This document discusses various methods for assessing embryo quality and selecting the best embryo for transfer, including traditional morphology assessment, kinetic/time-lapse imaging assessment, pre-implantation genetic testing, and 'omics' techniques. It emphasizes that traditional morphology alone provides limited information and that incorporating multiple parameters like developmental timing, fragmentation levels, and ploidy status can improve embryo selection and lead to higher implantation and pregnancy rates.

1. EMBRYO QUALITY ASSESSMENT
WHICH ONE TO CHOOSE???
DR RAHUL SEN (Ph.D)
VASUNDHARA HOSPITAL LIMITED

2. Embryo transfer

3. The biggest dilemma:
Selecting the ‘best’ embryo
WHICH ONE TO CHOOSE...??

4. DAY 0 1 2 3 4 5
early development later development
• Oocyte activation factor
• Organelle Factors
• Membrane – cytosolic
factors
• Cytoplasmic Factors
• Nuclear Factors
• DNA-fragmentation - integrity
Impact of sperm over embryo development
EMBRYONIC DEVELOPMENT IMPLANTATION RATES PREGNANCY RATES MISCARRIAGE

5. “History” of the embryo:
the quality of the oocyte

6. FERTILIZATION

7. EMBRYO ASSESSMENT IN LAB.

8. EMBRYO ASSESSMENT
NON-INVASIVE METHODS INVASIVE METHODS
• MORPHOLOGY (BY APPEARENCE)
• KINETIC (TIMED EVENTS OCCURRED
DURING DEVELOPMENT)
• TIME LAPSE IMAGING (MORPHO-
KINETIC)
• PRE-IMPLANTATION GENETIC TESTING
(PGT)
• OMICS

9. 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.

10. ZYGOTE SCORING
DAY-1 EMBRYO
SCOTT et. al., 2003 Reprod Biomed Online
ISTANBUL CONSENSUS, 2011. Human Reprod.
pn
NPB’s

11. ZYGOTE SCORING
DAY-1 EMBRYO
ISTANBUL CONSENSUS, 2011. Human Reprod.

12. 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.

13. CLEAVAGE STAGE EMBRYO SCORING
Observation
Expected stage of
development
Timing (hours post-
insemination)
Fertilization check Pronuclear stage 17 ± 1 h
Early cleavage check 2-cell stage
26 ± 1 h post-ICSI
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
(Early/Expanded)
116 ± 2 h
ISTANBUL CONSENSUS, 2011. Human Reprod.

14. CLEAVAGE STAGE EMBRYO SCORING
• Fragmentation – A fragment can be defined as anuclear, memberane
bound extracellular cytoplasmic structure.
(Mild <10%) (Moderate10-25%) (Severe >25%)
Symmetric
cleavage
Asymmetric
cleavage Multinucleation vacuolization
ISTANBUL CONSENSUS, 2011. Human Reprod.

15. representation of Stage Specific (green) and Non-Stage Specific (Yellow) embryos at
different stages of development.

16. Consensus Grading for Cleavage stage embryos
ISTANBUL CONSENSUS,
2011. Human Reprod.

17. 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.

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

19. IS THERE STILL
A PLACE FOR
MORPHOLOGY???

20. • 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.

21. • 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 - The new era of embryo selection

22. Time-lapse microscopy:
automated image acquisition every 5–20 min
Dynamic morphokinetic
assessment:
1. Pronuclear dynamics and
morphology
2. Duration of first cytokinesis
3. Reappearance of nuclei after
cleavage
4. Time to various cleavage stages
5. Duration of various cleavage
stages
6. Duration of cleavage cycles
7. Mitotic synchronicity
8. Time to morula, blastocyst and
hatching
Negative signs during
development
• Multinucleation
• Micronuclei
• Fragmentation
• Blastomere asymmetry
• Direct cleavage from 1-3 or 2-5 cells
• Reverse cleavage (blastomere fusion)
• Absent cleavage
• Chaotic cleavage
• Cell lysis
(Barrie et al. RBMonline April 2017)

23. 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

24. 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

27. • 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

28. 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

29. 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.

31. 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.

32. • 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.
SUMMARY

34. 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

35. TAKE HOME MESSAGE