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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
Redmond Production @ Pasadena
Sage IVF
Cooper Surgical, Trumbull
Sage IVF
Southern California Central Oregon
OUTLINE
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Metabolomics
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Morphology
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Scoring of embryos
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Embryo grading systems
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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; www.molecularbiometrics.com
What are Metabolomics/Metabolome?
Molecular Biometrics website
What are Metabolomics/Metabolome?
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.
Molecular Biometrics website
Information from the Embryo
Economics?
1.
2.
Actual cost to the patient How much more useful information is gained?
Metabolomic Profiling
Metabolomic Profiling
Raman vibrational biospectroscopy rapid, non-invasive analysis of spent culture medium
Metabolomic Profiling
Metabolomic Profiling
Day 3 ET Day 5 ET Diagnostic accuracy for predicting delivery or failure of implantation was 83%.
However, how much more added value is there compared to morphometric analysis?
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.
2.
> from 48 to 62% with 1 embryo with GES ≥70.
ET of > 1 embryo with GES ≥70 did 3.
4.
NOT > PR, but did > IR Same PR D3 v. D5 but IR > @ D5 as < embryos ETed.
Need to factor in MNBs
Graduated Embryo Score
FISCH JD et al. (2003) Fertil Steril 80:1352-1358
Ways to Increase Pregnancy Rates
By better embryo selection for ET www.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
Importance of Early Cleavage and Mononucleation
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 www.embryologists.com
Setting up Dishes
For ICSI Day -1, ie day before OPU
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.
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
D3 embryos from P=Cleavage medium Setting up Dishes 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.
Only culture 6 embryos in a dish and handle one dish at a time.
Scoring of Embryos D1, 16-18 h post insemination (pi): 2PN check 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
Scoring of Embryos D1, 25 h pi: early cleavage check 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
Scoring of Embryos D1, 25 h pi or if not cleaved, D2 40 - 44 h pi: fragmentation check Score for fragmentation.
Score = 30, if 0% fragments = 25, if <20% fragments = 0, if >20% fragments
Scoring of Embryos D2 40 - 44 h pi: Multinucleation check If any blastomere has ≥ 1 nucleus, ALL previous points are deducted.
Scoring of Embryos D3 64 - 67 h pi: D3 cell stage and fragmentation check No compaction, <20% fragmentation.
Score = 15 Some compaction, <20% fragmentation.
Score = 20 All of these D3 embryos show >20% fragmentation and get a score of 0 Fully compacted, <20% fragmentation.
Score = 20 If 7/8/9-cell, 0% fragmentation 8-cell, <20% fragmentation score= 20, if full to some compaction = 15, if no compaction If 7/9/≥10-cell, <20% fragmentation – score = 10
Scoring of Embryos D5/6 blastocyst scoring
1=eB(blastocoele <50% cavity) 3=FEB(100% cavity) & 4=>100% of cavity 5=hatching 2 = EB (>50% of cavity) 6= fully hatched
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
Graduated Embryo Score Day 1 1 1 or 2 2 3 Event 16-18 h pi 20=nucleoli aligned 0=no alignment 25 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 40-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 64-67 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 Angle’s System 16-18 h pi 20=nucleoli aligned 0=no alignment 25 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 40-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 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
Graduated Embryo Score Day 1 1 1 or 2 2 3 Event 16-18 h pi 20=nucleoli aligned 0=no alignment 25 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 40-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 64-67 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 TOTAL POINTS = 100 MAXIMUM Angle’s System 16-18 h pi 20=nucleoli aligned 0=no alignment 25 h pi 30=Symmetrical 2-cell 25=slight asymmetry, 2-cell 0=no cleavage 25 h or 40-44 h pi 30=0% frags 25=<20% frags 0=>20% frags 40-44 h pi Multinucleation; DEDUCT ALL PREVIOUS POINTS 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
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
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
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.
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.
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.