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Current gamete/embryo assessment based on morphology: - Strategies - A common language? - What are the limitations? Kersti Lundin Reproductive Medicine Sahlgrenska University Hospital Gothenburg Sweden
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Purpose of assessment
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Spermatogenesis Oogenesis 1st + 2nd meiosis Metamorphosis Cytoplasmic maturation 1st meiosis Capacitation Binding Acrosome reaction Penetration Fusion Decondensation Sperm aster Pronucleus 2nd meiosis Pronucleus Syngamy Cleavage
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”Quality / Normality” Scoring variables
? Consensus? Validation?
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Sperm selection Overall (sperm sample preparation):
Separation from seminal plasma By motility (swim-up) By discontiuous gradient centrifugation Individual sperm selection (ICSI): Selection by speed Selection by morphology – low magnification IMSI
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Individual sperm selection, low magnification morphology
De-selection of gross head, tail and/or neck 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
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IMSI morphology Selection of sperm based on: ”Normal” shape of nucleus
No or small vacuol-like structures of the head (< 4%) Correlation with sperm aneuploidy and chromatin condensation failure Time demanding method Expensive equipment
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Individual sperm selection, IMSI
Metaanalysis; 3 studies included (in 10 years!) => No clear evidences published (evidence based medicine, prospective randomized studies, enough power, identification of a specific category of patients) about the real efficacy of IMSI approach. Souza Setti et al 2010 Significantly improved IR for severe male factor patients (87+81, randomised) Balaban et al 2011 Significantly improved embryo quality in the presence of oocyte dysmorphisms ( patients, nonrandomised) Souza Setti et al 2012
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Oocytes - morphology Cytoplasmatic dysmorphisms may be associated with developmental potential Presumably affecting cellular functions, eg. cytoskeleton and signalling ”standard” IVF; lowered fertilisation potential Important to understand which individual factors that may affect the outcome
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The ”normal” (= fertilisable) oocyte?
Appropriate size Appropriate perivitelline space Single (intact?) polar body Appropriate zona thickness Healthy looking cytoplasm Poor predictors for fertilisation and development From Swain and Pool 2008
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Alpha & ESHRE, Hum Rep 2011, RBM online 2011
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Possible impact factors
Intracytoplasmatic; morphology Granulation, central (”clustering) vs. diffuse Vacuoles sER aggregation Refractile/necrotic bodies Color (”dark”) Cumulus oocyte complex Zona pellucida Shape Thickness
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Systematic 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
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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. 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 Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. For Permissions, please
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Inclusions /Vacuoles Fluid filled membrane- enclosed structures
Same composition of fluid as in 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)
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Inclusions /Refractile(necrotic) bodies
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
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”sER” aggregation of smooth endoplasmic 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
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Zona pellucida Microscopic morphology (light microscopy) – shape
No consensus Mechanical damage (zona splitting) High association with non-implantation
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What about things we do not see?
Genetic/chromosomal constitution Metabolism Respiration rate
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Summary oocyte morphology
Homogenous, light, smooth cytoplasm is associated with ”normal” oocyte morphology ”Clustering” and larger vacuoles associated with lowered 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 zona pellucida morphology … > half of all IVF oocytes show some sort of dysmorphism…. NB. Documentation!
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How do we define/find ”the best embryo”?
Does embryo ”quality” correlate to morphology assessment?
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Embryo development (cleavage, morphology)
Implantation Live birth Polarity / symmetry Polarity / symmetry Timing / Synchronisation Timing / Synchronisation Nuclear status / cytoplasmic status / metabolic status / environment / chromosomal status
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Chromosomal normality and embryo selection (n=144 embryos)
Ziebe et al 2003,
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What are we looking at? Day 1 (PN score) Early cleavage Day 2/3
Cytoplasm Number of cells Fragmentation Cell size Number of nuclei Day 5/6 ICM Trophectoderm Expansion What are we looking at?
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Embryo (a)symmetry Each cleavage results in daughter cells with uneven content of transcription factors
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Embryo asymmetry – good or bad?
Human embryos show asymmetric distribution of factors believed to be important for establishing embryonic axes / 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
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Chromosomal normality and blastomere size
Munné et al 2004, 2006
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Cell size and multinucleation
Cleavage Even Uneven Embryo multinuclearity (%) 1/13 (2.1) 5/11 (45.5) p=0.005 Cell size (µm3 x 106) 2 cell 4 cell Mononucleated Multinucleated p=<0.001 Hardarson et al 2001, Hnida et al 2004
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* All embryos transferred in a single cycle are of the same status
33% IR 39% IR 24% IR * All embryos transferred in a single cycle are of the same status Hardarson et al 2001
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Chromosomal normality and fragmentation
Munné et al 2004, 2006
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Summary; cell size and fragmentation
Uneven cell size: Unequal sized blastomeres (2-, 4-, 8- cells) correlates to aneuploidy, to multinucleation and to lower implantation rates 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
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Cleavage rate - number of cells
van Royen et al, 2002 – day 3 4 - 8/9 cells: 42% IR ≠ 4 – 8/9 cells: <33% IR Thurin et al 2005, (SET) – day 2, multicenter study (661 cycles) 4 cells: 28% IR ≠ 4 cells: 16% IR (p=0.013)
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Chromosomal normality and cleavage rate day 2
De los Santos et al ESHRE 2006, 447
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Chromosomal normality and cleavage rate day 3
42% IR: 9% 27% Magli et al 2001, van Royen et al 2002
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Summary; 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
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Visible nuclei 1 (0) / 4 4 / 4 IR 26% IR 4% Moriwaki et al 2004
IR 42% IR 22% Saldeen et al 2005 predictive factor (multivariate) Holte et al 2007
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Multinucleation/ Binucleation
Associated with lowered pregnancy and implantation rates Occurs in ~ 25-50% of embryos on day 2/3 Decreased incidence in good quality embryos ~ 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
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Embryo assessments in the lab
0 hours 16-18 hours 25-27 / hours 43-45 hours 67-69 hours Sequential scoring andTiming!! hours
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Common language?? WHY? Exchange of data Comparison of data / results
WE NEED: Documentation with a common structure Equal assessment
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Limitations? For a common scoring and a common language…
Assessments Nomenclature Timings IT systems Cost
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Assessement/selection
Increasingly important BUT Mainly subjective, dependent upon competence (training) accuracy consistency
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Embryo assessment variables
MNB Number of cells Fragmentation Cell size First cleavage Second cleavage Visible nuclei Cytoplasmic appearance Compaction Blastocyst grading None 4 / 8 < 20 (-30?)% Even sized < hours synchronised 1 visible nucleus / cell No vacuoles, no granulation - day 2, + day 3 Expansion, ICM, TC
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Are we scoring equally? Grade of fragmentation Blastomere size
Blastomere/fragment
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Interobserver and intraobserver variation in day 3 embryo grading Baxter AB, Mayer JF, Shipley SK and Catherino WH Fertil Steril 2006; 86:
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Design, Baxter 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
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Kappa statistics The Kappa is the ratio of the proportion of times the raters did agree to the proportion of 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
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Statistical Methods – Kappa Statistics
Terminology for the extent of agreement: kappa excellent kappa good kappa moderate kappa poor kappa very poor
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Results, Baxter et al Interobserver variability (median, range)
Kappa ( ) poor Intraobserver variability (median, range) Kappa ( ) good
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Conclusions, Baxter et al
Agreement is too low! Only use one embryologist for scoring ? Use consensus scoring from several embryologists ? Simplify the scoring system ?
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Interobserver agreement and intraobserver reproducibility of embryo quality assessments Arce JC, Ziebe, S, Lundin K, Janssens R, Helmgaard L and Sörensen P. Hum Rep 2006: 21;
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Mean agreement between Central Embryologists
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Agreement between Central and Local
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Consensus document / Embryology Atlas
Approx. 400 slides of oocytes, zygotes, early embryos and blastocysts Add nomenclature from consensus paper
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Thank you for your attention!
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