Embryo selection methods aim to improve IVF success rates but current options have limitations. [1] Embryo morphology is the most common assessment but can vary between labs and observers. [2] Genetic testing provides direct information but techniques like PGS remain controversial due to concerns about invasiveness and mosaicism. [3] Metabolomic analysis using techniques like near-infrared spectroscopy show promise as a non-invasive marker correlated with implantation potential but require more validation. Overall, no single method is ideal and a personalized, multifactorial approach may be needed along with advances in other areas like culture conditions, freezing, and endometrial receptivity.

1. Thomas FREOUR
Centre d’Assistance Médicale à la Procréation
CHU de Nantes, Nantes, France
EMBRYO SELECTION
Lyon, September 24th 2010

2. Framework
1. Introduction
2. Embryo morphology
3. Genomics
4. Embryo metabolism
5. Debate / Comments

3. INTRODUCTION (1)
• Increase use of ART around the world
• Results remain « disappointing » :
– Low implantation rate
– High multiple pregnancy rate
Need for better embryo selection method(s) to
improve implantation rates and subsequently lower
multiple pregnancy rate.

4. INTRODUCTION (2)
The literature available
Large amount of data from
cohort studies, cross
sectional studies …
But… relatively few studies with a
high level of evidence according to
evidence-based medicine criteria
(RCTs, meta analysis …)

5. INTRODUCTION (3)
The method of embryo quality assessment
depends on the end-point measure…
Cleavage rate
Rate of morphologically « top » embryos
Blastulation rate
Implantation rate
Pregnancy rate per transfer
Live birth rate per transfer
Number of oocytes/embryos necessary to
obtain a live birth
…

6. INTRODUCTION (4)
What would be the « ideal »
embryo quality assessment method ?
« Non invasive »
« Do no harm »
Reproducible
Objective
Rapid
Easy to use routinely
Cost-effective
No ethical question raised
Independent of other parameters

7. 1st Part
EMBRYO MORPHOLOGY

8. Embryo morphology :
Can you judge a book by its cover ?
Day 0 : Oocyte quality
• Nuclear maturity
• Cytoplasmic halo or viscosity (Ebner
T et al, 2003)
• Aggregation of smooth endoplasmic
reticulum (Ebner T et al, 2008)
• Zona refringence (Montag et al,
RBM, 2008)
• Spindle position assessment ?
(Woodward et al, RBM, 2008)

9. Embryo morphology :
Can you judge a book by its cover ?
Day 1: zygote
• Pronuclei score (Gianaroli et al, FS 2003 & HR
2007, Scott et al, HR, 2000)
• Cytoplasmic halo (Ebner T et al, 2003)
• Early syngamy (Edgar et al, 2004)
• Early cleavage (van Montfoort et al, 2004)
• Early cleavage & fragmentation rate (Ferrieres
et al, 2007; Hesters et al, 2008)

10. Embryo morphology :
Can you judge a book by its cover ?
Day 2 or 3 (Ziebe et al, 2003; Moriwaki et al, 2004, Holte et al, 2007;
Weitzman et al, 2010; Pelinck et al, 2010…)
• Number, size, regularity of blastomeres
• Fragmentation
• multinucleation
• Developmental speed

11. Embryo morphology :
Can you judge a book by its cover ?
Day 5 (Gardner DK et al, FS, 2000)
• Embryoblast / ICM
• Trophoblast cells

12. Embryo morphology
« Non invasive » ?
Reproducible
Objective
Rapid
Easy to use routinely
Cost-effective
No ethical question raised
Independent of other parameters

13. Limits of embryo morphology
All that glitters is not gold…

14. Limits ? Questions raised ?
• EBM : what about RCTs on embryo morphology ?
• Can morphology criteria be used in all patients ?
– Loss of correlation with implantation with increasing maternal age (Stensen et
al, 2010)
• Can morphology criteria be used in all labs ?
– Developmental speed varies according to culture conditions (media, [O2]…).
Should each laboratory build its own guidelines ?
• Intra and inter observer variability (Paternot et al, 2009; Arce et al, 2006)
• Real or theoretical non invasiveness ?
• No good correlation with genetic content : most embryos with good
morphology are chromosomally abnormal, especially in older women
However, correlations between morphology and implantation remain high,
although not absolute

15. Time-Lapse Cinematography :
the future of morphology ?
• Principle : dynamic morphometric
assessment
• allows precise measurement of 1st
cleavage time, PN fading …
• Prevents from taking the dishes out
of the incubator
• Can be associated with respirometry
Embryoscope ®
Primo Vision ®

16. 2nd Part
Genomics

17. Genetic content
Genomics / Transcriptomics
Does a good beginning make a good ending ?
Aneuploidy and embryo morphology : most
embryos with good morphology are chromosomally
abnormal, especially in older women (Magli,
Munne 2006)
• Need for systematic PGS ?

18. PGS
• Contradicting PGS results : most
studies reported a positive effect but 2
famous studies reported a detrimental
effect of PGD (Mastenbroek NEJM 2007,
Hardarson Hum Rep 2008)
• Explanations :
– Biological : mosaicism, self correction
– Technical ++ (misdiagnosis) : arrays, 1 cell
biopsy

19. PGS
How should PGS be performed to improve pregnancy rates ?
– Technical improvement (WGA CGH, SNP array) but
ethical concern about unexpected mutations
identification
– Blastocyst stage biopsy associated with vitrification
(Schoolcraft et al, 2010)
– Only if a reasonable number of zygotes/embryos
available (Munne S, 2010)

20. Gamete surroundings
Oocyte-cumulus dialogue
• Interactions between CCs and oocyte
mandatory for oocyte maturation
• Transcriptomic profile of cumulus cells has
been shown to reflect oocyte competence
and subsequent embryo quality and finally
implantation potential
• Target genes identified, potential
biomarkers currently under validation
(Assou et al, 2010; Adriaenssens et al, 2010)
Assou et al, MHR, 2010

21. Genomics
« Non invasive » ?
Reproducible
Objective
Rapid
Easy to use routinely
Cost-effective ?
No ethical question raised
Independent of other parameters

22. 3rd Part
Embryo Metabolism
Metabolomics

23. Embryo metabolism (1)
Aminoacids
• Early embryo development = intensive
metabolic turnover
• All AA (essential or not) are necessary for
early embryo development (degradation /
synthesis cycle)
• Correlation with blastocyst development
(Houghton et al, 2002)
• Correlation with implantation
(Brison et al, 2004)

24. Embryo metabolism (2)
Pyruvate and Glucose
• switch from lactate and pyruvate to glucose metabolism
around compaction
• Pyruvate (<Day 4) and glucose uptake (>Day 4) :
– largely studied in the 90’s
– Animal models (Gardner DK, 2000)
– not proved to be relevant in humans up to now
• Association of both could be correlated with blastocystis
formation (Gardner et al, 2000, 2001).
• Limits : technical aspects, correlation with implantation ?
• In the future : technical improvements
such as microfluidic chips allowing dynamic metabolioc
monitoring (Urbanski, 2008)

25. Embryo metabolism (3)
Metabolomic analysis
Botros et al, Mol Hum Rep, 2008
• Metabolome : reflects individual embryo
phenotype (uptake and release)
• Hypothese : differences in metabolism reflect
differences in implantation potential
• Technics : Near Infra Red (NIR) or RAMAN
Spectroscopy
• Prediction of embryo viability (and
subsequent implantation) according to
spectra-based algorithm
• Meta analysis confirms
correlation with implantation
(Botros L et al, 2008)

26. Correlation with
implantation
Independant of morphology
Independant of the day of
assessment
• Validated through multicenter blinded studies, RCTs ongoing ?
• Commercially available (Viametrics)
Embryo metabolism (4)
Metabolomic analysis : Viability scores / index
(Seli E et al, FS,2009)

27. Metabolomic analysis
« Non invasive »
Reproducible
Objective
Rapid
Easy to use routinely (extra equipment required)
Cost-effective ?
No ethical question raised
Independent of other parameters (morphology)

28. Embryo metabolism (5)
Respirometry
• Oxygen consumption of preimplantation
embryos has been shown to reflect
developmental competence and
subsequent implantation in animal models
(Lopes AS et al, Theriogenelogy, 2007).
• Preliminary work on human oocyte with the
Embryoscope® (Scott et al, RBMonline,
2008)
• No proof of efficiency on human embryos
up to now, clinical trials ongoing (Meseguer
M et al, IVI)

29. Embryo metabolism (6)
Secreted Factors
• sHLA-G : technical controversy (Sargent et al,
2007)
• Follicular IL-15 & GM-CSF (Lédée N et al, FS,
2010)

30. 4th Part
DEBATE / COMMENTS

31. The so-called « invasiveness » (1)
« First do no harm »
« Primum non nocere »
Morphology = « non invasive » ?
PGS/PGD = « invasive » ?

32. The so-called « invasiveness » (2)
Morphology assessment
• Morphology assessment means dishes
out of the incubator
• pH, osmolarity and temperature
modifications of the culture media each
time the incubator is opened
– Up to 5.6°C variation in microdrops (Kelly PB
et al, 2010)
– 30 min for temperature recovery and 8 min for
[O2] recovery in large incubators
– pH>7.4 in 2 minutes out of the incubator
• Consequences on embryo development
have not been evaluated…

33. The so-called « invasiveness » (3)
PGD/PGS
biopsy of 1 cell (De Vos et al, 2010) or trophoblast cells
(Schoolcraft et al, 2010) for PGS/ PGD by trained
embryologists in a reference laboratory do not affect
embryo developmental competence (Munne S et al,
2010)

34. Tailored embryo selection criteria ?
• Women’s age
• Ovarian stimulation protocol
– Mild vs conventional : benefits still
controversial… (Baart EB et al,
RBMonline, 2009; Verberg et al, Hum
Rep Update, 2009)
– Stimulation regimen (Weghofer et al,
HR, 2008; Ziebe et al, HR, 2007; Merit
Study) : different proportion of euploid
embryos, different implantation rates for
top quality embryos according to
stimulation regimen

35. Cost-effectiveness
• Must be evaluated on long term and a large
scale
• In countries where ART cycles are reimbursed,
every birth gained yields benefits for the society
even in very expensive cases (Evers, IFFS,
2010)
• Embryo quality assessment methods must also
be evaluated through socio-economical studies

36. That means embryo selection is
nothing without …
Performant
embryo
freezing
program
Performant
embryo
transfer
strategy
Performant
embryo
transfer
technique
Accurate uterine
receptivity and
timing of embryo
transfer

37. Performant embryo freezing strategy ?
• Technical restraints :
– Vitrification yields significantly higher
success rates than slow cooling
– Tolerated but not authorized yet in
France…
• Ethical and/or legal restraints :
– Cleavage stage embryo freezing
forbidden in Italy

38. Embryo [transfer strategy] selection
• Extended culture to the blastocyst stage is thought to result in
better embryo selection and higher implantation rates.
• BUT … is it suitable for every patients ? For every cycles ?
• Nomogram = statistical tool to generate graphical representation of
a predictive model
• Nomogram used to build a
predictive model of blastocyst
transfer cancellation
(Dessolle, Freour et al, 2010)
• Could help in reducing the
incidence of cancelled Day 5 transfers
• Currently undergoing external
multicenter validation

39. Quality control of Embryo transfer
• Embryo transfer is an operator-
dependent crucial step for
achieving pregnancy after IVF
• Quality control for learners and
for continuous monitoring of
performance
• Reference = senior gynaecologist
• Trainees :
1st : Reach acceptable
performance
2nd : maintain adequate level of
performance

40. In the future…
Improvement of the endometrium-
embryo dialog ?
• COH affects endometrial receptivity
(Haouzi et al, Biol Reprod, 2010;
Boomsma et al, Fertil Steril, 2010)
• Vitrification associated with embryo
transfer on spontaneous or hCG-primed
natural cycle could result in more
physiological endometrial implantation
window (Fatemi et al, Fertil Steril, 2010)
Haouzi et al, 2010
Boomsma et al, 2010

41. What we should not forget …

42. Last but not least…
chance, expertise, hazard …?

43. Conclusion
EMBRYO SELECTION IN THE IVF LAB
• It’s a long way from gametogenesis
to healthy baby delivery
• Improvement in embryo quality
assessment method(s) should not
result in higher multiple pregnancy
rates !!
• However, accurate embryo selection
in the IVF lab is required but not
sufficient to ensure higher
implantation rates
• The next frontier : select the right
embryo for the right endometrium

44. Take-Home Message ?
• No universally accepted and validated approach,
need for customized and individualized methods
• Numerous promising technological advances
(Omics)
• Need for EBM before stopping using morphology

45. Thank you for your attention