Should Every Embryo be Screened or Frozen?

What does the evidence say?

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

Contemporary Understanding of Maternal Age and Human Embryonic Aneuploidy

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Per

cen

t o

f Em

bry

os

Wh

ich

are

An

eup

loid

Age (yrs)

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

X

Chromosome #

Co

py

Nu

mb

er

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

Some Disagree with PGS

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”

• “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

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

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Normal Result

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ue

(%

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CIN III or Malignancy

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Abnormal Result

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CIN III or Malignancy

Is knowing the predictive value of a normal result sufficient?

Sherman et al 95:429-36 J Natl Cancer Inst (2003)

If they were the same it would likely be a rare coincidence…

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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

To have the opportunity for meaningful improvement, when you select for one criteria you most commonly deselect for another….

Transfer Based on Embryo Morphology

Abnl Abnl Nl Abnl Nl Nl

Transfer Based on Aneuploidy Screening and

Embryo Morphology

CCS changes the embryo selected 40% of the timeForman et al ASRM 2012

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

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22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 > 45

No

-Eu

plo

id B

last

s R

ate

(%

)

Age (yrs)

The No Transfer Rate with CCS

Franasiak et al – Fertil Steril 2014

N=15,169

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avai

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psy

(%

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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

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1.5

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2.5

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

Tris

om

y:M

on

oso

my

Rat

io

Age Group (yrs)

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

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< 35 35 - 38 - 41 - 43+

Clin

ical

Lo

ss R

ate

(%)

Maternal Age (yrs)

CCS - Delivery

No Screening - Delivery

CCS - Clinical Loss

No Screening - Clinical Loss

CCS - Chemical Los

No Screening - Chemical Loss

N=4,754 pregnancies

Scott KL et al – RMA

PGS Improves but Does Not Normalize Implantation and Delivery Rates in Older Women

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<25 26-29 30-34 35-37 38-40 41-42 43+

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ain

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Imp

lan

tati

on

Rat

e (

%)

Maternal Age (yrs)

N=28,567

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

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

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

Provided by a patient…

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

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100%

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

Pro

bab

ility

of

at le

ast

1 E

up

loid

Bla

sto

cyst

Aneuploidy Rate

2 Blastocyst

3 Blastocyst

4 Blastocyst

21%34%

55%64%

<35 35-37 38-40 41-42

Blastocyst Aneuploidy Rate by Age Group

Forman EJ et al., O-161

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

26%30%

13.2%

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5%

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25%

30%

35%

40%

45%

SET DET

Livebirth Rate - SET vs. DET

Twins

Singletons

0.5%

The Dropout Rate from IVF is Significant

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Dropout/cycle

Cumm Actual PR

Cycle Number

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rce

nt

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po

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)

66.3%

51.2%

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80%

Euploid SET Traditional DET

P=0.02

N=83 N=170

Same Delivery Rate:Randomized Controlled Trial

Forman EJ et al. Fertil Steril 2013

61%65%

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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

100%

52%

0%

48%

Single euploid blastocyst transfer Untested 2-blastocyst transfer

Singletons Multiples

P<0.001

Forman EJ et al. Fertil Steril 2013

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Single euploidblastocyst

transfer Grams

Untested 2-blastocyst

transfer Grams

Bir

thw

eig

ht

(Gra

ms)

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

•Mean Birthweight:3408 ± 562g – Single euploid2745 ± 743g – 2-Blastocyst

(P<0.001)

•Low birthweight (<2,500g):4.4% (2/45) – Single Euploid31.9% (22/69) – 2-Blastocyst

(P<0.001)

•Very low birthweight (<1,500g):0% (0/45) – Single Euploid7.2% (5/69) – 2-Blastocyst (P=0.06)

Single euploid

blastocyst transfer

Untested 2-blastocyst

transfer

Ongoing Pregnancy Rates Fresh vs. Frozen Transfers

37%

57%

43%

54%

ControlSET CCSSET

FreshET

FrozenET

66%oftransfers

50%oftransfers

P = 0.4 P = 0.7

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 age

Disability costs during bed rest

Loss of productivity in the work place

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

Overall Cost to Provide CareCCS with SET versus Conventional Treatment

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Costs 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

Do we ever recommend two embryo transfers?

Yes – but with caution…

Follow Up on Prospective Trials

Time Lapse Observations in the Embryology Laboratory

And others…..

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1st 2nd 3rd 4th

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

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Duration of the 2 cell stage (p=0.88)

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Duration of the 3 cell stage (p=0.12)

Time Lapse and AneuploidyTraditional Markers

Hong KH et al – in review

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Time from start of 1st cytokinesis to start of cavitation (p=0.01)

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Hong KH et al in review

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

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Transfer Outcome

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

Follow Up on Prospective Trials

Next Generation SequencingAligned Results

Reference sequence from

human genome database

Mu

ltip

le R

ea

ds (

de

pth

)

The Economics of NextGenA Major Factor for Accuracy

$$$$$$$

NextGen Sequencing Chip

NextGen Molecular Barcoding Reduced Costs

Embryo 1 Embryo 2

Barcode 1

Barcode 2

CTAAGGTAAC

TAAGGAGAAC

combine samples for a single sequencing chip

The Economics of NextGenA Major Factor for Accuracy

$$$$$$$/2

NextGen Sequencing Chip

The Economics of NextGenA Major Factor for Accuracy

$$$$$$$/4

NextGen Sequencing Chip

The Economics of NextGenA Major Factor for Accuracy

$$$$$$$/48

NextGen Sequencing Chip

The Economics of NextGenA Major Factor for Accuracy

$$$$$$$/96

NextGen Sequencing Chip

96 or more…

WGS(16 per chip)

chromosome

copy number

known trisomy

known monosomy

unpublished data

WGS(48 per chip)

chromosome

copy number

unpublished data

Targeted NGS (96 per chip)

chromosome

copy number

unpublished data

Embryo calibration results

chromosome

copy number

unpublished data

Chromosome specific cutoffs

1

0

2

3

4

NGS based copy number

on chr16

chr16 specific cutoffs

unpublished data

Embryonic Endometrial Synchrony

It take two…..

Embryonic-Endometrial Asynchrony Increases with Maternal Age

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Elev P Day 6 Blast OverallAsynch

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Cyc

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<30 31-34 35-40

• Retrospective

• 1,341 IVF cycles

• Thresholds for Asynchrony (either)– P >1.5 mg/mL on day of hCG – No blastulation prior to day 6

• Risk for asynchrony increases with maternal age

• Live birth predicted – Day 5 blastulation (P<0.0001)– P < 1.5 ng/mL (P=0.0002)

Shapiro BS et al Fertil Steril 2013 100:S287

P<0.01

(419) (436) (486)

Is it asynchrony or an intrinsic diminution in quality?

Late follicular rise in progesterone

• Retrospective study

• 4032 patients

• P4 ≥1.5ng/mL associated with lower ongoing pregnancy rates

Bosch E, et al. Hum Reprod. 2010 Aug;25(8):2092-100.

Progesterone and the Endometrial Transcriptome

Adapted from S. Young, MD, PhD

2.5 mg/d IM

Leuprolide acetate 1 mg/d td sc

40 mg/d IM

5 mg/d IM

Estradiol 0.2 mg/d td

10 mg/d IM

Progesterone Pharmacokinetics

Adapted from S. Young, MD, PhD

Progesterone and the Endometrial Transcriptome

Adapted from S. Young, MD, PhD

Progesterone and Impaired Implantation:A Pilot Study of Euploid Embryos

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Hours Relative to “Closure” of the Window

All patients had normal P levels prior to the administration of hCG

Beware of Interference in your P Assay

• Patients receiving DHEA have elevated DHEA-SO4 levels

• These levels may falsely elevate P levels

• Assay dependent

Forman - RMANJ

Natural Cycle

hCG

administration

Progesterone

Rise

Ovulation

Embryonic

Window of

Implantation

Endometrial

Window of

Implantation

time

Franasiak et al ASRM 2013

hCG

administration

timeEndometrial

Window of

Implantation

Embryonic

Window of

Implantation

embryo and endometrium synchrony -revisited

Progesterone

Rise

24h

Ovulation

Franasiak et al ASRM 2013

Fresh day 5 embryo transfer

44%

18%

64%

56%

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70%

<35 ≥35

D5 M-B1 D5 B2-B6

p <.001p <.001

Franasiak et al ASRM 2013

Fresh day 6 embryo transfer

p <.05

52%

32%

63%

48%

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60%

70%

<35 ≥35

D5 M-B1 D5 B2-B6

p <.005

Franasiak et al ASRM 2013

timeEndometrial

Window of

Implantation

Frozen synchronous cycle

Progesterone

Start

Older patients are more likely to have “slow” embryos

31%

46%

0%

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30%

40%

50%

60%

70%

<35 years old ≥35 years old

Proportion of "Slow" Blastocysts

P<0.0001

Forman et al ASRM 2013

Frozen day 6 embryo transfer

p =0.5

57%

37%

60%

42%

0%

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20%

30%

40%

50%

60%

70%

<35 ≥35

D5 M-B1 D5 B2-B6

p =0.3

Franasiak et al ASRM 2013

Obstetrical Outcomes Following Fresh versus Cryopreserved Embryo Transfer

• Fresh embryos at increased risk for• Preterm birth• Low birth weight• Small for gestational age

Wennerholm et al Hum Reprod 2013 28:2545-53

The supraphysiologic milieu which accompanies superovulation impact low birth weight risk

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TermLBW

PretermLBW

Fresh ET

Cryo ET

• Retrospective review of SART data

• 2004-2006

• 56,792 neonates

• Fresh embryo transfer at increased risk for LBW

%

The RMA New Jersey 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 LebiedzinskiOksana Bendarsky

Mariya RozovAnna CzyrsznicKay 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

FAEECRebekah

ZimmermanBrynn Levy

Lindsey McBainHeather Garnsey Andrew Behrens Sylvia Kloskowski

Erica Vuu

Clinical ResearchChristine RedaTalia MetzgarJennifer Tyler

SupportAndrew Ruiz

Nancy NiemaseckRMANJ Nurses