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

2. Purpose of assessment

3. 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

4. ”Quality / Normality” Scoring variables?
Consensus?
Validation?

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. Alpha & ESHRE, Hum Rep 2011, RBM online 2011

12. 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

13. 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

14. 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

15. 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)

16. 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

17. ”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

18. Zona pellucida Microscopic morphology (light microscopy) – shape
No consensus
Mechanical damage (zona splitting)
High association with non-implantation

19. What about things we do not see?
Genetic/chromosomal constitution
Metabolism
Respiration rate

20. 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!

21. How do we define/find ”the best embryo”?
Does embryo ”quality” correlate to morphology assessment?

22. Embryo development (cleavage, morphology)
Implantation
Live birth
Polarity / symmetry
Polarity / symmetry
Timing / Synchronisation
Timing / Synchronisation
Nuclear status / cytoplasmic status / metabolic status / environment / chromosomal status

23. Chromosomal normality and embryo selection (n=144 embryos)
Ziebe et al 2003,

24. 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?

25. Embryo (a)symmetry
Each cleavage results in daughter cells with uneven content of transcription factors

26. 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

27. Chromosomal normality and blastomere size
Munné et al 2004, 2006

28. 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

29. * 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

30. Chromosomal normality and fragmentation
Munné et al 2004, 2006

31. 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

32. 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)

33. Chromosomal normality and cleavage rate day 2
De los Santos et al ESHRE 2006, 447

34. Chromosomal normality and cleavage rate day 3
42%
IR: 9%
27%
Magli et al 2001, van Royen et al 2002

35. 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

36. 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

37. 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

38. Embryo assessments in the lab
0 hours
16-18 hours
25-27 / hours
43-45 hours
67-69 hours
Sequential scoring
andTiming!!
hours

39. Common language?? WHY? Exchange of data Comparison of data / results
WE NEED:
Documentation with a common structure
Equal assessment

40. Limitations? For a common scoring and a common language…
Assessments
Nomenclature
Timings
IT systems
Cost

41. Assessement/selection
Increasingly important BUT
Mainly subjective, dependent upon
competence (training)
accuracy
consistency

42. 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

43. Are we scoring equally? Grade of fragmentation Blastomere size
Blastomere/fragment

44. Interobserver and intraobserver variation in day 3 embryo grading Baxter AB, Mayer JF, Shipley SK and Catherino WH Fertil Steril 2006; 86:

45. 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

46. 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

47. Statistical Methods – Kappa Statistics
Terminology for the extent of agreement:
kappa excellent
kappa good
kappa moderate
kappa poor
kappa very poor

48. Results, Baxter et al Interobserver variability (median, range)
Kappa ( ) poor
Intraobserver variability (median, range)
Kappa ( ) good

49. Conclusions, Baxter et al
Agreement is too low!
Only use one embryologist for scoring ?
Use consensus scoring from several embryologists ?
Simplify the scoring system ?

50. 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;

51. Mean agreement between Central Embryologists

52. Agreement between Central and Local

53. Consensus document / Embryology Atlas
Approx. 400 slides of oocytes, zygotes, early embryos and blastocysts
Add nomenclature from consensus paper

54. Thank you for your attention!