Embryos biopsied at cleavage stageEmbryos biopsied at cleavage stage

•• Copy number of all chromosomes tested using a microarrayCopy number of all chromosomes tested using a microarray

•• Embryos transferred after biopsy, but before aneuploidyEmbryos transferred after biopsy, but before aneuploidyresultresult

Other new data using comprehensive aneuploidyOther new data using comprehensive aneuploidyscreening methodsscreening methods

Retrospective analysis of IVF outcome revealed the following:Retrospective analysis of IVF outcome revealed the following:

•• 0/31 aneuploid embryos produced a child0/31 aneuploid embryos produced a child

•• 26 implantation failure, 5 miscarriages26 implantation failure, 5 miscarriages

•• 100% negative predictive value100% negative predictive value

•• After CGH pregnancy rates were above those typicallyAfter CGH pregnancy rates were above those typicallyachieved for all age groups.achieved for all age groups.

•• However, pregnancy rates were lower for older patients due toHowever, pregnancy rates were lower for older patients due tothe 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 aChromosomally normal embryos from older patients have asimilar chance of producing a child as those derived fromsimilar chance of producing a child as those derived fromyoung patients.young patients.

•• Aneuploidy is likely to be the principal factor causing reducedAneuploidy is likely to be the principal factor causing reducedIVF success with advancing maternal ageIVF success with advancing maternal age

ConclusionsConclusions

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

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

QuestionsQuestions

•• How much of the observed benefit for blastocyst screening isHow much of the observed benefit for blastocyst screening isdue to transfer in a subsequent cycle?due to transfer in a subsequent cycle?

•• Aneuploidy explains most of the decline in IVF success withAneuploidy explains most of the decline in IVF success withadvancing maternal age. What explains the remainder?advancing maternal age. What explains the remainder?

Understanding aneuploidy andUnderstanding aneuploidy andnoninvasive methods of testingnoninvasive methods of testing

Identifying new viability markersIdentifying new viability markers ––gene expressiongene expression

Analysis of gene expression:Analysis of gene expression:

may provide detailed information concerning viabilitymay provide detailed information concerning viability

may reveal the basis of underlying biological problemsmay reveal the basis of underlying biological problems

indicates the processes occurring within a cellindicates the processes occurring within a cell

disturbances of expression associated with morphologicaldisturbances of expression associated with morphologicalabnormalitiesabnormalities

Not even transfer of morphologically normal euploidNot even transfer of morphologically normal euploidembryos can guarantee a pregnancyembryos can guarantee a pregnancy

Pre-genome activation2-cells, 3-cells, early 4-cells

Oocytes

Morulae

Blastocysts

(hatching)

2-10 cell embryos

Gene expression, stage & morphology

Wells et al., Hum. Reprod. & Fertil. Steril. (2005)

4-10 cell embryo withmultinucleation

Wells et al., Hum. Reprod. & Fertil. Steril. (2005)

Gene expression, stage & morphology

Pre-genome activation2-cells, 3-cells, early 4-cells

Oocytes

Morulae

Blastocysts

(hatching)

2-10 cell embryos

BRCA1-Roles in repair of

DNA damage(chromosomebreakage) andchromosomesegregation

Genes with lowGenes with lowlevels oflevels of

expressionexpression

Genes with highGenes with highlevels oflevels of

expressionexpression

Genes showingGenes showingdifferentialdifferentialexpressionexpression

Gene expression microarraysGene expression microarrays

Gene expression microarrayGene expression microarray-- simultaneous analysis of 1000s ofsimultaneous analysis of 1000s ofgenesgenes

Supported by educational grant from EMD SeronoSupported by educational grant from EMD Serono

•• Identify malfunctioning cellular mechanisms resultingIdentify malfunctioning cellular mechanisms resultingin aneuploidyin aneuploidy

•• Identify new nonIdentify new non--invasive markers of oocyte aneuploidyinvasive markers of oocyte aneuploidy

Aneuploidy and oocyte/cumulus cell gene expressionAneuploidy and oocyte/cumulus cell gene expression

•• The origin of aneuploidy is poorly understoodThe origin of aneuploidy is poorly understood

Polarbody

Reverse transcription

mRNA converted to cDNA

RNA purification

Second strand cDNA synthesis

Double stranded cDNA produced

In vitro transcription

ds-cDNA used as a templatefor RNA synthesis

Cell lysis

Whole genome amplification

Cytogenetic analysis

CGH

Reverse transcription

RNA converted to cDNA

Second strand cDNAsynthesis

ds-cDNA produced

1st round of amplification

In vitro transcription andcRNA labelling

ds-cDNA used as a templatefor RNA synthesis

Microarray analysis

2nd round of amplification

Cumuluscells

Oocyte

Real-time PCR

ResultsResults

261 genes with statistically significant differences261 genes with statistically significant differences(213 genes p<0.05; 48 genes p<0.01)(213 genes p<0.05; 48 genes p<0.01)

Altered genes include several involved with spindle dynamics,Altered genes include several involved with spindle dynamics,chromatin structure and cell cycle regulationchromatin structure and cell cycle regulation

Several genes producingSeveral genes producing cell surfacecell surface oror secretedsecreted proteins areproteins aredifferentially expresseddifferentially expressed

Aneuploidy and the oocyte transcriptomeAneuploidy and the oocyte transcriptome

Oocyte aneuploidy and the cumulus cell transcriptomeOocyte aneuploidy and the cumulus cell transcriptome

•• Comparison of cumulus cells associated with aneuploid orComparison of cumulus cells associated with aneuploid ornormal oocytes:normal oocytes:

Differential gene expression for 752 genesDifferential gene expression for 752 genes(125 p<0.01, 627 p<0.05)(125 p<0.01, 627 p<0.05)

•• Principal processes alteredPrincipal processes altered……....

ResultsResults

Cellular pathways altered in cumulus cellsCellular pathways altered in cumulus cellsassociated with aneuploid oocytesassociated with aneuploid oocytes

HormonesHormones

Stress pathwaysStress pathways

HypoxiaHypoxia

Nutritional deprivationNutritional deprivation

Cumulus cellCumulus cellproliferationproliferation

Cumulus cellCumulus cellapoptosisapoptosis

DifferentiationDifferentiation

*Known association with oocyte quality*Known association with oocyte quality

AgeAge AneuploidyAneuploidy

ConclusionsConclusions

Aneuploidy and cumulus cell gene expressionAneuploidy and cumulus cell gene expression

•• Support of specific pathways (in vivo/in vitro) may reduceSupport of specific pathways (in vivo/in vitro) may reduceaneuploidy. Reduce maternal age affectaneuploidy. Reduce maternal age affect

•• Follicular environment influences oocyte aneuploidy ratesFollicular environment influences oocyte aneuploidy rates

•• Targets for indirect detection of chromosome anomaliesTargets for indirect detection of chromosome anomalies14 genes have differential expression (P<0.01)14 genes have differential expression (P<0.01)and fold change >4and fold change >4

•• Altered genes link aneuploidy and ageAltered genes link aneuploidy and age--related hormonalrelated hormonalchangeschanges

Dagan.Wells@obs-gyn.ox.ac.uk