1. Will Metabolomic Profiling Replace Accurate Morphological Analysis for Selecting Human Embryos? Patrick Quinn PhD, HCLD patrick.quinn@coopersurgical.com Sage In-vitro Fertilization, Inc Redmond, Oregon, USA

2. DISCLOSURE I am Vice President of research and Development at Sage In-Vitro Fertilization, Inc. We produce a range of commercial ART media products

3. Sage IVF Production @ Pasadena Redmond Cooper Surgical, Trumbull

4. Sage IVF Southern California Central Oregon

5. OUTLINE Metabolomics Morphology Scoring of embryos Embryo grading systems Added value?

6. Ways to Increase Pregnancy Rates By better embryo selection for ET But we also want to reduce multiple pregnancies by decreasing the number of embryos transferred

7. Ways to Increase Pregnancy Rates By better embryo selection for ET

8. Ways to Increase Pregnancy Rates By better embryo selection for ET

9. Ways to Increase Pregnancy Rates By better embryo selection for ET

10. What are Metabolomics/Metabolome? “The complete set of the human metabolome consists of (approximately) 2,500 small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample.” Molecular Biometrics website; www.molecularbiometrics.com

11. What are Metabolomics/Metabolome? Molecular Biometrics website

12. What are Metabolomics/Metabolome? Molecular Biometrics website The absorption of NIR radiation by organic molecules is due to overtone and combination bands primarily of O-H, C-H, N-H and C=O groups whose fundamental molecular stretching and bending absorb in the mid-IR region.

13. Information from the Embryo Economics? 1.Actual cost to the patient 2.How much more useful information is gained? Molecular Biometrics website

14. Metabolomic Profiling

15. Raman vibrational biospectroscopy rapid, non-invasive analysis of spent culture medium

16. Metabolomic Profiling

17. Diagnostic accuracy for predicting delivery or failure of implantation was 83%. However, how much more added value is there compared to morphometric analysis? Day 3 ET Day 5 ET

18. Metabolomic versus Morphology Accuracy

19. Metabolomics &/or Morphology? Selection of embryo potential morphological parameters v. metabolomicic profile The use of embryo markers secreted into culture media, eg sHLA-G, and metabolic activity, the so- called metabolomic profile of an embryo, will have to be compared to current assessment of embryo potential using morphological and developmental parameters to determine whether the newer but more expensive techniques provide significantly added value.

20. Ways to Increase Pregnancy Rates By better embryo selection for ET

21. Ways to Increase Pregnancy Rates By better embryo selection for ET Ongoing preg rate 1.> from 48 to 62% with 1 embryo with GES ≥70. 2.ET of > 1 embryo with GES ≥70 did NOT > PR, but did > IR 3.Same PR D3 v. D5 but IR > @ D5 as < embryos ETed. 4.Need to factor in MNBs

22. Graduated Embryo Score FISCH JD et al. (2003) Fertil Steril 80:1352-1358

23. Ways to Increase Pregnancy Rates By better embryo selection for ET www.embryologist.comwww.embryologist.com 2006 Uses a modified Graduated Embryo Score 16-18h post insemination: 20 pts if pronuclei aligned 25 h if early cleavage: 30 pts if 2 symmetrical blastomeres 25 pts if asymmetrical 25h – Fragmentation : 30 pts if none 25 pts if slight 0 pts if high 40-43h – Multinucleation: lose all points if present

24. Importance of Early Cleavage and Mononucleation

26. Graduated Embryo Score With at least 2 embryos per ET ANGLE MJ (2006) using two concurrent sequential media systems improves pregnancy outcomes. The Clinical Embryologist 9: Issue 1:5-11, available at www.embryologists.com

27. Setting up Dishes Place 6 x 10 uL drops of QA Protein Plus Cleavage Medium (Sage Ref # ART- 1526) in the dish and 2 x 30 uL drops for washing in the dish - see diagram below. = 10 uL drops used for culture of oocytes after ICSI = 30 uL drops used for washing oocytes after ICSI Immediately cover the drops with 9 mL of oil and place the dish in the CO2 incubator. Do no more than two dishes at a time. For ICSI Day -1, ie day before OPU

28. Setting up Dishes Time for equilibration of dishes to obtain full saturation of medium with CO 2 and optimal pH: Overnight or 4 h minimum Need a minimum of 4 h to equilibrate

29. Setting up Dishes Only culture 6 embryos in a dish and handle one dish at a time. On D3 between 10.00 am and 2.00 pm, place embryos for extended culture into individual drops of medium after washing through the 30 uL drops. D3 embryos from P=Cleavage medium

30. Scoring of Embryos Score for alignment of pronuclei and nucleoli (also know as nucleolar precursor bodies – npb). A good zygote should also have a clear halo of cytoplasm just under the cytoplasmic membrane. Score = 20, if nucleoli aligned = 0, if NOT aligned D1, 16-18 h post insemination (pi): 2PN check

31. Scoring of Embryos Score for early cleavage to 2-cell and symmetry of blastomeres. Score = 30, if 2-cell and symmetrical = 25, if 2-cell and slight asymmetry = 0, if no cleavage D1, 25 h pi: early cleavage check

32. Scoring of Embryos Score for fragmentation. Score = 30, if 0% fragments = 25, if <20% fragments = 0, if >20% fragments D1, 25 h pi or if not cleaved, D2 40 - 44 h pi: fragmentation check

33. Scoring of Embryos If any blastomere has ≥ 1 nucleus, ALL previous points are deducted. D2 40 - 44 h pi: Multinucleation check

34. Scoring of Embryos If 7/8/9-cell, 0% fragmentation 8-cell, <20% fragmentation If 7/9/≥10-cell, <20% fragmentation – score = 10 D3 64 - 67 h pi: D3 cell stage and fragmentation check No compaction, <20% fragmentation. Score = 15 Some compaction, <20% fragmentation. Score = 20 Fully compacted, <20% fragmentation. Score = 20 All of these D3 embryos show >20% fragmentation and get a score of 0 score= 20, if full to some compaction = 15, if no compaction

35. Scoring of Embryos D5/6 blastocyst scoring 1=eB(blastocoele <50% cavity) 2 = EB (>50% of cavity) 3=FEB(100% cavity) & 4=>100% of cavity 5=hatching 6= fully hatched

36. Scoring of Embryos D5/6 blastocyst scoring The inner cell mass (ICM) and trophectoderm cell layers are scored as: A=tightly packed, many cells B=loosely grouped, several cells C=few cells

37. Graduated Embryo Score DayEventAngle’s System 116-18 h pi 20=nucleoli aligned 0=no alignment 16-18 h pi 20=nucleoli aligned 0=no alignment 125 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 25 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 1 or 2 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 240-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 40-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 364-67 h pi 20=7/8/9-cell, 0% frags, some/full compaction 20=8-cell, <20% frags, some/full compaction 15= for both of the above if NO compaction 10=7/9/≥10-cell, <20% frags 64-67 h pi 20=7/8/9-cell, 0% frags 20=8-cell, <20% frags 10=7/9/≥10-cell, <20% frags NO COMPACTION DATA TOTAL POINTS = 100 MAXIMUM

38. Graduated Embryo Score DayEventAngle’s System 116-18 h pi 20=nucleoli aligned 0=no alignment 16-18 h pi 20=nucleoli aligned 0=no alignment 125 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 25 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 1 or 2 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 240-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 40-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 364-67 h pi 20=7/8/9-cell, 0% frags, some/full compaction 20=8-cell, <20% frags, some/full compaction 15= for both of the above if NO compaction 10=7/9/≥10-cell, <20% frags 64-67 h pi 20=7/8/9-cell, 0% frags 20=8-cell, <20% frags 10=7/9/≥10-cell, <20% frags NO COMPACTION DATA TOTAL POINTS = 100 MAXIMUM

39. Graduated Embryo Score With at least 2 embryos per ET ANGLE MJ (2006) using two concurrent sequential media systems improves pregnancy outcomes. The Clinical Embryologist 9: Issue 1:5-11, available at www.embryologists.com

40. Reduction in number of embryos transferred 1.Need enough good quality embryos to select from, eg ≥3 good quality 8-cells on D3. 2.Probably need to extend culture to be able to select more good quality embryos for ET, eg D3 instead of D2, D5/6 Blastocyst ET instead of D3. 3.Use a good embryo scoring system based on: * rate of development * amount of fragmentation * absence of multinucleation

41.  Benefits of Metabolomic Profiling 1.We await a definitive trial of MBP in which patients are prospectively randomized to have their embryos chosen for ET by: a. Morphology only – graduated embryo score b. MBP c. Morphology + MBP 2.Ideally, this would be a Single Embryo Transfer (SET) study.

42.  Benefits of Metabolomic Profiling 3.Would need an assessment of the statistical significance of the difference between the two diagnostic tests. 4.Valid statistical comparisons of the two methods as well as statistical analyses of consistency across sites and subgroups will be necessary.

43.  Benefits of Metabolomic Profiling Where would it be best used? * Does it predict pregnancy or non-pregnancy better? * Only in patients with a number of ‘good’ embryos. * Thawed cryopreserved embryos. Again, studies would be needed to assess the added value of MBP v. morphology scoring in these circumstances.

44.  THANK YOU!