EMBRYO QUALITY ASSESSMENT

WHICH ONE TO CHOOSE???

DR RAHUL SEN (Ph.D)

VASUNDHARA HOSPITAL LIMITED
Embryo transfer
 The biggest dilemma:
Selecting the ‘best’ embryo

WHICH ONE TO CHOOSE...??

 Impact of sperm over embryo development

DAY 0 1 2 3 4 5

early development later development

• Oocyte activation factor

• Cytoplasmic Factors
• Organelle Factors
• Nuclear Factors
• Membrane – cytosolic
factors • DNA-fragmentation - integrity

EMBRYONIC DEVELOPMENT IMPLANTATION RATES PREGNANCY RATES MISCARRIAGE

“History” of the embryo:
the quality of the oocyte
FERTILIZATION
EMBRYO ASSESSMENT IN LAB.
 EMBRYO ASSESSMENT
NON-INVASIVE METHODS INVASIVE METHODS
• MORPHOLOGY (BY APPEARENCE) • PRE-IMPLANTATION GENETIC TESTING
• KINETIC (TIMED EVENTS OCCURRED (PGT)
DURING DEVELOPMENT) • OMICS
• TIME LAPSE IMAGING (MORPHO-
KINETIC)
The istanbul consesnsus (2011) workshop came
with an ultimate goal to establish a common
criteria and terminology for grading zygotes,
embryos that would be amenable to routine IVF
laboratory.
 ZYGOTE SCORING
DAY-1 EMBRYO

pn

NPB’s
SCOTT et. al., 2003 Reprod Biomed Online

ISTANBUL CONSENSUS, 2011. Human Reprod.

ZYGOTE SCORING
DAY-1 EMBRYO

ISTANBUL CONSENSUS, 2011. Human Reprod.

 CLEAVAGE STAGE EMBRYO SCORING
• Growth Rate / Cleavage speed / Kinetics
• Degree of Fragmentation
• Additional parameters
Symmetric/ Asymmetric cleavage
Stage specific cleavage pattern
Multinucleation
Vacuolization
Spatial Distribution of cells
Cytoplasmic anomalies

ISTANBUL CONSENSUS, 2011. Human Reprod.

CLEAVAGE STAGE EMBRYO SCORING
Expected stage of Timing (hours post-
Observation development insemination)

Fertilization check Pronuclear stage 17 ± 1 h

26 ± 1 h post-ICSI
Early cleavage check 2-cell stage
28 ± 1 h post-IVF

Day 2 assessment 4-cell stage 44 ± 1 h

Day 3 assessment 8-cell stage or more 68 ± 1 h
Day 4 assessment Morula 92 ± 2 h
Day 5 assessment Blastocyst 116 ± 2 h
(Early/Expanded)

ISTANBUL CONSENSUS, 2011. Human Reprod.

 CLEAVAGE STAGE EMBRYO SCORING
• Fragmentation – A fragment can be defined as anuclear, memberane
bound extracellular cytoplasmic structure.
(Mild <10%) (Moderate10-25%) (Severe >25%)

Symmetric Asymmetric
cleavage Multinucleation vacuolization
cleavage

ISTANBUL CONSENSUS, 2011. Human Reprod.

representation of Stage Specific (green) and Non-Stage Specific (Yellow) embryos at
different stages of development.
Consensus Grading for Cleavage stage embryos

ISTANBUL CONSENSUS,
2011. Human Reprod.
 BLASTOCYST GRADING DAY-5

•An optimal embryo at this stage of development, will be a fully expanded with an
ICM (easily discernible and consisting of many cells) with trophectoderm comprises
Of many cells forming a cohesive epithelium.

•In literature it is agreed that ICM and TE has high prognostic value for implantation
and fetal development.

ISTANBUL CONSENSUS, 2011. Human Reprod.

BLASTOCYST GRADING DAY-5

ISTANBUL CONSENSUS, 2011. Human Reprod.

IS THERE STILL
A PLACE FOR
MORPHOLOGY???
• Morphology has been assisting embryologists for
many years to improvise embryo selection.

• With more detailed information concerning cleavage

patterns, kinetic events in development has the
potential to improve outcomes.
 TIME LAPSE - The new era of embryo selection
• Time lapse provides a continuous monitoring system for embryo
development as a powerful tool in embryo selection.

• Embryo development is a dynamic process that has been documented with

great specificity.

• TLM for embryo selection is one such technique that allows selection & de-
selection of embryos based on various morphokinetic parameters.
 Time-lapse microscopy:
automated image acquisition every 5–20 min

Dynamic morphokinetic Negative signs during

assessment: development

1. Pronuclear dynamics and • Multinucleation

morphology • Micronuclei
2. Duration of first cytokinesis • Fragmentation
3. Reappearance of nuclei after • Blastomere asymmetry
cleavage • Direct cleavage from 1-3 or 2-5 cells
4. Time to various cleavage stages • Reverse cleavage (blastomere fusion)
5. Duration of various cleavage • Absent cleavage
stages • Chaotic cleavage
6. Duration of cleavage cycles • Cell lysis
7. Mitotic synchronicity (Barrie et al. RBMonline April 2017)
8. Time to morula, blastocyst and
hatching
 Time Lapse: Advantages

• Embryo assessment occurs without removing dishes from the

incubator

 Embryos are not exposed to changes in light, humidity,

temperature, pH and gas phase during development

• Less sample handling

• Some TLM systems also have software programmes for

computer-assisted analysis

• Excellent quality control and research tool & also allows off-site
data analysis
 Time Lapse: Potential Concerns
• Safety regarding use in routine laboratory workflow need is concern,

• Conflicting results: no single parameter correlates with clinical outcome

• Cannot distinguish between aneuploid and euploid embryos

– Yang et al., 2014: observed 10 morphokinetic parameters & compared

with CGH screening

– Rienzi et al., 2015: 16 morphokinetic parameters from ICSI to

completed blastulation compared with 24-chromosome PCR screening

 Evidence of potential increase in IR, CPR, LBR is still

conflicting

 Cost Effectiveness
• Correlations between gross morphology and implantation potential can be weak
and inaccurate

• Embryo morphology changes with time

– Time lapse has shown that fragments can be absorbed
• Embryos with fragments do sometimes implant
• Aneuploidy ????

….. bad morphology may mean

“bad quality”

Good morphology does not always mean high

developmental capacity
 Need of the Hour..
• Other criteria must be considered to identify
and select the embryo with highest possible
implantation potential and possibility of live
birth
 Standard embryo evaluations does not reveal embryos with the
wrong number of chromosomes.

• Chromosome errors (aneuploidy) in human embryos are a major

cause of IVF failure, miscarriage, obstetric complications, stillbirth
and can lead to the birth of affected children.

• Accurate technology for detecting chromosomally-normal

(euploid) embryos is now available.

• Ideally, one euploid embryo should be transferred after embryo

biopsy and chromosome screening for aneuploidy.
 Metabolomics
• Metabolites show fluctuations in response to biological
perturbations/ interventions or environmental state of the
cell and have a direct correlation with any abnormal
changes.

• Since Metabolome reflects the genotype, physiology and

environment; Metabolomics offers a unique opportunity to
look at genotype-phenotype as well as genotype-
envirotype relationship.

• Metabolites are ultimate results of cellular pathways;

taking into account changes in genome, transcriptome,
proteome as well as metabolic influences.

• Offers estimation with retention of embryo viability.

SUMMARY
• Number of scoring systems have been proposed, but
lack of standardization makes interpretation of
results difficult.
• Traditional embryo evaluation systems are simple,
non-invasive, cost-effective & mainstay in majority of
IVF laboratories.
• Embryo selection based on combinations of
morphology scores at different stages of embryonic
development may be more effective
• Time lapse would certainly provide a powerful tool to
ascertain both cleavage rates and morphology.
 TAKE HOME MESSAGE
• It is imperative that critical morpho-kinetic parameters are
used to improve outcomes.

• When the volume of fragmentation exceeds 25%,

implantation and pregnancy rates reported to be low.

• Genetic assessment can provide the additional advantage of

selecting a chromosomally normal embryo

• Strong n robust embryo scoring systems selects embryos with

highest implantation potential paves way to single embryo
transfer.

• Follow 2011 Istanbul consensus of zygote, embryo assessment

TAKE HOME MESSAGE