The impact of embryo quality on the cumulative success rates ...
Current gamete/embryo assessment based on morphology: - Strategies - A common language? - What are the limitations? Kersti Lundin Reproductive Medicine Sahlgrenska University Hospital Gothenburg Sweden Purpose of assessment Spermatogenesis
1st + 2nd meiosis Metamorphosis a p a C Oogenesis Cytoplasmic maturation 1st meiosis n o i
t cita Binding Acrosome reaction Penetration Fusion Decondensation 2nd meiosis Sperm aster Pronucleus Pronucleus Syngamy
Cleavage Quality / Normality Scoring variables Consensus? Validation? 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 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
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 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 (332 + 332 patients, nonrandomised) Souza Setti et al 2012 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 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 Alpha & ESHRE, Hum Rep 2011, RBM online 2011 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 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 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: [email protected] Inclusions /Vacuoles Fluid filled membraneenclosed 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 < 14m is believed to be of no consequence, larger vacuoles may interfere with spatial development (eg. function of tubuli) Inclusions /Refractile(necrotic )
/Refractile( 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
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 Zona pellucida Microscopic morphology (light microscopy) shape No consensus Mechanical damage (zona splitting) High association with non-implantation What about things we do not see? Genetic/chromosomal constitution
Metabolism Respiration rate 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! How do we define/find the best embryo? Does embryo quality correlate to morphology assessment? Embryo development (cleavage, morphology) Implantation Live birth Polarity / symmetry
Timing / Synchronisation Nuclear status / cytoplasmic status / metabolic status / environment / chromosomal status Chromosomal normality and embryo selection (n=144 embryos) Percentage Embryo classification 100
90 80 70 60 50 40 30 20 10 0 >50% normal 100% normal
Total Transf erable Selected Ziebe et al 2003, 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 Embryo (a)symmetry Each cleavage results in daughter cells with uneven content of transcription factors
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 Chromosomal normality and blastomere size 45
40 35 30 25 20 15 10 5 0 uneven sized even sized Munn et al 2004, 2006
Cell size and multinucleation Cleavage Even Uneven Embryo multinuclearity (%) 1/13 (2.1) 5/11 (45.5) p=0.005 Cell size (m3 x 106) Mononucleated 0.118 Multinucleated 0.203
2 cell 0.210 4 cell 0.314 Hardarson et al 2001, p=<0.001 Hnida et al 2004 33% IR 39% IR 24% IR
* All embryos transferred in a single cycle are of the same status Hardarson et al 2001 Chromosomal normality and fragmentation 70 60 50 40 30
20 10 0 0-5 6-15 16-25 26-35 >35 % fragments
Munn et al 2004, 2006 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, Munn et al 2004, 2006, Holte 2007 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) Chromosomal normality and cleavage rate day 2 70
3 4 5 6 De los Santos et al ESHRE 2006, 447 Chromosomal normality and cleavage rate day 3 50 40
30 42% IR: 9% 27% 20 10 0 <5 cells
7-8 cells >9 cells Magli et al 2001, van Royen et al 2002 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
Visible nuclei 4/4 IR 26% IR 4% IR 42% IR 22% predictive factor (multivariate) 1 (0) / 4 Moriwaki et al 2004 Saldeen et al 2005 Holte et al 200
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 0 hours
Embryo assessments in the lab 16-18 hours 25-27 / 27-29 hours 43-45 hours 67-69 hours Sequential scoring andTiming!!
115-117 hours Common language?? WHY? Exchange of data Comparison of data / results WE NEED: Documentation with a common structure Equal assessment
Limitations? For a common scoring and a common language Assessments Nomenclature Timings IT systems Cost Assessement/selection Increasingly important BUT Mainly subjective, dependent upon
No vacuoles, no granulation - day 2, + day 3 Expansion, ICM, TC Are we scoring equally? Grade of fragmentation Blastomere size Blastomere/fragment
Interobserver and intraobserver variation in day 3 embryo grading Baxter AB, Mayer JF, Shipley SK and Catherino WH Fertil Steril 2006; 86: 1608-1615 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 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 Statistical Methods Kappa Statistics Terminology for the extent of agreement:
kappa 0.8 -1 kappa 0.6-0.79 kappa 0.4-0.59 kappa 0.2-0.39 kappa 0-0.19 excellent good moderate poor very poor Results, Baxter 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, Baxter et al Agreement is too low! Only use one embryologist for scoring ? Use consensus scoring from several embryologists ? Simplify the scoring system ? Interobserver agreement and intraobserver
reproducibility of embryo quality assessments Arce JC, Ziebe, S, Lundin K, Janssens R, Helmgaard L and Srensen P. Hum Rep 2006: 21; 2141-2148 Mean agreement between Central Embryologists 1 Cleavage stage 0,8 Blastomere
uniformity 0,6 Degree of fragmentation 0,4 Multinucleation 0,2 Cytoplasmic
appearance 0 Day 1 Day 2 Day 3 Agreement between Central and Local 1 Cleavage stage
0,8 Blastomere uniformity 0,6 Degree of fragmentation 0,4 Multinucleation
0,2 Cytoplasmic appearance 0 Day 1 Day 2 Day 3 Consensus document /
Embryology Atlas Approx. 400 slides of oocytes, zygotes, early embryos and blastocysts Add nomenclature from consensus paper Thank you for your attention!
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