zsolt peter nagy reproductive biology associates atlanta ... · infant complications from multiple...
TRANSCRIPT
-
Declared being a member of company advisory board (ORIGIO UNISENSE)
Zsolt Peter Nagy Reproductive Biology Associates Atlanta, GA, USA
-
A review of the promises
and pitfalls of oocyte and
embryo metabolomics
Zsolt Peter NAGY, M.D., PhD, EMB
Scientific and Laboratory Director – Reproductive Biology Associates,
Atlanta, USA
Associate Professor at Eastern Virginia Medical School MS PG
-
Disclosure Scientific Advisory Board
•Molecular Biometrics
•Origio (Medicult/Humagen/MidAtlantic)
•Unisense
•EMD Serono and Merck
Associate Editor, Human Reproduction and
Reproductive Biomedicine Online
Founder: My Egg Bank LLC.
-
The goal of assessing embryos
is to identify those that are
viable and can contribute to a
healthy (singleton) pregnancy
after fresh (or frozen) transfer
-
Assessing embryo quality is
probably the most critical and
most challenging task that is
performed in the human in-
vitro fertilization laboratory
-
Transfer
Which Embryo to choose?
Best morphology
Non Viable by “Omics”
Transfer
Transfer?
Best by time lapse
-
Outline
• Background and history
• Current methods
• PGD/S and genomics in ART
• Emerging techniques and ‘-omics’
• Advantages, disadvantages and
controversies
-
Contribution of ART to all deliveries
The Economist, July 2008
-
Contribution of ART to all deliveries
Proportion of all singletons 3.2%
Proportion of all twins 38.1%
Proportion of all triplets 79.6%
Ombelet, et al, 2005
-
Infant Complications
from Multiple Pregnancy
Singleton Twin Triplet
Ave. Week @ Birth 39 wks 36 wks 32 wks
% Very Premature 1.7% 14% 41%
Ave. Birth Weight 3357 gms 2390 gms 1735 gms
% Severe Handicap 1.9% 3.4% 5.7%
% Infant Mortality
1.1% 6.6% 19.7%
Expense $15K $30K $152K
Oleszczuk et al. 2003; Callahan et al. New Engl J Med 1994;331:244
-
Historic and Current Approaches
of Embryo Assessment
-
Historic and Current Approaches
of Embryo Assessment
Day 1
Day 2
Day 3
Day 4
Day 5
Oocyte quality: zona, cytoplasm, PB’s
2PN Assessments - Z scoring
Early cleavage/ Multinucleation
Genetic screen ( PGD); metabolic evaluation,
morphologic evaluation; extended culture decision
?
Selection blastocyst for transfer,
embryo cyopreservation, PGD
-
Traditional Embryo Assessment
(day-2 / day-3)
A B C D
Even Blastomeres
Uneven Blastomeres
No Fragmentation
Increased Fragmentation
-
Sibling Embryos
•Uneven Cells
•Fragments
•Multinucleation
•Vacuoles
•Even Cells
•No Fragments
Sibling Embryos
•Poorly Expanded
•Poor Inner Cell Mass
•Thick Zona
•Under Developed Trophectoderm
Traditional Embryo Assessment (day-3 to day-5)
•Fully Hatched
•Distinct Inner Cell Mass
•Well Developed
Trophectoderm
-
Poor Prognosis Good Prognosis
• 1978: -- 0.4 %
• 1997: 5 % 25 %
• 2007:
cleavage 15 % 45 %
blastocyst 35 % 65 %
Implantation Rates in IVF
Overcoming some inefficiencies….
-
SART Summary 1996–>2005 Women < 35y/o using Autologous Oocytes
Year 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 % Change **
# IC x 1000 * 23 25 28 30 34 36 38 40 37 37 62% +
LBR / IC (%) 29 31 32 32 33 35 37 37 37 37 29% +MPR (%) 43 44 43 41 40 42 40 39 38 37 13% -
Twin (%) nr 31 31 33 32 33 33 33 33 33 7% +>Twin (%) nr 14 13 9 9 8 7 6 5 4 69% -
Ave # / ET 3.9 3.7 3.4 3 2.9 2.8 2.7 2.6 2.5 2.4 38% -
Stillman JAMA 2007
-
ESHRE Consensus Conference Risks and Complications in ART
“the essential aim of IVF is the
birth of a single healthy child,
with a twin pregnancy regarded
as a complication”
Land & Evers. Hum Reprod 2003
One solution: Transfer of 1 Embryo
-
Need for Optimal Embryo Selection
• Reduce number of embryos
transferred
• Maintain high pregnancy rates per
cycle
Contemporary goals of IVF
-
- Invasive
- D0/D1 - PB
- D2/D3 - blastomere
- Blastocyst - trophectoderm
- FISH / SKY / CGH / aCGH
- Outcomes?
PGS: A Failed Approach?
-
Positive effect
Gianaroli et al. 1999 Munne et al 1999
Gianaroli et al 2001a Gianaroli et al. 2001b
Munne et al. 2003 Gianaroli et al. 2004 Munne et al. 2005
Munne et al 2006 Verlinsky et al. 2005
Colls et al. 2007 Garrisi et al. 2009 Rubio et al. 2009
No effect Werlin et al. 2003
Platteau et al. 2005 Mersereau et al. 2008 Schoolcraft et al. 2009
Stevens et al.,2004 Staessen et al. 2004 Jansen et al. 2008 Debrock et al 2009
Blockeel et al 2008 Meyer et al 2009
Negative effect Mastenbroek et al. 2007
Hardarson et al. 2008
Despite large studies indicating the advantages of aneuploidy screening, the
notion that PGS for infertility is beneficial is not shared uniformly.
Randomized
controlled trials
Contradicting PGD results using day 3 biopsy and FISH
-
Blastocyst stage biopsy with aCGH:
The Answer?
1. Biopsy less embryos, but the most robust ones.
2. Obtain more than one cell – a clear advantage in any testing
3. The issues of mosaicism are markedly reduced.
4. Concordance between chromosomes in TE and ICM
5. There is no rush to ship and diagnose
6. Transfer is to a natural endometrium
7. You transfer one embryo at a time, per natural cycle.
8. Multiples are markedly reduced
9. Hyperstim – becomes much less of a problem
10. The molecular data are cleaner, clearer
- May not be suitable to all patients
- RCTs needed to demonstrate benefit
-
Alternative Embryo Assessment Approaches
Possible Targets to Use for Testing
Morphology - Birefringence (SpindleView)
- EmbryoScope/Monitoring System
Metabolic Activity - Pyruvate/Glucose uptake
- Amino acids *
- Oxygen consumption (Respirometry)
Constituents - Genome
- Transcriptome
- Proteome *
- Metabolome *
Secreted Factors - PAF
- HLAg
- “Secretome” *
-
Polscope/Oosight
Spindle morphology
ZP Birefringence
-
Polscope / SpindleView
Montag et al., 2007 RBMonlin
This study concludes that oocyte zona birefringence is a good selection criterion and a good predictive
criterion for embryo implantation potential.
-
Time Lapse Embryo Development
-
17 Hrs 27 Hrs 44 Hrs 70 Hrs
Time-Lapse: 1500+ Images over 3 days per embryo
Normal Incubator and Microscope: 4 Observations
Time-Lapse Advantage
More
Observations
Better Selection
Less Disturbance
Better Delopment
-
What is it all about ?
-
Embryo development in Embryoscope
-
N=279
Standard
incubator
Embryo evaluation:
4 discrete time points.
Time-lapse information NOT
used to improve selection
Embryo evaluation:
4 discrete time points.
N=77
Time-lapse
incubator
(EmbryoScope)
hCG positive
64%
hCG positive
59%
Standard incubator (N=279)
49% 51%
Time-lapse incubator (N=77)
45% 55%
Pregnancy Outcomes
Inclusion criteria:
- Cycles with transfer
- ICSI
- >3 aspirated oocytes
No significant difference
Clinical pregnancy
Clinical pregnancy
ASRM abstract 2010
-
Target
Molecule
Method of
analysis
Embryonic
stage tested
Clinical
practicality Outcome References
Pyruvate
Ultramicrofluo
rescence
Day 0-5
High
technicality,
less practical.
Contrasting
results
(Hardy, Hooper et al. 1989;
Gott, Hardy et al. 1990;
Conaghan, Handyside et al.
1993; Gardner, Lane et al.
2001; Jones, Trounson et
al. 2001)
Glucose
Ultramicrofluo
rescence
Oocytes, Day 0-5
embryos
High
technicality,
less practical.
Contrasting
results
(Hardy, Hooper et al. 1989;
Gott, Hardy et al. 1990;
Gardner, Lane et al. 2001;
Jones, Trounson et al.
2001)
Single or specific molecule targeting
-
Single or specific molecule targeting
Glucose consumption of single post-compaction human embryos is predictive of embryo sex and live-birth outcome Gardner et al., HR, 2011
Day-5
Day-4
-
Single or specific molecule targeting
Target
Molecule
Method of
analysis
Embryonic
stage tested
Clinical
practicality Outcome References
Oxygen
Microspectrop
hotometry
Respirometry
Oocytes,
blastocysts
Oocytes
High
technicality,
impractical. Expensive
equipment.
Acquired oxygen
consumption rates
Respiration rates
correlated to
maturation and
viability of oocytes.
(Magnusson,
Hillensjo et al. 1986)
(Scott, Berntsen et al.
2008)
HLA-G
Enzyme-
linked
immunoabsorb
ent assay
Follicular
fluid,
Day 0-5
High
technicality,
impractical
Contrasting
findings.
(Fuzzi, Rizzo et al.
2002; Warner,
Lampton et al. 2008;
Tabiasco, Perrier
d'Hauterive et al.
2009)
Leptin
Enzyme-
linked
immunoabsorb
ent assay
Day 5
embryos
High
technicality,
impractical
Positive correlation
between leptin
secretion and
blastocyst
development
(Gonzalez,
Caballero-Campo et
al. 2000)
-
Groups of molecules targeted
Target
Molecule
Method of
analysis
Embryonic
stage
tested
Clinical
practicality Outcome
Selected
references
Protein
comple-
ment
Surface-
enhanced laser
desorption
ionization time-
of-flight mass
spectrometry
Protein
microarray
Day 5
embryos
Day 5
embryos
High
technicality,
impractical.
Expensive
equipment.
High
technicality,
impractical.
Expensive
equipment.
Protein profiles
are related to
blastocyst
morphology.
Implantation
potential
corresponds to
specific protein
secretion levels.
(Katz-Jaffe,
Gardner et al.
2006)
(Dominguez,
Gadea et al.
2008)
-
Groups of molecules targeted
Target
Molecule
Method of
analysis
Embryonic
stage tested
Clinical
practicality Outcome
Selected
references
Metabo-
lomic
comple-
ment
Non-optical
spectroscopy
(Proton nuclear
magnetic
resonance)
Vibrational
spectroscopy
(Near infrared;
Raman)
Day 3
embryos
Oocytes,
Day 3-5
embryos
High
technicality,
impractical.
Expensive
equipment.
Simple, rapid
procedure,
inexpensive,
high
practicality
for clinical
setting.
Metabolomic
profile correlates
with reproductive
potential of
embryos.
Oocyte viability
score correlates to
developmental
potential. Embryo
viability score
predicts
pregnancy
independent of
morphology.
(Seli, et al.
2008)
(Nagy et al.
2009)
(Agrawal et
al. 2006;
Seli, et al.;
2006; Seli et
al. 2007;
Scott et al,
2008;
Vergouw et
al. 2008)
-
Genome genes
Transcriptome mRNA
Proteome proteins
Metabolome metabolites
~25,000
~100,000
~1,000,000
~2,500
Flowchart of “Omics” - Metabolomics
Function/Phenotype
-
Techniques: Anion Exchange Chromatography / time-of-flight mass spectrometry
Results: Several up-regulated and down-regulated proteins were detected in embryos
Conclusions: Protein expression profiles relate to morphology
Open bars, degenerating embryos; solid bars, developing blastocysts.
Katz-Jaffe. Human embryo proteome. Fertil Steril 2006
m/z
(kDa)
P Potential ID candidates
9.7 0.0009
Heparin-binding EGF-like
growth factor precursor (HB-
EGF) (MW = 9,729.32)
14.2 0.008 Cystatin-9-like precursor
(MW = 14,239.95)
9.9 0.008 CART (MW = 9,941.53)
NADH-ubiquinone oxireductase (MW =
9,965.71)
9.1 0.004 Beta-catenin-interacting
protein 1 (MW = 9,170.35)
Cytochrome c oxidase
subunit VIIIa 3, (MW =
9,174.71)
19.8 0.01 Caspase-1 precursor (MW =
19,843.81)
5 0.01
Inhibitor of growth protein 1
ING1-like tumor suppressor
protein, X-linked (MW =
5,076.86 Da)
Proteomics
-
Technique: High performance liquid chromatography.
Results: The turnover of three amino acids, Asn, Gly and Leu, was significantly correlated with a clinical
pregnancy and live birth. (asparagine; glycine; leucine)
Conclusions: Non-invasive assay of amino acid turnover has the potential to improve significantly the
prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.
Amino Acid Uptake
Brison, D.R. et al. Hum. Reprod. 2004 19:2319-2324;
Box plots of the 18 individual amino acids showing the medians (thicker lines), inter-quartile ranges (boxes), ranges (whiskers; excluding outliers) and outlying observations
(open circles) for amino acid appearance in the culture medium
-
Metabolomics Biomarker Spectral Signatures (by NIR)
Rel
ati
ve
Ab
sorb
an
ce
600 700 800 900 1000 0
0.02
0.04
0.06
0.08 OH
R-OH
Heme
NH
CH
What is measured?
Wavelength (nm)
-
Algorithm Development and Blind Assessment
Step 1: Develop Predictive Algorithms on Day 2, 3 or 5 Embryo Culture Media with known Fetal Cardiac Activity outcome
Step 2: Develop Predictive Formula Viability Score =
a(Wa) + b(Wb) + g(Wg) + d(Wd)
Wavelength (nm)
Step 3: Blind Validation on unknown outcomes
-
Blind validation using the Day 2 prediction algorithm
on 176 day 2 cleavage stage embryos that underwent
SET.
N = 44 in each column
% F
CA
Po
siti
ve
y = 5.9x + 13.5 R² = 0.8338
0
5
10
15
20
25
30
35
40
1 2 3 4
P = 0.0086 when comparing the mean viability scores of FCA positive and negative
Increasing viability scores in each quartile
FCA +ve rates in relation to
Quartiles of increasing
Viability Scores
Data on file, courtesy of D. Sakkas Molecular Biometrics Inc.
-
Blind validation using a Day 3 prediction
algorithm on 448 day 3 cleavage stage embryos
that underwent SET.
N = 112 in each column
% F
CA
Po
siti
ve
Increasing viability scores in each quartile
FCA +ve rates in relation to
Quartiles of increasing
Viability Scores
y = 4.8236x + 3.2801 R² = 0.9915
0
5
10
15
20
25
1 2 3 4
Data on file, courtesy of D. Sakkas Molecular Biometrics Inc.
-
MOLECULAR BIOMETRICS DAY 5 ALGORITHM
Blindly Validated on Day 5 Single Embryo Transfers
(N = 133) from Clinics A, B and C
% F
CA
Po
siti
ve
y = 10.3x - 4 R² = 0.8949
0
5
10
15
20
25
30
35
40
1 2 3 4
% F
CA
Po
siti
ve y = 20.7x + 6 R² = 0.8175
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4
% F
CA
Po
siti
ve
1 to 4 are Increasing viability scores
A B C
FCA +ve rates are plotted in relation to Quartiles of increasing Viability Scores for each Clinic
y = 12.5x - 2.5 R² = 0.9542
0
5
10
15
20
25
30
35
40
45
50
1 2 3 4
-
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
A B C D
ViaTest-E™ Score 0.3
Embryos of same Morphology
% I
mp
lan
tati
on
Rate
aft
er
SE
T
SET Implantation Rates of Day 3 Embryos Comparing the Same
Morphology Grade and Metabolomic Score of < or > than 0.3
(98) (54) (16) (11) (74) (51) (11) (13)
(number of SET in parentheses)
Morphology Alone
-
Hardarson ESHRE
2011
Ongoing pregnancy rate:
Group Day 2 Day 5
NIR 31.0% 39.0%
Control 26.5% 45.0%
Prospective RCT Results (n=327, ITT Analysis)
-
Conclusions
- Morphological evaluation of embryo is still
the principal approach.
- Novel techniques may provide additional
information that can help to select viable
embryos.
- Novel approach must be simple, easy to
implement and provide useful information.
- Future RCT need to validate the efficiency
of any novel technique.