1. KED - Society of Clinical Embryologists 1 st Symposium, Istanbul Alan Thornhill The Bridge Centre, London, UK December 5 th 2012 Assessment of Gamete/Embryo Viability by the Utilization of new PGS (Preimplantation Genetic Screening) Techniques or PGS: an Embryologist’s Perspective OR I am doing PGS anyway Can you give me any tips?

3. Implantation rate Wells and Fragouli, unpublished Aneuploidy rate No transfer Evidence: Clinical Value of ACGH?

4. Bridge Success Rates LIVE BIRTH RATE post PGD ANEUPLOIDY screening Using ARRAY CGH in 40 – 45 yr Maternal Age Group Year PGD-A array CGH 2010 22% 2011 22% *Equivalent UK national rates for this age group are 10-13%

5. New Technologies – The Rise of array CGH

6. 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 Evidence At Last?

7. 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 Why PGS? Do Nothing Option

8. 1 st polar body - Single cell (small) No role in further development Fragmented Biopsy sample 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

9. Laser or mechanical Timing ICSI required Biopsy Procedure 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

10. Entire MII cohort analyzed High Biopsy & diagnostic workload/costs Proportion will not fertilize Biopsy – Practical Issues 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

11. High result rate (>90%) No paternal/meiosis II/post-zygotic information Biopsy – Diagnostic Issues High result rate (>90%) No paternal/post-zygotic information High result rate (>90%) Chromosomal mosaicism common Extremely high result rate (>>95%) SIGNIFICANCE OF MOSAICISM?

12. 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

13. And Neither Is PGS….. So What Happens In The Real World? Life is never that simple…..

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

15. 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

16. Summary of Net Gains and Losses in The Aneuploid Zygotes Source of errorGainsLossesTotal (%) Meiosis I 443377 (34) Meiosis II 5547102 (45) Paternal or chromosome loss 183048 (21) ESHRE data 2010

17. Case studies: PB1+2 *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 41 year old (AMA) 2nd attempt (1 st PB1 – LB) 11 x oocytes/7 x 2pns Results: 6 x abnormal, 1 x normal*

18. RECIPROCAL GAIN/LOSS – PB1+2

19. Case studies: PB1+2 Reported as abnormal Mosaicism in TE/ICM? Counsel for risk of cri-du-chat Need for data on mosaicism 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

20. Case studies: Trophectoderm Reliability of aCGH? *5AB – discard/re-biopsy/et? Question diagnostic lab Repeat test? Follow-up 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!)

21. Single blastomere analysis: Complex pattern of gains and losses

22. Algorithms and reporting 22 Automated calling Estimation of confidence in aneuploidy call

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

24. 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 (NEW) Indications - 24 chromosome test

25. 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 i – What patients/providers need to know

26. 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 Summary ii – how to provide good PGS

27. The Bridge Centre Alan Handyside Michael Summers Karen Sage Shaun Rogers Bluegnome Tony Gordon Reprogenetics uk Dagan Wells Acknowledgements