- 1. Thomas FREOUR Centre dAssistance Mdicale la Procration 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) :
largely studied in the 90s 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 (Lde 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 5.6C
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 ? Womens 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 Its 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
