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!