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

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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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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)

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Prediction of Ovarian Response Before COS

Bio

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EvidenceLevel1a

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• Avoid over-aggressive stimulation in ‘true’ high responders

• Avoid over-conservative stimulation in ‘false’ high respondersE

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Ovari

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• Avoid over-conservative stimulation in ‘true’ DOR

• Avoid over-aggressive stimulation in ‘false’ DOR

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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

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EvidenceLevel2a

Ris

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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

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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

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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

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In addition, Vitrification is simpler andfaster than Slow Freezing

AbdelFahez et al . Reproductive BioMedicine Online (2010) 20, 209– 222

EvidenceLevel1a

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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

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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

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Gametes and

Embryos

Morphological biomarkers

Metabolic based-

biomarkers

Genetic-based

biomarkers

Proteomic-based

biomarkers

The ART Laboratory

Tomorrow

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Sperm Quality Biomarkers

• Hyaruronic Acid Binding

• Polarization Microscopy

• MSOME

• Electroforetic Sperm Isolation

• Magnetic-activated Cell Sorting

• Microfluids

• Microarray Technology

• ProteomicsSp

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Normalcy of Sperm Chromatin Content

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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

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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%)*

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Esteves, 16

Oocyte Quality BiomarkersO

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• 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

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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)

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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)

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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

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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

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Non-Invasive

Katz-Jaffe & McReynolds, Fertil Steril 2013;99:1073-77

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Embryo Quality Biomarkers

Metabolic Profile (2010):• Via-Metrics™ (Molecular Biometrics, USA)

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Non-Invasive

Advocated as a Highly

Sensitive Method of

Metabolomics Analysis

by NIR Spectroscopy

Market withdrawal due to instrument inability to

perform accurate measurements

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Embryo Quality Biomarkers

Morphokinetics (2010):

Image capture over time

Combination of morphological, dynamic and quantitative

information about developmental events

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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

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Embryo Quality Biomarkers

Stage-top Incubator

Tokai-Hit, JapanInCu-Cell Live™

Sanyo, JapanBioStation™

Nikon, Japan

Several Time-lapse Technologies Available:

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Embryo Quality BiomarkersT

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Primo-Vision™

Cryo-InnovationLtd., Hungary

EmbryoScope version C™ Nanorespirometer + Time-lapse videomicrography

Unisense Fertilitech, Denmark

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Embryo Quality BiomarkersT

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Eeva™ (Videomicrography + Computer Vision Software)

Auxogyn, USA

Cell tracking and prediction software (measure of time embryo takes

to achieve specific milestones):

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Clinical Translation:

Where we are

Bio

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th

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RT

Lab

Time-lapse

Technology

Micro-array

CGH

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Clinical Translation:

Where we are going

Bio

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th

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Lab Sperm and

Oocyte

Selection

Using

Biomarkers

Embryo

Selection by

Real-time

Secretome +

Morphokinetics

AnalysisMicrofluidic Platform for Embryo Culture

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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

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