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.