Santiago Munné, PhD

PGS with fresh embryo transfer Fresh embryo

Reprogenetics Laboratories US: Livingston (NJ), Los Angeles (CA), Chicago (IL), Portland (OR), Boca Raton (FL) / Europe:

Barcelona (Spain), Oxford (UK), Hamburg (Germany) / Asia: Kobe (Japan), Macao, Abu Dhabi (UAE) / Latin America: Lima (Peru), Buenos Aires (Argentina), Sao Paulo (Brazil), DF (Mexico)

Why PGS?

Most loss of implantation is caused by chromosome abnormalities

Reprogenetics data: 96 centers, >3500 cycles, >19,000 blastocysts analyzed by aCGH to 9/2013

0%10%20%30%40%50%60%70%80%90%

100%

<35 35-37 38-40 41-42

% euploid no imp% euploid implan% aneuploid

Euploidy decreases with age but not with cohort size

N = 4,747 cycles and 29,803 embryos, up to 12/2013. Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data.

# of blastocysts

% normal embryos egg

donors <35

years 35-37 years

38-40 years

41-42 years

>42 years

1-3 58% 61% 51% 39% 22% 13%

4-6 62% 60% 52% 38% 23% 17%

7-10 65% 62% 51% 36% 21% 14%

>10 68% 63% 55% 37% 25% n/a

The PGS hypothesis - updated

• 50% of blastocysts are aneuploid, • Aneuploidy increases with maternal age • Maternal age is inversely proportional to implantation • The error rate of PGS with CCS is low (<2%) • And blastocyst biopsy is non-detrimental

Therefore PGS with CCS and blastocyst biopsy:

• Improves Implantation rates • Eliminates the Maternal age effect on implantation

CCS: comprehensive chromosome analysis, such as array CGH, qPCR, Karyomaping, NGS

WHEN TO BIOPSY

Biopsy stage: before arrays

• 30% postmeiotic abnormalities undetected

• Triple amount of cells to analyze vs. blastocysts

•30%a to 60%b loss of implantation potential

• PGD can compensate the damage but does not reach its potential

• Centers not proficient in blastocyst culture until recently

• Applied clinically in 2005 c

• First positive results in 2010 d

a Scott et al (2013), b Mastenbroek et al. (2007), c McArthur et al. (2005), d Schoolcraft et al. (2010)

blastocyst biopsy: Advantages

Advantages:

• More DNA: less no results

• Less mosaicism = low error rate

• Reduced impact of embryo biopsy

• Less embryos to process

• Facilitates single embryo transfer

• Frozen cycle: Uterine environment optimized after thaw

Disadvantages: • Not all embryos reach blastocyst the same day

Evolution of PGS: Reprogenetics data

0

1000

2000

3000

4000

5000

6000

7000

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

Num

ber o

f Cas

es

Year

aCGH day 5

aCGH day 3

CGH

FISH 12

FISH 5-9

Reprogenetics Laboratories: 42,000 PGD procedures up to 3/2014

COMPREHENSIVE

CHROMOSOME ANALYSIS

aCGH advantages

• All 24 chromosome aneuploidies and translocations detected.

• Results in <16 hours: allows for day 5 biopsy and 10am day 6 transfer

• Parental DNA not required: ad hoc decisions possible. • ICSI not required.

46,XX+7-10

aCGH validation: reanalysis of blastocysts

Fragouli et al. (2011) Hum. Reprod. 26: 480-90, Colls et al. (2013) ASRM P-168, Capalbo et al. (2013) Hum Reprod, in press, Wells et al. (2013) ASRM O-435 and unpublished data from Reprogenetics

11% of embryos were mosaic, explaining the 2.4% error rate

Reanalysis method

Confirmed Euploid

Confirmed abnormal

TOTAL

Fragouli et al 2011 FISH, aCGH 23/25 27/27 50/52

Capalbo et al. 2013 FISH 19/20 50/50 69/70

Colls et al. 2013 FISH, aCGH 7/7 39/40 46/47

Wells et al. 2013 Next Gen. Sequencing

23/23 67/67 90/90

Total 96% Sensitivity

99.5% Specificity

1.6% Error rate

Biopsy Reception Results by qPCR: day 5 day 5, 6pm day 6, am aCGH: day 5 day 5, 6pm day 6, noon NGS: day 5 day 5, 6pm day 6, noon SNPs: day 5 day 5, noon day 6, 6pm

Speed of different techniques

Differences between PGD methods

PCR PCR + SNP Next Gen aCGH arrays* Sequencing Detects aneuploidy no yes yes yes Detects gene defects yes yes yes yes Detects mitotic errors no yes no yes >2 month of Preparation yes yes no no Requires affected proband no no yes no # genomes / run 0 0 0 1 * * Karyomapping using BlueGnome ** with NextSeq

NGS: Capacity vs. Depth

samples genome depth of / run coverage coverage Genome 1 100% x 30 PGS * 16- 96 ≤ 10% x1 to x3

* Output: MiSeq = PGM << NextSeq

Wells, Kaur, Rico, Grifo, Anderson, Sherlock, Taylor, Munne (2013) ESHRE, Yin et al (2013) Biol Reprod 88, 69

78 blastocysts previously diagnosed by aCGH were reanalyzed by NGS in a blinded experiment.

21/21 euploid 90/90 aneuploid > 99% concordant

validation of Next Generation Sequencing (NGS)

Allen Kung et al. (2014) ESHRE and Reprogenetics unpublished data

Overall clinical results with PGD v2

• Blastocyst biopsy • Comprehensive chromosome analysis

IMPLANTATION RATES IN RCT STUDIES USING PGS v2:

Meta analysis of RCTs

Control PGS Yang et al. 2012 46% 69% Scott et al. 2013 63% 80% Forman et al. 2013 40% 58% TOTAL 53% 73% P<0.001

maternal age effect disappears with full

chromosomes analysis

aCGH eliminates the negative effect of maternal age on implantation

* SART 2011 ** Harton, Munné et al. (2013) Fertil Steril. And unpublished data to 8/2013. N >800 blast biopsies

0%

10%

20%

30%

40%

50%

60%

<35 35-37 39-40 41-42 >42

SARTblastocystPGS (aCGH) **

No PGS *

Impl

anta

tion

rate

Maternal age

n/a

Miscarriage rate after blastocyst biopsy

0%5%

10%15%20%25%30%35%40%

<35 35-37 38-40 41-42

Compared to SART: Compared to other studies:

Pregnan cies

age SAB

This study

307 34.9 7.5%

Scott et al. 2013

72 32.2 8.3%

*SART, ** Harton et al. (2013) Fertil Steril, and unpublished data

No PGS * PGS **

One clear advantage of freezing:

EMBRYO BANKING

Reprogenetics data, unpublished >350 cycles of embryo banking, average age 39.9

In embryo banking aneuploidy rates Remain constant

1st cycle

2nd cycle

3rd cycle

Total

Euploidy rate 29% 29% 27% 28% # blastocyst produced 2.4 3.0 2.5 6.3 # euploid blastocysts 0.7 0.9 0.7 2.2

N = 3,571 cycles and 19,356 embryos, up to 8/2013. Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data.

# of blastocysts

% of patients with normal embryos egg

donors <35

years 35-37 years

38-40 years

41-42 years

>42 years

1-3 86% 85% 72% 60% 58% 24%

4-6 95% 97% 95% 88% 69% 54%

7-10 100% 99% 96% 92% 85% 65%

>7-10 100% 100% 98% 98% 92% 83%

Embryo banking for low responders or bad prognosis patients

26

Success of SET by Euploid Cohort Size

No. Euploid Embryos

CPR

1 23/55 (41.8%) 2 13/27 (48.1%) 3 9/19 (47.4%) 4 16/21 (76.2%) 5 8/11 (72.7%) 6 6/8 (75.0%)

>7 11/15 (78.6%)

p < 0.01

S. Morin, K. Melzer, J. Grifo, P. Colls, Z. Zheng, S. Munné (2014) JARG

Cycles needed to accumulate 3 euploid blasts

Age Av. blastocysts Av. euploid

# cycles to achieve 95% patients w/ 3

euploid

35-39 5.2 2.5 2.7

40-42 4.2 1.1 5.8

Advantages of embryo banking

• Less patient “fatigue”: less drop out from

cycle to cycle.

• Cheaper PGD: One fee per package of IVF cycles

• Facilitates “guaranteed baby” plans

Fresh or Frozen?

Roque et al., 2012

RR=1.31 [1.10-1.56]

Meta-Analysis

Courtesy: Dr. Racowsky

Does COS affects endometrial receptivity? Non-PGD Studies

The only effect is on embryos developing to blastocyst on day 6, which in PGS with fresh transfer they are frozen.

Does COS affects endometrial receptivity? Non-PGD Studies

Is it worthy to biopsy day 6 blastocysts?

Reprogenetics, unpublished

The differences between day 5 biopsy and fresh transfer vs. day 5-6 biopsy and vitrification is that the later includes day 6 biopsies:

- Day-5 morulas were cultured to day-6 and biopsied if reached blastocyst

- SET of blastocysts either biopsied on day 5 or on day 6, thawed transfer

Day 5 blastocyst

Day 6 blastocyst

Implantation 61% 60% N.S.

Euploidy 56% 42% P<0.025

SET SET SET fresh FET FET Control Control PGS-CCS Cycles 118 272 347 Maternal age 36.1 36.8 37.9 p<0.05 Implantation rate (FHT) 49.2% 52.6% 65.1% p<0.05 SAB 14.3% 14.4% 4.6% p<0.05 Ongoing pregnancy rate 40.7% 43.8% 60.0% p<0.05

Schoolcraft et al. Fertil Steril 2013;100:615–9

Controlled studies comparing both treatments

Difference between control and PGS: not due to FET

Meta-analysis

Anderson et al. (2013) ASRM O-120, Barrit et al. (2013) ASRM abstract P-167, Forman et al. (2013) Fertil Steril

# cycles

Ongoing pregnancy rate /

transfer

FET fresh day 6 FET

fresh day 6

Anderson et al. 2013 38 46 55% 66% NS Hill et al. 2013 143 99 56% 45% NS Forman et al. 2013 27 62 55% 65% NS Total 208 207 56% 56% NS

day 6 transfer and the window of implantation

No studies published, but some recommend: • Replace before noon day 6 but… • If last progesterone before trigger is elevated then

freeze: the endometrium is advanced and the window of implantation could be missed.

• Each center needs to determine their threshold. One

center recommends 1.5 or over to freeze.

What IVF centers are doing in the US?

2013 2014 Q1 Day 3 fresh 18.1% 20.9% Day 5 fresh 14.3% 12.4% Day 5 frozen 67.5% 66.6%

Total PGD procedures

4359 1460

In Germany is mostly PB biopsy, and in Spain mostly day 3

Fresh: pros and cons

Pros - Day 5 blastocysts implant equally well on d6 or FET - Lower cost (no freezing) - Some centers biopsy day 5 morulas - International patients: no need to come back for transfer - INSTANT SATISFACTION Cons - Day 6 blastocysts need to be frozen anyway - Does not allow for embryo banking

Frozen: pros and cons

Pros - Day 5 blastocysts implant equally well on d6 or FET - Day 6 blastocysts implant better frozen - Allows for embryo banking for poor prognosis patients - Better uterine receptivity in aggressive stimulations

Cons - Higher cost (freezing) - International patients need to come back for transfer - Delayed gratification

Ongoing RCT:Fresh vs. frozen using NGS

Procedure: all patients undergo blastocyst biopsy and analysis by Next Gen Sequencing. Group comparison: Patients randomized to day 6 transfer or to FET. Location: Oregon Reproductive Medicine, Portland, OR.

munne@reprogenetics.com www.reprogenetics.com

Scientists Jacques Cohen, PhD (US) Santiago Munne, PhD (US) Dagan Wells, PhD (UK) Renata Prates (US) Samer Alfarawati (UK) Souraya Jaroudi (UAE) Tomas Escudero (US) Mireia Sandalinas, PhD (Spain) Luis Guzman, PhD (Peru) J. Horcajadas, PhD (Latin Am.) M. Konstantinidis, PhD (US) N’Neka Goodall (US) Allen Kung (US) Lia Ribustello (US)

Lab & Medical Directors Pere Colls, PhD (US) Carles Gimenez, PhD (Spain) Elpida Fragouli, PhD (UK) Karsten Held, MD (Germany) Tetsuo Otani, MD (Japan) Muriel Roche, PhD (Japan) Braulio Peramo, MD (UAE) Ahmed Yesilyurt, MD (Turkey) Xuezhong Zeng, MD (China) Francisco Rocha (Mexico)

Reprogenetics

Embryologists Kelly Ketterson Catherine Welch Tim Schimmel Genetic Councilors Jill Fischer Amy Jordan Erin Mills G. Manassero, MD