Comprehensive Chromosome Screening in IVF: Enhancing Outcomes While Reducing the Burden of Care

Richard T. Scott, Jr, MD, HCLDClinical and Scientific Director,

Reproductive Medicine Associates of New JerseyProfessor and Director, Reproductive Endocrinology

Robert Wood Johnson Medical School, Rutgers University

aCGH (Lab 1)

SNP array (Lab 3)

qPCR (Lab 2)

46,XY

45,XY,-8

Disclosures

• Board Member – FEC

• No compensation of any kind

• No financial interest in any test or diagnostic platform

The Road to Clinically Effective Preimplantation Genetics

John Rock, MD“According to Specifications”

1937Robert Edwards,

PhD“Sexing Rabbit

Blastocysts”

1967

Leeanda Wilton, PhD“Cleavage Bx”

1986

Marilyn MonkMurine Model

PGD Lesch-Nyhan

1987

Yury Verlinsky, PhD

“PB Biopsy”

1988

Audrey Muggleton-Harris, PhD

“Blastocyst Bx”

19881989Elena

Kontogianni, PhD

“PCR of Y in single

blastomeres”

Alan Handyside, PhDSuccessful

Clinical PGD for X Linked Disorders

1990

Eric Forman, MDEnhanced Safety,

Reduced Cost

2013

Santiago Munne, PhD

First FISH PGD

1993

Nathan Treff, PhDValidated 24 Chr .Platform

2008

What do people think about when you say IVF…

Efficiency of Human Reproduction through IVF

0

20

40

60

80

100

%

N=132,874 Mature Follicles

Retrospective Eval.

Proportion leading to delivery:

7% of Follicles

13% of Embryos

21% of 8 Cells

42% of Blastocysts

What might explain the high failure rate?

Estimating implantation potentialBest practices - morphology

X

1 2 3 4 5 6 7

XX

X X XX

Applying Genomics to Clinical EmbryologyIn an ideal world, identifying implantation potential- the “BEST” test would reflect the true state of nature.

0

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Su

stai

ned

Imp

lan

tati

on

Rat

e (%

)

Embryo Number

X

1 2 3 4 5 6 7

X

X

X X X

X

Transfer decisions would be extremely simple

Contemporary Understanding of Maternal Age and Human Embryonic Aneuploidy

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 450

10

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Age (yrs)

Perc

ent

of E

mbr

yos

Whi

ch a

re A

neup

loid

Franasiak et al – Fertil Steril 2014

N=15,169

Is transferring an aneuploid embryo clinically useful?

Gain of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19

2021

22

XChromosome #

Copy

Num

ber

What are the “Burdens” of CCS

Thus the real questions are:

1. Safely attaining embryonic DNA

2. Predictive values of the techniques

3. Proportion of euploid embryos that will fail

4. Cost effectiveness

Does Embryo Biopsy Impact the Developmental Potential of the Oocyte

Routine IVF Care through Retrieval

Identify mature oocytes ICSI, culture, and select 2 best embryos for transfer

One embryo randomized to undergo biopsy

Transfer the embryos

Implantation, Maternal serum sampling for free fetal DNA and Fingerprinting

Cell submitted for eventual aneuploidy screening and fingerprinting

N=113 pairs; 226 embryos

Overall implantation rates

39% reduction insignificant

27% (mean maternal age 32) reported by Gutierrez-Mateo, C., et al. Fertility and sterility 92, 1544-1556 (2009)

In our opinion, day 3 biopsy will soon be of historic interest only

aCGH enhances delivery rates – an RCT

• RCT

• Age– All < 35– Mean age of 31

• Sample Size– 55 aCGH– 48 control

• Significant improvement in outcomes

• Answers one of the four critical validation questionsMonosomy:Trisomy Ratio of 2

Scott et al Fertility and Sterility 2013; 100:697-703

0

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< 32 33-35 36-38 39-40 41-42

%

Age (yrs)

With greater experience, actual negative

predictive value is ~98.8%

Scott et al Fertil Steril 2012; 97:870-5

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 > 450

10

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100Chart Title

Age (yrs)

No-

Eupl

oid

Blas

ts R

ate

(%)

The No Transfer Rate with CCS

Franasiak et al – Fertil Steril 2014

N=15,169

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 360

5

10

15

20

25

Prop

ortio

n of

Cas

es w

ith th

is

num

ber

of b

last

ocys

ts a

vaila

ble

for

Biop

sy (%

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 360

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Cum

ulati

ve P

ropo

rtion

of C

ases

w

ith th

is n

umbe

r of

bla

stoc

ysts

av

aila

ble

for

Biop

sy (%

)

Number of Blastocyts

52% of cases had 3 or fewer evaluable embryos

How Many Embryos Do Patient Undergoing CCS Have?

Franasiak et al – Fertil Steril 2014

N=15,169

Trisomy:Monosomy Ratio by Age

<35 35-37 38-40 41-42 43+0

0.5

1

1.5

2

2.5

3

Age Group (yrs)

Tris

omy:

Mon

osom

y Ra

tio

Franasiak et al – Fertil Steril 2014Key Indicator for QA of your assay

N=15,169 Ratios consistent across nine programs

Clinical ExperienceMisdiagnoses

• 4974 embryos

• 2976 gestations (62.1%)

• 10 errors– 1 tetraploid– 2 monosomies– 7 trisomies

• 3168 transfers

• 2354 ongoing / delivered (72.1%)

• Mean age 38.4 years

• 10 errors– 7 found in losses– 3 found in ongoing preg.

Mosaicism evaluated in 4 samples – 100% mosaic

Clinical Error RatePer embryo 0.2%

Per transfer 0.3%Per ongoing pregnancy 0.1%

Consolidated Pregnancy OutcomesProportion of All Pregnancies

< 35 35 - 38 - 41 - 43+0

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90

CCS - Delivery

No Screening - Delivery

CCS - Clinical Loss

No Screening - Clinical Loss

CCS - Chemical Los

No Screening - Chemical Loss

Maternal Age (yrs)

Clin

ical

Los

s Rat

e (%

)

N=4,754 pregnancies

Scott KL et al – RMA

Kulkarni D et al, New Engl J Med, 2014. PMID: 24304051

Kulkarni D et al, New Engl J Med, 2014. PMID: 24304051

With >2 blastocysts, even patients at high aneuploidy risk are very likely to have a euploid blastocyst

10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 60% 65% 70% 75% 80%0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

2 Blastocyst3 Blastocyst4 Blastocyst

Aneuploidy Rate

Prob

abili

ty o

f at l

east

1 E

uplo

id B

last

ocys

t

<35 35-37 38-40 41-42

21%34%

55%64%

Blastocyst Aneuploidy Rate by Age Group

Forman EJ et al., O-161

Simplified Calculation of Sustained Implantation Rates

Transfer Order:

30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 603032343638404244464850525456586062646668707274767880828486

1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2

Sustained Implantation Rate (%)

Deliv

ery

Rate

(%)

• Start with delivery rate• Draw horizontal line to transfer order• Draw vertical line to attain SIR• Interpolate as needed

Singleton Term Delivery: The Ideal IVF Outcome

• IVF twin pregnancies are at an increased risk of:– Preeclampsia (2-fold risk increase)1

– Extreme prematurity (7.4-fold increase delivery <32 wks) 2

– NICU admission (3.8-fold increased risk)2

– Perinatal Death (2-fold increase)2

• Two IVF singleton deliveries have better obstetrical outcomes than one IVF twin delivery3

1. ASRM Practice Committee, Fertil Steril, 2012. PMID: 221923522. Pinborg A, et al., Acta Obstet Gynecol Scand, 2004. PMID: 154881253. Sazonova A ,et al., Fertil Steril, 2013. PMID: 23219009

The Perception that Patients Love Multiples if They are Born Healthy is not Well Validated

Provided by a patient…

FRESH SET RESULTS IN LOWER DELIVERY RATES THAN DOUBLE EMBRYO TRANSFER (DET)

• Cochrane Review of 6 randomized trials from 1999-2006 (N = 1,257)

• Young, good prognosis patients with “top quality” embryos available

• Slightly more singletons after DET

Pandian Z et al., Cochrane Database Syst Rev, 2009. PMID: 19370588

SET DET0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

26%30%

13.2%

Livebirth Rate - SET vs. DET

TwinsSingletons

0.5%

The Dropout Rate from IVF is Significant

1 2 3 4 5 6 70%

10%

20%

30%

40%

50%

60%

70%

Dropout/cycleCumm Actual PR

Cycle Number

Perc

ent D

ropo

ut

Source: Schroder AK: Cumulative pregnancy rates and drop out rates of a German IVF programme: 4, 102 cycles in 2,130 patients. RBM Online (2004) 8:600-606

Can1 ≥ 2?

CCS Results in Higher Implantation Rates

Implantation = cardiac activity at time of discharge to obstetrical care (~9 weeks)

Euploid SET Traditional DET0%

10%

20%

30%

40%

50%

60%

70%

80%

66.3%

51.2%

P=0.02

N=83 N=170

Same Delivery Rate:Randomized Controlled Trial

Forman EJ et al. Fertil Steril 2013

0%

10%

20%

30%

40%

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61%65%

Delivery Rate Per Patient (n=175)Single euploid blastocyst transfer (N=89)Untested 2-blastocyst transfer (N=86)

P=0.5

P<0.001

Eliminates Multiples

Single euploid blastocyst transfer Untested 2-blastocyst transfer

100%

52%

0%

48%

Singletons Multiples

P<0.001

Forman EJ et al. Fertil Steril 2013

Single euploid blastocyst transfer Grams

Untested 2- blastocyst transfer Grams

1000150020002500300035004000450050005500

Bir

thw

eig

ht

(Gra

ms)

Better Obstetrical Outcomes are Attained CCS/eSET than Conventional Two Embryo Transfer

•Mean Birthweight: 3408 ± 562g – Single euploid 2745 ± 743g – 2-Blastocyst

(P<0.001)

•Low birthweight (<2,500g): 4.4% (2/45) – Single Euploid 31.9% (22/69) – 2-Blastocyst

(P<0.001)

•Very low birthweight (<1,500g): 0% (0/45) – Single Euploid 7.2% (5/69) – 2-Blastocyst (P=0.06)Single

euploid blastocyst transfer

Untested 2-blastocyst

transfer

Better Obstetrical OutcomesReduced Risk of NICU Admission

Untested 2-Embryo Transfer: 479 total days spent in NICU

Euploid eSET: 93 total days spent in NICU

Forman EJ et al, Am J Obstet Gynecol, 2014. PMID: 24145186

Fewer “Big Ticket” Hospital Admissions

• Mean total hospital and IVF charges per delivery:– $73,407 (euploid eSET)– $111,488 (untested 2-ET)

• ~33% reduction in costs per delivery

• A $38,000 difference per IVF delivery would represent $1.9 B savings on health care costs with ~50,000 IVF deliveries annually

Forman EJ et al

Obstetrical Costs for 100 PatientsCurrent Standard Of Care

Costs per Delivery*

Singleton $21,458

Twins $104,831

Triplets $407,199

Does not include:Pediatric costs after 28 days of ageDisability costs during bed restLoss of productivity in the work place

Lemos et al Am J Obstet Gynecol 2013; 209:586

Overall Cost to Provide Care CCS with SET versus Conventional Treatment

CCS / SET National Avg

Regional Avg

$0

$10,000

$20,000

$30,000

$40,000

$50,000

$60,000

$70,000

$80,000Costs per Delivery*• Use actual cost data

• Inclusive of all IVF costs including– IVF cycle costs– CCS costs– Medication costs

• Delivery costs and subsequent hospital stay through 28 days of life

Natural versus Stimulated Folliculogenesis and Embryonic

Aneuploidy• Prospective

observational

• Historic Control

• Ages 21-49 years

• Follicular diameter at retrieval ~ 21 mm

• hCG induced follicular maturation

<30 30+ 35+ 40+ 42+ 45+0

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Stimulated Natural

Ane

uplo

id (%

)

P=NS

The Natural Cycle Study

Hong ASRM 2014

Patient Acceptance of eSET

CCS Unscreened0%

10%

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DETeSET

Franasiak - RMANJ 2013-2015

Follow Up on Prospective Trials

The Future How NGS Works

• Amplification of limited starting material• Millions of small fragments are generated…

Embryo Biopsy

Next Generation SequencingAlignment

• Aligned relative to a reference genome

Next Generation SequencingDepth of Coverage

• Assemble all the fragments• The number of fragments which “fit” into a given area of the

genome defines the DEPTH• High number of fragments allows each base pair to be

sequenced/ evaluated thousands of times in a single assay

dept

h

Embryo 1 Embryo 3Embryo 2

D1152 D1152

W1282 W1282

D1152

W1282

Cystic Fibrosis Case 1 – Point Mutations

Normal Carrier Affected

Absent Present Present

Absent Absent Present

Universal Primer with new UP reference (SNPs replaced with Ns), on 54 samples. Some small improvements.

Coverage per sample is on next page. Known aneuploidies in red; known XX in cyan.

Next Gen Sequencing for Aneuploidy Screening

Accuracy (n=54)

Per Chromosome: 99.93% (1295/1296)Per Cell: 98.14% (53/54)

Known Mosaic

Time Lapse Observations in the Embryology Laboratory

And others…..

1st 2nd 3rd 4th0

102030405060708090

100ICSI to start of 1st cytokinesis (p=0.61)

Quartile

% E

uplo

id

1st 2nd 3rd 4th0

102030405060708090

100Duration of the 2 cell stage (p=0.88)

Quartile

% E

uplo

id

1st 2nd 3rd 4th0

102030405060708090

100Duration of the 3 cell stage (p=0.12)

Quartile

% E

uplo

id

Time Lapse and AneuploidyTraditional Markers

Hong KH et al – in review

Can Time Lapse Help Distinguish Which Euploid Blasts will Deliver from those Destined to Fail?

Failed

Succesful

1

1.5

2

2.5

3

3.5

4

4.5

5

Transfer Outcome

sy

n_

rm:

tim

e f

rom

sy

ng

am

y t

o 1

st

cy

tok

ine

sis

NO: None of the 5 traditional parameters or 5 additional blast related parameters prognosticate outcome

Temporal data evaluated:

• 5 conventional endpoints through cleavage stage

• Additional temporal endpoints from extended culture:

• First compaction• Morula formation• First cavitation• Blastocyst Expansion• First contraction

Duplications and Deletions

• Arrays versus metaphase spread at time of amniocentesis

• Wopner et al NEJM December 2012

• Population mostly advanced maternal age

• Duplications and Deletions– Evaluated 80 dup/del’s with known clinical phenotype– 1 in 72 fetus’s had a clinically significant dup/del!– Risk considerably higher than for ongoing aneuploid gestation

• Possible to simultaneously screen for these dup/del’s and aneuploidy with multiple platforms

• Extensive validation required

Some Disagree with PGS

• All embryo selection techniques are detrimental• Inappropriate to use “Implantation Rates” as an endpoint• “it can be questioned whether all patients will ever be able to understand all of the complexities

concerning PGS”• “cost-effectiveness is being forgotten”• “In our view evaluating the (cost-)effectiveness of medical treatments is by far the greatest

challenge in current day medicine, especially in an era where health care costs continue to increase to the extent where they are the number one item of expense for many governments across the globe”

• “evidence is now accumulating that all embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairing, and maybe even improving, the cumulative pregnancy rate of that IVF cycle”

• Embryo selection should therefore not be used to select out embryos, but only to determine the order in which the embryos will be transferred, as the time to pregnancy can be improved by embryo selection, if embryos with the highest implantation potential are transferred first.

• Culturing to the blastocyst may be harmful

Chromosome Pairing Before Segregation is Essential to Allow Crossing Over – Even with Balanced Translocations

Normal Translocation Carrier

Normal Chromosome Segregation

Normal

Translocations - Chromosome Segregation

Alternate Segregation

Translocations - Chromosome Segregation

Adjacent-1 Segregation

Translocations - Chromosome Segregation

Adjacent-2 Segregation

Normal

Normal Balanced Unbalanced Unbalanced

Unbalanced Unbalanced Unbalanced Unbalanced Unbalanced

Alternate Segregation Adjacent-1 Segregation Adjacent-2 Segregation

3:1 Segregation 3:1 Segregation 3:1 Segregation 3:1 Segregation 4:0 Segregation

The RMA New Jersey Research TeamScientific Director

Richard T. Scott

PhysiciansPaul Bergh

Michael BohrerMaria CostantiniMichael Drews

Eric FormanRita Gulati

Doreen HockKathleen Hong

Thomas KimMarcy Maguire

Thomas MolinaroJamie Morris

Eli RybakWendy Schillings

Shefali Shastri

FellowsMarie Werner

Jason FranasiakCaroline Juneau

The Treff LaboratoryNathan Treff

Xin TaoAgnes LonczakDavid GabrieleChelsea BohrerTori Gartmond

Margaret Lebiedzinski Oksana Bendarsky

Mariya RozovAnna Czyrsznic Kay Green, MDMeir Olcha, MDDavid Goodrich

BioinformaticsDeanne TaylorJessica Landis

Yuanchao Zhang

Clinical EmbryologyKathleen Upham

Xinying LiRosanna Pangasnan

Tian ZhaoMichael ChengHokyung Lee

Ayesha WinslowAngela Romaniello

Stephanie MilneSarah DunnLauren Rary

Kristen PauleyDesiree GreeneSerhan Ozensoy

Erin DubellSusan Ng

Maria MorelJessica Wall

FECRebekah

ZimmermanBrynn Levy

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Clinical ResearchChristine RedaTalia MetzgarJennifer Tyler

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