KED - Society of Clinical Embryologists 1 st Symposium, Istanbul Assessment of Gamete/Embryo Viability by the Utilization of new PGS (Preimplantation Genetic or

PGS: an Embryologist’s Perspective

December 5 th 2012

I am doing PGS anyway Can you give me any tips?

Evidence: Clinical Value of ACGH?

100 90 80 30 20 10 0 70 60 50 40 30-34 35-38 39-42 43-50 Implantation rate Aneuploidy rate No transfer Wells and Fragouli, unpublished

Bridge Success Rates

LIVE BIRTH RATE post PGD ANEUPLOIDY screening Using ARRAY CGH in 40 – 45 yr Maternal Age Group Year 2010 2011 PGD-A array CGH

22% 22% *Equivalent UK national rates for this age group are 10-13%

New Technologies – The Rise of array CGH

Evidence At Last?

• First randomised trial* of blastocyst PGS with aCGH • Women under 35 years (good prognosis) • eSET • • Day-5 (blastocyst) biopsy

All cycles had embryo transfer (day 6)

• No PGS: embryo transfer on morphology (n=48) Pregnancy rate**: 41.7% • PGS (aCGH): chromosomally normal embryos transferred (n=55) Pregnancy rate**:

69.1%

*Yang et al (2012) **On going pregnancy rate ≥20 weeks/cycle started

Why PGS? Do Nothing Option

• Retain all embryos • No harm to embryo • No upfront cost to patient • Transfer all embryos sequentially Why Not?

• Risk of abnormality NOT accepted • Miscarriage NOT Trivial • Repeated failure NOT Accepted • Multiple cycles (inc. FET) costs Time and Money

Biopsy sample

• 1 st polar body - Single cell (small) • No role in further development • Fragmented • 2 nd polar body - Single cell (small) • Haploid, Incomplete extrusion • No role in further development • Nucleated blastomere – single cell (large) • Representative of embryo? mosaicism • critical role in further development • Trophectoderm – 6-10 cells • Representative of FUTURE BABY?

• Mosaicism

Biopsy Procedure

• Laser or mechanical • Timing • ICSI required • Laser or mechanical • Timing (Sequential or simultaneous) • Chemical, Laser or mechanical • Safety? Reduced implantation potential • Laser or mechanical • Need to see AND AVOID ICM • Laser effects on sample?

• Need for vitrification post-biopsy

Biopsy – Practical Issues

• Entire MII cohort analyzed • High Biopsy & diagnostic workload/costs • Proportion will not fertilize • Entire 2pn cohort analyzed • High Biopsy & diagnostic workload/costs • Proportion will not develop well • Good quality ‘day 3’ embryos only • Chance of no biopsy • Medium Biopsy & diagnostic workload/costs • Good quality blastocysts d5/6 only • Moderate chance of no biopsy • Lower Biopsy & diagnostic workload/costs

Biopsy – Diagnostic Issues

• High result rate (>90%) • No paternal/meiosis II/post-zygotic information • High result rate (>90%) • No paternal/post-zygotic information • High result rate (>90%) • Chromosomal mosaicism common • Extremely high result rate (>>95%) • SIGNIFICANCE OF MOSAICISM?

What is the aim of PGS?

….to

transfer embryos with the correct amount of genetic material

to

increase the chance

of having a healthy baby &

reduce the risk

of having a pregnancy with a chromosomal abnormality ..identify patients with high risk of aneuploid gametes/embryos to inform future treatment

Life is never that simple…..

And Neither I s PGS…..

So What Happens In The Real World?

Case studies: PB1 only

• 49 year old • 2ND attempt IVF/ICSI • FAVOURABLE AMH/AFC • Results: 11 OOCYTES ALL WITH MULTIPLE ANEUPLOIDIES Extensive PRE- AND POST-TEST counselling Suggested egg donation Last chance/diagnostic closure FOLLOW-UP OF EMBRYOS CONFIRMED PB1 RESULTS

New IVF test – Array CGH – produces baby Oliver, offering hope to infertile 13 previous failed IVF cycles 7/9 first polar bodies aneuploid September 2 nd 2009

Summary of Net Gains and Losses in The Aneuploid Zygotes

Source of error Meiosis I Meiosis II Paternal or chromosome loss

ESHRE data 2010

Gains

44 55 18

Losses

33 47 30

Total (%)

77 (34) 102 (45) 48 (21)

Case studies: PB1+2

• 41 year old (AMA) • 2nd attempt (1 st PB1 – LB) • 11 x oocytes/7 x 2pns • Results: 6 x abnormal, 1 x normal* *Reciprocal gain and loss (chr 9 + 13) Reported ‘normal’ (no plots/correction?) Counsel for risk of patau’s syndrome Need for data on outcomes of G/L

Need for truth data

RECIPROCAL GAIN/LOSS – PB1+2

Case studies: PB1+2

• 40 year old (AMA) • 1st attempt (top – trisomy 21) • 6 x oocytes/5 x 2pns • Results: • 1x Normal, 2 x Abnormal, 2 x Normal (but no result for PB2) – D5 rebiopsy • Partial deletion in chr 5 Reported as abnormal Mosaicism in TE/ICM?

Counsel for risk of cri-du-chat Need for data on mosaicism

Case studies: Trophectoderm

• • • • • • • 38 year old RIF (4 x IVF) Frozen d3 (5) + D5 (1) thawed for TE BIOPSY Results: DELAYED DEVELOPMENT (no Fresh ET) 2 x Euploid, 2 x Aneuploid 1 x multiple aneuploid* (every chromosome!) Reliability of aCGH?

*5AB – discard/re-biopsy/et?

Question diagnostic lab Repeat test? Follow-up

Single blastomere analysis: Complex pattern of gains and losses

Algorithms and reporting

Estimation of confidence in aneuploidy call Automated calling 22

Karyomapping and 24sure from a single cell SurePlex amplification

• Cells disaggregated from the remainder of embryo.

Data supplied with courtesy of Professor Alan Handyside, London Bridge Fertility Centre.

FISH probes 13, 16, 18, 21, 22 • Trisomy 17 • Monosomy 18, 21, 22 FISH probes X, Y, 21 • Trisomy X • Monosomy 21 23

(NEW) Indications - 24 chromosome test • Advanced maternal age (>35 yr) • Most embryos aneuploid • No embryo transfer in most cycles • Prognostic for chance of pregnancy with the patient’s own eggs • Optimal age range for embryos selection 37 to 43 years of age • eSBT (<35 yr) good prognosis patients at high risk of twins • Moderate incidence of aneuploidy • All cycles with ≥1 aneuploid blastocyst • All cycles with ≥1 euploid blastocyst for transfer • Severe male factor infertility • Idiopathic repeat IVF failure • Miscarriage risk/previous abnormal/TOP

Summary i – What patients/providers need to know

FACTS

• Aneuploidy INCIDENCE c.

50%

affects all ages • Aneuploidy

main cause

of failed implantation • Aneuploidy testing

CANNOT

change the cumulative pregnancy rate

POTENTIAL BENEFITS

• Prevent transfer or storage of aneuploid/non-viable eggs/embryos • Identify AMA women with good/poor prognosis for a livebirth using their own eggs • Increase pregnancy/live birth rate/ET (

eSBT

) • Reduce time to live birth or resolution

Summary ii – how to provide good PGS

•

Genetic Counsellor

provides Face to face, telephone, email support for clinicians/ embryologists/patients throughout process • Single point of contact for patients • Excellent patient information & consent forms • Secondary reports (combine embryology and diagnostic information) • Time-lapse incubation?

• Treat every egg/embryo as important • Question diagnostic team

Acknowledgements

The Bridge Centre

• Alan Handyside • Michael Summers • Karen Sage • Shaun Rogers

Bluegnome

• Tony Gordon

Reprogenetics uk

• Dagan Wells