Will Metabolomic Profiling Replace Accurate Morphological Analysis for Selecting Human Embryos? Patrick Quinn PhD, HCLD Sage In-vitro Fertilization, Inc Redmond, Oregon, USA DISCLOSURE I am Vice President of research and Development at Sage In-Vitro Fertilization, Inc. We produce a range of commercial ART media products Sage IVF Pasadena Redmond Cooper Surgical, Trumbull Sage IVF Southern California Central Oregon OUTLINE Metabolomics Morphology Scoring of embryos Embryo grading systems Added value? 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 Ways to Increase Pregnancy Rates By better embryo selection for ET Ways to Increase Pregnancy Rates By better embryo selection for ET Ways to Increase Pregnancy Rates By better embryo selection for ET 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; What are Metabolomics/Metabolome? Molecular Biometrics website 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. Information from the Embryo Economics? 1.Actual cost to the patient 2.How much more useful information is gained? Molecular Biometrics website Metabolomic Profiling Raman vibrational biospectroscopy rapid, non-invasive analysis of spent culture medium Metabolomic Profiling 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 Metabolomic versus Morphology Accuracy 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. Ways to Increase Pregnancy Rates By better embryo selection for ET 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 Graduated Embryo Score FISCH JD et al. (2003) Fertil Steril 80: Ways to Increase Pregnancy Rates By better embryo selection for ET 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 Importance of Early Cleavage and Mononucleation 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 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 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 Setting up Dishes Only culture 6 embryos in a dish and handle one dish at a time. On D3 between 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 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, h post insemination (pi): 2PN check 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 Scoring of Embryos Score for fragmentation. Score = 30, if 0% fragments = 25, if 20% fragments D1, 25 h pi or if not cleaved, D h pi: fragmentation check Scoring of Embryos If any blastomere has 1 nucleus, ALL previous points are deducted. D h pi: Multinucleation check Scoring of Embryos If 7/8/9-cell, 0% fragmentation 8-cell,