future perspectives in the art lab
TRANSCRIPT
Future Perspectives in
the ART Lab
Sandro Esteves, M.D., Ph.D.
Director, ANDROFERT
Center for Male Reproduction & Infertility
Campinas, BRAZIL
Fertility Experts Latinamerica Meeting 1
Mexico City, April 11-12, 2013
Esteves, 2
Maximize beneficial effects of treatment;
Minimize complications and risks
Redifinition of Success:
SET leading to one healthy live birth
Central Paradigm
Individualization of ovarian stimulation
Optimalendometrial receptivity
High-quality gametes and
embryos
Esteves, 3
Defining what is a “valid” biomarker in the context of reproductive medicine
Overview of biomarkers with potential application in the ART lab
Clinical translation: where are we and where are we going?
What is in it for me?
A Valid Biomarker in Reproductive
Medicine Provides Realistic
Prognostic Information
Esteves, 4 Adapted from: ASRM Practice Committee, Fertil Steril 2012;98:147
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Bio
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Outcome Assessed
FalsePositive
(B)
FalseNegative
(C)
TrueNegative
(D)
TruePositive
(A)
Sensitivity (A/A+C)
Specificity (D/B+D)
Predictive Value(A/A+B)
Accuracy(Sens./1-Spec.= Area
under the ROC curve)
Esteves, 5
Prediction of Ovarian Response Before COS
Bio
ma
rkers
EvidenceLevel1a
Esteves, 6
• Avoid over-aggressive stimulation in ‘true’ high responders
• Avoid over-conservative stimulation in ‘false’ high respondersE
xce
ssiv
e
Ovari
an
R
es
po
ns
e
• Avoid over-conservative stimulation in ‘true’ DOR
• Avoid over-aggressive stimulation in ‘false’ DOR
Dim
inis
hed
O
va
ria
n
Rese
rve
(DO
R)
Why Do We Need Biomarkers to
Predict Ovarian Response to
COS?
Low-starting
FSH dose (150 UI)
AMH (ng/mL) >2.1¶
GnRH Agonist GnRH
Antagonist
Days of Stimulation 13 (12-14) 9 (8-11)*
No. Oocytes (n) 14 (10-19) 10 (8.5-13.5)*
OHSS requiring
hospitalization
20 (13.9%) 0 (0%)*
Cancellation 4 (2.7%) 1 (2.9%)
CPR per transfer 40.1% 63.6%*
¶DSL assay; Adapted from Nelson SM et al . Anti-Müllerian hormone-based approach to
controlled ovarian stimulation for assisted conception. Hum Reprod. 2009; 24(4):867-75.
*P ≤ 0.01
Esteves, 7
EvidenceLevel2a
Ris
k o
r E
xc
es
siv
e
Re
sp
on
se
InterventionMeta-analytic
StudiesPopulation
Effect on
Pregnancy
Rec-hLH
supplementation
to rec-hFSH
Mochtar et al, 20071
Bosdou et al, 20122
Hill et al, 20123
Poor
responders1,2
Age ≥35 yrs3
Higher OPR1
Higher LBR2
Higher CPR3
Growth Hormone
Kyrou et al,20091
Kolibianakis et al, 20092
Duffy et al, 20103
Poor
responders
Higher LBR1,2,3
Higher PR2
Higher CPR3
Testosterone Bosdou et al , 2012Poor
responders
Higher LBR
Higher CPR
Kolibianakis et al, Hum Reprod Update 2009,15:613-22; Kyrou et al, 2009;91: 749–66; Duffy et al,
Cochrane Database Syst Rev 2010;1:CD000099; Mochtar MH et al. Cochrane Database Syst Rev.
2007,2:CD005070; Bosdou JK et al, Hum Reprod Update 2012;8:127-45; Hill MJ et al. Fertil Steril
2012;97:1108-4.
Level
1a
Esteves, 8
Dim
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d O
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Re
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Esteves, 9
The ART Laboratory
Today
ART Lab
Morphology-based Gametes and
Embryos Selection
FISH-based
PGD/PGS
Air Quality Control
Vitrification
Cleavage-stage and Blastocyst
Culture
Esteves, 10
Embryo Freezing
● Five RCT: VITRI vs. Slow Freezing
●765 cycles
●Better outcomes with Vitrification:
CPR = 39% x 33%; OR: 1.55; 95% CI: 1.03-2.32
OPR = 35% x 27%; OR: 1.82; 95% CI: 1.04-3.20
IR = 29% x 24%; OR: 1.49, 95% CI: 1.03-2.15
Vit
rifi
ca
tio
n
In addition, Vitrification is simpler andfaster than Slow Freezing
AbdelFahez et al . Reproductive BioMedicine Online (2010) 20, 209– 222
EvidenceLevel1a
Esteves, 11
Embryo Culture
● Eight RCT: Blastocyst vs Cleavage-
stage Transfers; 1,654 patients
●Better outcomes with Blastocyst ET:
LBR = 35% x 28%; OR: 1.39; 95% CI: 1.10-1.76
CPR = 39% x 33%; OR: 1.27; 95% CI: 1.03-1.55
Bla
sto
cy
st
Tra
ns
fer
Improved ability to select embryos, but...
1. Risky for pts. with few embryos
2. Prolonged culture associated with imprinting, epigeneticdisorders, pre-term birth
3. High rate of aneuploidy in blastocysts
Manipalviratn et al, 2009; Kallen et al, 2009; Munné et al., 2012
Papanikolaou E et al. Hum Reprod 2008; 23: 91–99;
EvidenceLevel1a
Esteves, 12
Bio
ma
rke
rs
in t
he A
RT
Lab
Gametes and
Embryos
Morphological biomarkers
Metabolic based-
biomarkers
Genetic-based
biomarkers
Proteomic-based
biomarkers
The ART Laboratory
Tomorrow
Esteves, 13
Sperm Quality Biomarkers
• Hyaruronic Acid Binding
• Polarization Microscopy
• MSOME
• Electroforetic Sperm Isolation
• Magnetic-activated Cell Sorting
• Microfluids
• Microarray Technology
• ProteomicsSp
erm
Se
lec
tio
n
Tech
niq
ues
Normalcy of Sperm Chromatin Content
Esteves, 14
DNA Integrity is the Key Sperm
Biomarker
Current non-invasive sperm selection techniques
cannot directly assess sperm DNA fragmentation
Dyes are used to reach the nucleus,
using fixed specimens
In general, labor-intensive techniques
Recent progress (Enciso et al, 2012):
• New synthetic peptide (DWI)
• Derived from p53 protein
• Affinity to various DNA lesions
• Rapid and inexpensive
• Still unable to penetrate intact
sperm membranes
Sp
erm
Se
lec
tio
n
Tech
niq
ues
Alternative has been
TESA-ICSI
Sperm % TUNEL + % CPR %IR
Ejaculated (N=18) 23.6 ± 5.1 6 2
Testicular (N=18) 4.8 ± 3.6 44 21
P value <0.001 <0.05 <0.01
Greco E et al. Hum Reprod 2005;20:226–30
*Absolute differences between two specimens ranging from -3.3% to -56.3%.
Moskovtsev et al. Fertil Steril 2010; 93(4): 1142–6.
DNA damage in Testicular Spermatozoa (13.3%)
is three-fold lower compared with Ejaculated
Spermatozoa (39.7%)*
Esteves, 15
Esteves, 16
Oocyte Quality BiomarkersO
oc
yte
Se
lec
tio
n
Te
ch
niq
ues
• Polarization Microscopy (Polscope)
• Oxygen Consumption (Embryoscope)
• Microarray Technology
Cumulus cells gene expression (mRNA transcripts)
• Molecular Mining of Follicular Fluid
Amino acids, metabolites, peptides, proteins
Mass Spectroscopy, Raman Spectroscopy
Nuclear Magnetic Resonance
Esteves, 17
Embryo Quality Biomarkers
Rationale: >50% in vitro-produced embryos are abnormal
Developmental Stage:
• Cleavage-stage Embryo Biopsy (most used)
• Polar Body (single allelic copy)
• Blastocyst (trophectoderm cells)
Em
bry
o S
ele
cti
on
T
ech
niq
ues
InvasiveBiopsy:
Techniques:
• FISH (single-cell test; technical limitations)
• PCR (DNA amplification-based approach)
• CGH (combination of molecular and cytogenetic)
• Single-nucleotide Polymorphism Micro-array
• Next-generation Sequencing (single gene)
• Quantitative Real-time PCR (qPCR)
Esteves, 18
Em
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ele
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ech
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ues
Biopsy:
Polar body, Day-3 embryo and
Trophectoderm cells
OPR: 59% vs 38% controls (p<0.001)Munné et al, Fertil Steril 2010
PGS sure™ (Blue Genome, UK)
Micro-array CGH solution to
count all chromosomes in <12h
Micro-array lab
hardware
Embryo Quality Biomarkers
Esteves, 19
Embryo Quality Biomarkers
Metabolic Profile:
• Glucose and pyruvate uptake
• Amino acid turnover
• Oxygen consumption
Proteomics
• Mass Spectroscopy
• Raman Spectroscopy
• Nuclear Magnetic Resonance
Morphokinetics
• Time-lapse microscopy
Em
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Non-Invasive
Katz-Jaffe & McReynolds, Fertil Steril 2013;99:1073-77
Esteves, 20
Embryo Quality Biomarkers
Metabolic Profile (2010):• Via-Metrics™ (Molecular Biometrics, USA)
Em
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o S
ele
cti
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ech
niq
ues
Non-Invasive
Advocated as a Highly
Sensitive Method of
Metabolomics Analysis
by NIR Spectroscopy
Market withdrawal due to instrument inability to
perform accurate measurements
Esteves, 21
Embryo Quality Biomarkers
Morphokinetics (2010):
Image capture over time
Combination of morphological, dynamic and quantitative
information about developmental events
Em
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ues
Non-Invasive
Principle:
1st cytokynesis (within 14 6 min)
Time between 1st and 2nd mitosis (11.1 2.2 h)
Time between 2nd and 3rd mitosis (1.0 1.6 h)
Payne et al, 1997; Lemmen et al, 2008; Wong et al, 2010*; Meseguer et al, 2011;
Hashimoto et al, 2012
Esteves, 22
Embryo Quality Biomarkers
Stage-top Incubator
Tokai-Hit, JapanInCu-Cell Live™
Sanyo, JapanBioStation™
Nikon, Japan
Several Time-lapse Technologies Available:
Tim
e-l
ap
se
T
ech
no
log
ies
Esteves, 23
Embryo Quality BiomarkersT
ime-l
ap
se
T
ech
no
log
ies
Primo-Vision™
Cryo-InnovationLtd., Hungary
EmbryoScope version C™ Nanorespirometer + Time-lapse videomicrography
Unisense Fertilitech, Denmark
Esteves, 24
Embryo Quality BiomarkersT
ime-l
ap
se
T
ech
no
log
ies
Eeva™ (Videomicrography + Computer Vision Software)
Auxogyn, USA
Cell tracking and prediction software (measure of time embryo takes
to achieve specific milestones):
Esteves, 25
Clinical Translation:
Where we are
Bio
ma
rke
rs in
th
e A
RT
Lab
Time-lapse
Technology
Micro-array
CGH
Esteves, 26
Clinical Translation:
Where we are going
Bio
ma
rke
rs in
th
e A
RT
Lab Sperm and
Oocyte
Selection
Using
Biomarkers
Embryo
Selection by
Real-time
Secretome +
Morphokinetics
AnalysisMicrofluidic Platform for Embryo Culture
Esteves, 27
The Biomarkers Era has arrived. Several markers under investigation and some already translated
Valid biomarkers are highly sensitive and specific, and have high predictive value
For application at a global level, ART lab’s biomarkers/ technologies should be VEELI:
ValidatedEasy to useEasy to replicateLow costImprove outcomes
Future Perspectives in
the ART LabC
on
clu
sio
ns
Esteves, 28