Current gamete/embryo Current gamete/embryo assessment based on assessment based on morphology morphology : : - Strategies - Strategies - A common language? - A common language? - What are the limitations? - What are the limitations? Kersti Lundin Reproductive Medicine Sahlgrenska University Hospital Gothenburg Sweden
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Current gamete/embryo assessment based on morphology: - Strategies - A common language? - What are the limitations? Kersti Lundin Reproductive Medicine.
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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?
• 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
• 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
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.
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)
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
* * 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
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
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