RGH ILLUMINA: Preimplantation Genetic Screening: Scientific Evidence, Alan Thornhill

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New Concepts in Preimplantation
Genetic Screening
Professor Alan Thornhill
Market Development
Illumina Inc.
15th December 2016

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IVF outcomes: Overall Poor with Huge Variation
378k
192k
28k
180k
60k
250k
80k
104k
57k
20k
20k
1.5 million IVF cycles globally per year
Only about 1 in 3 take home baby rate
2010-2014 (1% increase in LB YOY)
21% versus 67% - CP/cycle started

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Maternal Age, Aneuploidy, and Fertility
1. CDC, 2013 ART National Summary Presentation, http://www.cdc.gov/art/reports/2013/national-summary.html; Figures 14-16
2. Franasiak JM, Forman EJ, Hong KH, et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with
comprehensive chromosomal screening. Fertil Steril. 2014;101(3):656-663.e1.
Aneuploidy is a major cause of the age-related decline in fertility1,2
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
24 25 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
% Live Birth, Miscarriage, Aneuploidy vs. Age
Live Birth % (CDC) Miscarriage % (CDC) D5 Aneuploidy %(Franasiak)
Age (years)

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The Embryologist’s Dilemma....
Pick me ... No … pick me,
don’t worry about
a couple of
fragments
No … I’m the one …
I’ve got nice nuclei
Pick me ...
Courtesy of Dr S.Troup/Prof C Kingsland. Hewitt Fertility Centre, Liverpool Women’s Hospital
Worldwide this process occurs ~ 50,000 times daily .….

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Aneuploidy in Embryos – The Facts
Aneuploidy = incorrect # of chromosomes
(eg Trisomy 21/Down’s syndrome)
Main reason for failed implantation and
failed development to term
Present in women of all ages (~50%)
Increases with maternal age
All chromosomes represented at day 5*
Development to blastocyst stage does not
eliminate specific aneuploidies
Similar incidence between natural and
stimulated cycles** (STREAM study?)
Ref: S Munne. Preimplantation Genetic Diagnosis for Aneuploidy and Translocations Using Array Comparative Genomic Hybridization. Curr
Genomics. 2012 Sep; 13(6): 463–470.
*Franasiak et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies
evaluated with comprehensive chromosomal screening. Fertil Steril 2014 101:656-63
**Ata B, Kaplan B, Danzer H, et al. Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed
Online. 2012;24(6):614-620.

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Benefits of PGS

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Next Generation IVF
Improving Clinical Outcomes by PGS
Avoid transfer + storage of aneuploid embryos
Mitigates the effect of maternal age1
Increase Implantation Rates 2,3
Increase pregnancy & live birth rates/embryo transfer 3,4
Reduce miscarriage rate 2,4,5
Select euploid blastocysts to support single-embryo transfer
(reduce multiple pregnancy and birth, especially twins) 2,3,4,5
Reduce time to live birth? Costs?
1. Harton G, Munné S, Surrey M, et al. Diminished effect of maternal age on implantation after Preimplantation Genetic Diagnosis with array comparative genomic
hybridization. Fertil Steril. 2013; 100(6):1695-703.
2. Grifo JA, Hodes-Wertz B, Lee HL, Amperloquio E, Clarke-Williams M, Adler A. Single thawed euploid embryo transfer improves IVF pregnancy, miscarriage, and multiple
gestation outcomes and has similar implantation rates as egg donation. J. Assist. Reprod. Genet. 2013; 30, 259–64.
3. Scott RT, Upham KM, Forman EF, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases IVF IRs and
delivery rates: a randomized controlled trial. Fertil. Steril. 2013; 100(3):697-703
4. Yang Z, Liu J, Collins GC, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF
patients: results from a randomized pilot study. Mol. Cytogenet. 2012; 5(1):24
5. Forman EJ, Hong KH, Ferry KM, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013; 100(1):100-7.

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Clinical Evidence Supporting PGS

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0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
30-34 35-37 38-40 41-42
Implantation
Aneuploidy
Miscarriage
Harton GL, Munne S, Surrey M, et al. Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative
genomic hybridization. Fertil Steril. 2013;100(6):1695-1703.
PGS Helps Mitigate Maternal Age Effect
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
30-34 35-37 38-40 41-42
Implantation
Aneuploidy
Miscarriage
Selective transfer of euploid embryos diminishes the adverse effect of
maternal age (up to 42 years) on implantation1

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Improved IVF outcomes with 24sure®
PGS – good prognosis patients
Ongoing pregnancy (> 20 weeks)
41.7%
Morphology
Alone
69.1%
aCGH PGS
+
Morphology
For Research Use Only. Not for use in diagnostic procedures.
Ref: Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good
prognosis IVF patients: results from a randomized pilot study.
Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, Peck AC, Sills ES, Salem RD.
Mol Cytogenet. 2012 May 2;5(1):24. doi: 10.1186/1755-8166-5-24
*Young, good prognosis patients; n=103, age<35, first-time IVF, no history of miscarriage

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1. Forman EJ, Hong KH, Ferry KM, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013;100(1):100-
107.e101.

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PGS Enables More Efficient Elective Single
Embryo Transfer (eSET)
1. Forman EJ, Hong KH, Ferry KM, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013;100(1):100-107.e101.
61% 65%
SINGLE EUPLOID
BLASTOCYST
TRANSFER (N=89)
UNTESTED 2-
BLASTOCYST
TRANSFER (N=86)
100%
52%
0%
48%
SINGLE EUPLOID
BLASTOCYST TRANSFER
(N=54)
UNTESTED 2-BLASTOCYST
TRANSFER (N=56)
Singletons
Multiples
Similar ongoing pregnancy rates.
Single euploid transfer dramatically reduced the risk of a multiple gestation.1
Patients less than 43 years of age
Fetal Number in Ongoing PregnanciesOngoing Pregnancy

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Whitney et al (2016) JARG Advance online
• An observational, retrospective cohort study of first transfer
outcomes was performed in two groups:
Group 1 (PGS) all blastocysts were biopsied on days 5/6,
vitrified and array CGH (24Sure™) performed
Group 2 (no PGS) included the first transfer on day 3 or
day 5 from non-PGS fresh cycles or cycles in which all
blastocysts had been vitrified
• Clinical pregnancies and implantation were confirmed by
ultrasound and live birth information obtained

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Whitney et al (2016) JARG Advance online

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43 41 37 12
64 32 35 15
PGS n =
No PGS n =
Whitney et al (2016) JARG Advance online

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Meta-analyses and Real world Data

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Lee E, Illingworth P, Wilton L, Chambers GM. The clinical effectiveness of preimplantation genetic diagnosis for aneuploidy
in all 24 chromosomes (PGD-A): systematic review. Hum Reprod. 2015;30(2):473-483.

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PGS RCTs in Young Women with Good Prognosis
Ongoing Pregnancy
0
0.5
1
1.5
2
2.5
Yang, 2012 Forman, 2013 Scott, 2013 Combined RCT
RiskRatio
Randomized Controlled Trials
PGS vs. Routine Care
Ongoing Pregnancy >20 Weeks
FavorsPGS
FavorsControl
Ongoing pregnancy 1.39x more likely with PGS in RCTs
Lee E, Illingworth P, Wilton L, Chambers GM. The clinical effectiveness of preimplantation genetic diagnosis for aneuploidy
in all 24 chromosomes (PGD-A): systematic review. Hum Reprod. 2015;30(2):473-483.

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Meta-analysis of published studies on PGS using comprehensive
chromosome screening (CCS) versus routine care in embryo selection
Determine clinical and sustained implantation rates
In-depth evaluation of the available evidence for this new form of PGS
Dahdouh EM, Balayla J, García-Velasco JA. Comprehensive chromosome screening improves embryo selection: a meta-
analysis. 2015; 104(6):1503-12.

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Matched Cohort Studies of PGS in AMA Women
Clinical Implantation Rate (IR)
0
1
2
3
4
5
6
7
Sher, 2009 Schoolcraft,
2010
Fishel, 2011 Forman, 2012 Keltz, 2013 Greco, 2014 Combined OS
RiskRatio
Observational Studies
PGS vs. Routine Care
Clinical IR (Gestational Sacs/#ET)
Dahdouh EM, Balayla J, García-Velasco JA. Comprehensive chromosome screening improves embryo selection: a meta-analysis. Fertil Steril. 2015;104(6):1503-1512.
FavorsPGS
FavorsControl
Clinical IR 1.78x more likely with PGS in
observational studies

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Matched Cohort Studies of PGS in AMA Women
Ongoing Pregnancy
Dahdouh EM, Balayla J, García-Velasco JA. Comprehensive chromosome screening improves embryo selection: a meta-analysis. Fertil Steril. 2015;104(6):1503-1512.
0
1
2
3
4
5
Sher, 2009 Forman, 2012 Lee, 2015 Feichtinger, 2015 Combined OS
RiskRatio
PGS vs. Routine Care
Ongoing Pregnancy >20 weeks
FavorsPGS
FavorsControl
Ongoing pregnancy 1.75x more likely with PGS in
observational studies
Observational Studies

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Chang J, Boulet SL, Jeng G, Flowers L, Kissin DM. Outcomes of in vitro fertilization with preimplantation genetic diagnosis: an analysis of
the Unites States Assisted Reproductive Technology Surveillance Data, 2011-2012. Fertil Steril. 2016;105(2):394-400.
~107,000 fresh, non-donor cycles with blastocyst transfer from clinics
with ≥1 PGD cycle
– 5,471 cycles w/ PGD Aneuploidy (same as PGS) vs. 97,069 non-PGD
cycles
– 2,859 PGD Other & 1,503 PGD Genetics cycles
For women who underwent IVF with PGD Aneuploidy (PGS):
Decreased risk of miscarriage in women ≥35 years of age
Increased chance of live birth in women >37 years of age

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PGS – Does choice of technology matter?

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Evolution of PGS Technology
- Which is the best?
FISH
24sure ® VeriSeqTM PGS
Arrays SequencingTechnique
Product
Data points 5 3,000 1,000,000
Vysis
Aneuvysion
For Research Use Only. Not for use in diagnostic procedures

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Advantages of NGS vs aCGH for PGS
(courtesy of Dr Tony Gordon, Genesis Genetics)
Whole copy chromosome changes – no real advantage
Sub chromosomal changes – smaller gains and losses –
NGS more sensitive and specific
Mosaic samples
– 50% detection limit aCGH
– 25% detection limit NGS*
Noisy samples – NGS more able to determine signal vs noise
Detect common 69XXY and 69XYY triploid – 70% all triploids
Scalable and cost-effective for large sample volume
* Mosaic embryos have reduced implantation potential but can produce normal live births

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1. Bolton H, Graham SJL, Van der Aa N, et al. Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal
developmental potential. Nat Commun. 2016;7:11165.
2. Taylor TH, Gitlin S a., Patrick JL, Crain JL, Wilson JM, Griffin DK. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism
in humans. Hum Reprod Update. 2014;20(4):571-581.
Mosaicism in Embryos
Mechanisms by which chromosomal mosaicism can arise in embryonic
development include errors during:2
– Normal cell division (mitotic) in the first few cell divisions
 Mitotic errors later in development can also lead to mosaicism
 Can be confined to certain fetal/placental tissues or organism systems
– Production of sperm or egg cells (meiotic error) and the error is corrected (rescued)

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Mosaicism in human embryos
Is it real?
Can all technology reliably detect and measure it?
What is the incidence?
What is the clinical impact?
Are there any professional guidelines? PGDIS,
COGEN (TBC)

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Best Practice Reporting Working Group
PGDIS Bologna, Italy May 2016
Genoma, Rome, Italy
Francesca Spinella, Francesco Fiorentino
IVI, Valencia, Spain
Carmen Rubio
Repromeda, Brno, Czech Republic
Jakub Horák, Kateřina Veselá
Safe Fertility Clinic, Bangkok, Thailand
Sunanta Nabu, Wiwat Quangkananurug
Repromed, Dulwich, South Australia
Deirdre Zander-Fox
Genesis Genetics, London, UK
Mike Large, Leoni Xanthopoulou, Tony Gordon
Bridge Centre, London, UK
Alan Handyside, Christian Ottolini, Michael Summers

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Euploid
45%
Multiple aneuploidies
± other abns
14%
Karyotype wide abns
1%
Mosaic (11%) or segmental abns (7%) only
18%
Single aneuploidy
± other abns
21%
All data from labs D-G n=1952
Trophectoderm biopsy data from all labs reporting
mosaic aneuploidy

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COGEN Data on Mosaicism Incidence?
15k sample study presented at COGEN showed 18% mosaicism
Genesis, Repro also have large scale data sets
Author to give talk at ILMN
31
Courtesy of Genesis Genetics, A Cooper Surgical company

32. 3232
Courtesy of Genesis Genetics, A Cooper Surgical company

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The Future

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ClinicalTrials.gov NCT02268786
Study Details Locations
Patient Details
Protocol
Multicenter
International
Blinded
RCT
9 Laboratories
34 Clinical Sites
Aus, Can,
UK, USA
Women aged 25–40 years
Moderate prognosis
≥2 blastocysts at day 5/6
Randomization (day 5/6)
Vitrification
Single Embryo Transfer
Intervention Arm
Trophectoderm biopsy
VeriSeq PGS system
Comparator Arm
Standard morphological
assessment
For Research Use Only. Not for use in diagnostic procedures.

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Other Clinical Trials Ongoing:
IVI : Advanced maternal age – day 3 biopsy – 24Sure
(submitted)
IVI: Male Factor Infertility – day 3 – 24Sure (interim analysis)
Richard Scott – NGS
Richard Scott – Poor prognosis
China – BEACON trial – NGS
IVF Sweden – day 5 – 24Sure
ESTEEM – polar body – 24Sure

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“ Whilst we recognize that as yet much of the published data do not meet
the very highest level of medical analysis, there is now a significant, and
increasing body of scientific literature supporting the recommendation of
PGS to patients undergoing IVF, for the reasons stated above. Based on
available data PGS should no longer be considered as an
experimental procedure. We therefore believe that PGS should be
part of the discussion with all patients considering/undergoing IVF
treatment ”
http://www.ivf-worldwide.com/cogen/general/cogen-statement.html
COGEN PGS Statement (2015)
http://www.ivf-worldwide.com/cogen/general/cogen-statement.html
ASRM, CFAS, Czech Republic………..BFS/HFEA?

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Take Home Messages …..
Aneuploidy is a problem in all IVF patient populations
aCGH/NGS screening of 24 chromosomes is highly
accurate and reproducible (but clear advantages with NGS)
All published RCTs to date using 24 chromosome screening
are positive (with more studies ongoing)
STAR study (and other) results will add to this body of data
Need to further understand mosaicism
Poor IVF is not rescued by PGS

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Thank You!