Post on 12-Jan-2016
Current gamete/embryo assessment Current gamete/embryo assessment based on based on morphologymorphology: :
- Strategies - Strategies - A common language?- A common language?
- What are the limitations?- What are the limitations?
Kersti LundinReproductive Medicine
Sahlgrenska University HospitalGothenburg
Sweden
Purpose of assessmentPurpose of assessment
• 1st + 2nd meiosis1st + 2nd meiosis• MetamorphosisMetamorphosis
• Cytoplasmic maturationCytoplasmic maturation
• 1st meiosis1st meiosis
• 2nd meiosis• Pronucleus
• Decondensation• Sperm aster• Pronucleus
SpermatogenesisSpermatogenesis OogenesisOogenesis
Fusion
Capacitation
SyngamySyngamy
Penetration
BindingAcrosome reaction
CleavageCleavage
””Quality / Normality”Quality / Normality”
Scoring variablesScoring variables
Consensus? Consensus? Validation?Validation?
Sperm selectionSperm selection
• OverallOverall (sperm sample preparation): (sperm sample preparation):– Separation from seminal plasmaSeparation from seminal plasma– By motility (swim-up) By motility (swim-up) – By discontiuous gradient centrifugationBy discontiuous gradient centrifugation
• Individual sperm selection (ICSI):– Selection by speed– Selection by morphology
– low magnification– IMSIIMSI
Individual sperm selection, low Individual sperm selection, low magnification morphologymagnification morphology
• De-De-selection of gross head, tail and/or neck selection of gross head, tail and/or neck and midpiece abnormalities and midpiece abnormalities
• Lower fertilisation rates but no difference in PR and IR depending on overall sperm morphology, but no consensus for low magnification individual selection
IMSI morphologyIMSI morphology
• Selection of sperm based on:Selection of sperm based on:– ””Normal” shape of nucleusNormal” shape of nucleus– No or small vacuol-like structures of the head No or small vacuol-like structures of the head
(< 4%)(< 4%)– Correlation with sperm aneuploidy and Correlation with sperm aneuploidy and
chromatin condensation failurechromatin condensation failure
• Time demanding method• Expensive equipment
Individual sperm selection, IMSIIndividual sperm selection, IMSI
• Metaanalysis; 3 studies included (in 10 years!)Metaanalysis; 3 studies included (in 10 years!)
=> No clear evidences published (evidence based => No clear evidences published (evidence based medicine, prospective randomized studies, enough medicine, prospective randomized studies, enough power, identification of a specific category of power, identification of a specific category of patients) about the real efficacy of IMSI approach.patients) about the real efficacy of IMSI approach.
Souza Setti et al 2010Souza Setti et al 2010
Balaban et al 2011
Significantly improved IR for severe male factor patients (87+81, randomised)
Significantly improved embryo quality in the presence of oocyte dysmorphisms (332 + 332 patients, nonrandomised) Souza Setti et al 2012Souza Setti et al 2012
• Cytoplasmatic dysmorphisms may be associated Cytoplasmatic dysmorphisms may be associated with developmental potentialwith developmental potential
• Presumably affecting cellular functions, eg. Presumably affecting cellular functions, eg. cytoskeleton and signallingcytoskeleton and signalling
• ””standard” IVF; lowered fertilisation potentialstandard” IVF; lowered fertilisation potential
Important to understand which individual factors that may affect the outcome
Oocytes - morphologyOocytes - morphology
The ”normal” (= The ”normal” (= fertilisablefertilisable) oocyte?) oocyte?
• Appropriate sizeAppropriate size• Appropriate perivitelline Appropriate perivitelline
spacespace• Single (intact?) polar bodySingle (intact?) polar body• Appropriate zona thicknessAppropriate zona thickness• Healthy looking cytoplasmHealthy looking cytoplasm
Poor predictors for fertilisation and development
From Swain and Pool 2008
Alpha & ESHRE, Hum Rep 2011, RBM online 2011
Possible impact factorsPossible impact factorsIntracytoplasmatic; morphology• Granulation, central (”clustering) vs. diffuseGranulation, central (”clustering) vs. diffuse• VacuolesVacuoles• sER aggregationsER aggregation• Refractile/necrotic bodiesRefractile/necrotic bodies• Color (”dark”)Color (”dark”)
Cumulus oocyte complex
Zona pellucida • Shape• Thickness
Systematic review – oocyte qualitySystematic review – oocyte quality• 50 relevant articles were identified• 33 analysed a single feature, 9 observed multiple features and
investigated the effect of these features individually, 8 summarized the effect of individual features.
• Investigated structures were the following: meiotic spindle (15 papers), zona pellucida (15 papers), vacuoles or refractile bodies (14 papers), polar body shape (12 papers), oocyte shape (10 papers), dark cytoplasm or diffuse granulation (12 papers), perivitelline space (11 papers), central cytoplasmic granulation (8 papers), cumulus-oocyte complex (6 papers) and cytoplasm viscosity and membrane resistance characteristics (2 papers).
• No clear tendency in recent publications to a general increase in predictive value of morphological features was found. These contradicting data underline the importance of more intensive and coordinated research to reach a consensus and fully exploit the predictive potential of morphological examination of human oocytes.
Rienzi et al, 2011
Different human oocyte morphological abnormalities (arrows) observed by light microscopy (400× magnification): (A) diffuse cytoplasmic granularity, (B) centrally located cytoplasmic granular area, (C) smooth endoplasmic reticulum clusters, (D) vacuoles, (E) abnormal zona
pellucida shape, (F) large perivitelline space with fragments.
Rienzi L et al. Hum. Reprod. Update 2011;17:34-45
© The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. For Permissions, please email: journals.permissions@oxfordjournals.org
Inclusions /VacuolesInclusions /Vacuoles• Fluid filled membrane- Fluid filled membrane-
enclosed structuresenclosed structures• Same composition of fluid as Same composition of fluid as
in perivitelline spacein perivitelline space• Believed to arise either
spontaneously, or through fusion of vesicles from sER and/or the Golgi structure
• < 14µm is believed to be of no consequence, larger vacuoles may interfere with spatial development (eg. function of tubuli)
Inclusions /Refractile(Inclusions /Refractile(necrotic) ) bodiesbodies
• Incorporation and aggregation of (mainly) membranes
• Not shown to have any impact on fertilisation or developmental potential
De Sutter et al, 1996 Balaban et al, 1998 Ebner et al, 2001
””sER”sER”aggregation of smooth aggregation of smooth endoplasmic reticulumendoplasmic reticulum
• Smooth endoplasmic reticula synthesise lipids and steroids, and regulates calcium levels
• Can in some oocytes aggregate, seen as a disc-like structure, not membrane- enclosed
• Not known why they arise (certain association with stimulation/high levels of estradiol?)
• Shown that these oocytes have changes in eg. calcium signalling and mitochondrial function
Otsuki et al, 2004 Jonathan van Blerkom Akarsu et al, 2009
Zona pellucidaZona pellucida
• Microscopic morphology (light microscopy) – Microscopic morphology (light microscopy) – shapeshape– No consensus
• Mechanical damage (zona splitting)– High association with non-implantation
What about things we do not What about things we do not seesee? ?
• Genetic/chromosomal constitutionGenetic/chromosomal constitution
• MetabolismMetabolism
• Respiration rateRespiration rate
Summary oocyte morphologySummary oocyte morphology• Homogenous, light, smooth cytoplasm is Homogenous, light, smooth cytoplasm is
associated with ”normal” oocyte morphologyassociated with ”normal” oocyte morphology
• ””Clustering” and larger vacuoles associated with Clustering” and larger vacuoles associated with lowered development and implantation potentiallowered development and implantation potential
• Do not use / inseminate oocytes with aggregation of smooth endoplasmic reticulum
• Do not use / inseminate giant oocytes
• No consensus regarding other characteristics or for No consensus regarding other characteristics or for zona pellucida morphologyzona pellucida morphology
• … > half of all IVF oocytes show some sort of dysmorphism….
NB. Documentation!
How do we define/find ”the best embryo”?
Does embryo ”quality” correlate to morphologymorphology assessment?
Nuclear status / cytoplasmic status / metabolic status / environment /
chromosomal status
Embryo development (cleavage, Embryo development (cleavage, morphology)morphology)
ImplantationImplantation
Live birthLive birth
Polarity / symmetry Timing / SynchronisationPolarity / symmetryPolarity / symmetry Timing / SynchronisationTiming / Synchronisation
Embryo classification
010
2030
4050
6070
8090
100
Total Transf erable Selected
Perc
enta
ge
> 50% normal
100% normal
Chromosomal normality and embryo Chromosomal normality and embryo selection (n=144 embryos)selection (n=144 embryos)
Ziebe et al 2003,
What are we What are we looking at?looking at?
• Day 1 Day 1
– (PN score)(PN score)
– Early cleavageEarly cleavage
• Day 2/3
– Cytoplasm
– Number of cells
– Fragmentation
– Cell size
– Number of nuclei
• Day 5/6
– ICM
– Trophectoderm
– Expansion
Embryo (a)symmetryEmbryo (a)symmetry
• Each cleavage results in daughter cells with uneven content of transcription factors
Embryo asymmetry – good or bad?Embryo asymmetry – good or bad?
• Human embryos show asymmetric Human embryos show asymmetric distribution of factors believed to be distribution of factors believed to be important for establishing embryonic important for establishing embryonic axes / positional identityaxes / positional identity = GOOD
• Loss of blastomeres or part of blastomeres (fragmentation) or incorrect distribution of material (uneven sized) might impair the correct establishment of axis = BAD
Chromosomal normality and Chromosomal normality and blastomere sizeblastomere size
Munné et al 2004, 2006
05
1015202530354045
uneven sized even sized
Hardarson et al 2001, Hnida et al 2004
Cell size and multinucleationCell size and multinucleation
CleavageCleavage Even Even UnevenUneven
Embryo multinuclearity (%)Embryo multinuclearity (%)1/13 (2.1)1/13 (2.1) 5/11 (45.5) 5/11 (45.5)
p=0.005p=0.005
Cell size (µm3 x 106) 2 cell 4 cell Mononucleated 0.2100.118Multinucleated 0.3140.203
p=<0.001
39% IR
24% IR
33% IR
* * All embryos transferred in a All embryos transferred in a single cycle are of the same single cycle are of the same
statusstatus Hardarson et al 2001
Chromosomal normality and Chromosomal normality and fragmentationfragmentation
Munné et al 2004, 2006
0
10
20
30
40
50
60
70
0-5 6-15 16-25 26-35 >35
% fragments
Summary; cell size and Summary; cell size and fragmentationfragmentation
Fragmentation:
No studies (multivariate) show an independent predictive influence of fragmentation (up to 20 (-30)%) for implantation
Van Royen et al 2001, Munné et al 2004, 2006, Holte 2007
Uneven cell size:Uneven cell size:
Unequal sized blastomeres (2-, 4-, 8- cells) Unequal sized blastomeres (2-, 4-, 8- cells) correlates to aneuploidy, to multinucleation and correlates to aneuploidy, to multinucleation and to lower implantation ratesto lower implantation rates
Cleavage rate - number of cellsCleavage rate - number of cells
van Royen et al, 2002 – day 3van Royen et al, 2002 – day 3
4 - 8/9 cells: 4 - 8/9 cells: 42% IR 42% IR
≠ ≠ 4 – 8/9 cells:4 – 8/9 cells: <33% IR<33% IR
Thurin et al 2005, (SET) – day Thurin et al 2005, (SET) – day 2, 2, multicenter study (661 cycles)multicenter study (661 cycles)
4 cells:4 cells: 28%28% IR IR ≠ ≠ 4 cells:4 cells: 16%16% IR IR
(p=0.013)(p=0.013)
0
10
20
30
40
50
60
70
2 3 4 5 ≥ 6
Normality rate
Blastocyst rate
Chromosomal normality and Chromosomal normality and cleavage rate day 2cleavage rate day 2
De los Santos et al ESHRE 2006, 447
Chromosomal normality and Chromosomal normality and cleavage rate day 3cleavage rate day 3
Magli et al 2001, van Royen et al 2002
0
10
20
30
40
50
<5 cells 7-8 cells >9 cells
27%9%
42%IR:
Summary; number of cellsSummary; number of cells
• Number of cells day 2 and day 3– Should follow a ”normal” cleavage pattern– Correlates to blastocyst rates– Correlates to pregnancy/implantation rates– Correlates to aneuploidy rates
Visible nucleiVisible nuclei
4 / 4 1 (0) / 4
IR 26% IR 4% Moriwaki et al 2004IR 42% IR 22% Saldeen et al 2005predictive factor (multivariate) Holte et al 2007
Multinucleation/ BinucleationMultinucleation/ Binucleation
Associated with lowered Associated with lowered pregnancy and implantation pregnancy and implantation ratesrates
Occurs in Occurs in ~~ 25-50% of embryos 25-50% of embryos on day 2/3on day 2/3
Decreased incidence in good Decreased incidence in good quality embryos quality embryos ~~ 15% 15%
Palmstierna et al 1997, Kligman et al 1996, Jackson et al 1998, van Royen et al 2001, 2003,Hardarson, 2001, Hnida et al 2004
0 hours
16-18 hours
25-27 / 27-29 hours
43-45 hours
67-69 hours
115-117 hours
Embryo assessments Embryo assessments in the labin the lab
Sequential scoring andTiming!!
Common language??Common language??
• WHY?WHY?
• Exchange of dataExchange of data
• Comparison of data / resultsComparison of data / results
• WE NEED:
• Documentation with a common structure
• Equal assessment
Limitations?Limitations?For a common scoring and a For a common scoring and a
common language…common language…
• Assessments
• Nomenclature
• Timings
• IT systems
• Cost
Assessement/selectionAssessement/selection
• Increasingly importantIncreasingly important BUTBUT
• Mainly subjective, dependent upon– competence (training)– accuracy– consistency
• MNB• Number of cells • Fragmentation • Cell size• First cleavage• Second cleavage • Visible nuclei • Cytoplasmic appearance
• Compaction• Blastocyst grading
• NoneNone• 4 / 84 / 8• < 20 (-30?)%< 20 (-30?)%• Even sizedEven sized• < 25-27 hours < 25-27 hours • synchronisedsynchronised• 1 visible nucleus / cell1 visible nucleus / cell
• No vacuoles, no No vacuoles, no granulationgranulation
• - day 2, + day 3- day 2, + day 3• Expansion, ICM, TCExpansion, ICM, TC
Embryo assessment variablesEmbryo assessment variables
Are we scoring equally?Are we scoring equally?
Grade of fragmentationBlastomere size Blastomere/fragment
Interobserver and intraobserver variation in day Interobserver and intraobserver variation in day 3 embryo grading3 embryo grading
Baxter AB, Mayer JF, Shipley SK and Catherino WHBaxter AB, Mayer JF, Shipley SK and Catherino WHFertil Steril 2006; 86: 1608-1615Fertil Steril 2006; 86: 1608-1615
Design, Design, Baxter et alBaxter et al
• 26 embryologists at ASRM in Philadelphia
• 35 embryos video recorded
(interobserver variation)
• 7 embryos shown several times
(intraobserver variation)
• Scale with 5 embryo grades (Veeck)
• Kappa values used for statistics
Kappa statisticsKappa statistics
• The Kappa is the ratio of the proportion of The Kappa is the ratio of the proportion of times the raters did agree to the proportion of times the raters did agree to the proportion of times the raters were expected to agree. times the raters were expected to agree.
•K=1 means perfect agreement as to what was expected
• K=0 means that agreement is not different from chance
Statistical Methods – Kappa StatisticsStatistical Methods – Kappa Statistics
Terminology for the extent of agreement:Terminology for the extent of agreement:
– kappa 0.8 -1 kappa 0.8 -1 excellentexcellent– kappa 0.6-0.79 kappa 0.6-0.79 good good – kappa 0.4-0.59kappa 0.4-0.59 moderate moderate – kappa 0.2-0.39kappa 0.2-0.39 poor poor – kappa 0-0.19 kappa 0-0.19 very poorvery poor
Results, Results, Baxter et alBaxter et al
• Interobserver variability (median, range)
Kappa 0.24 (0.03-0.49) poor
• Intraobserver variability (median, range)
Kappa 0.69 (0.44-1.00) good
Conclusions, Conclusions, Baxter et alBaxter et al
Agreement is too low!Agreement is too low!
• Only use one embryologist for scoring ?
• Use consensus scoring from several
embryologists ?
• Simplify the scoring system ?
Interobserver agreement and intraobserver Interobserver agreement and intraobserver reproducibility of embryo quality reproducibility of embryo quality assessmentsassessments
Arce JC, Ziebe, S, Lundin K, Janssens R, Helmgaard L Arce JC, Ziebe, S, Lundin K, Janssens R, Helmgaard L and Sörensen P. Hum Rep 2006: 21; 2141-2148and Sörensen P. Hum Rep 2006: 21; 2141-2148
Mean agreement between Central Mean agreement between Central EmbryologistsEmbryologists
0
0,2
0,4
0,6
0,8
1
Day 1 Day 2 Day 3
Cleavage stage
Blastomereuniformity
Degree offragmentation
Multinucleation
Cytoplasmicappearance
Agreement between Central and Agreement between Central and LocalLocal
0
0,2
0,4
0,6
0,8
1
Day 1 Day 2 Day 3
Cleavage stage
Blastomereuniformity
Degree offragmentation
Multinucleation
Cytoplasmicappearance
Consensus document / Consensus document / Embryology AtlasEmbryology Atlas
Approx. 400 slides of Approx. 400 slides of oocytes, zygotes, early oocytes, zygotes, early embryos and embryos and blastocystsblastocysts
Add nomenclature from Add nomenclature from consensus paperconsensus paper
Thank you for your attention!Thank you for your attention!