Emma Lingham Learning outcomes

April 2023

LO2 Evaluate embryo

competency using a variety of

Clinical decision morphological, physiological and

AI selection methods.

making
(embryo grading)

MCE5103 : Theory ART Laboratory Processes (embryology)

What’s this?
Note

The first 14 slides were covered in detail in the

Insemination, fertilisation and early cleavage lecture
They are included here as a reference

This tutorial will start from Day 4 (Slide 15)

 Maturity assessment
Key concepts
▪ Three types of oocyte collected: GV, MI, MII
▪ We expect 80-90% of oocytes to be mature
▪ Only MII oocytes have the capacity to be fertilised

IMMATURE MATURE

Image from: ESHRE Atlas of Embryology

 Maturity assessment
Key concepts
▪ Three types of oocyte collected: GV, MI, MII
▪ We expect 80-90% of oocytes to be mature
▪ Only MII oocytes have the capacity to be fertilised

Image from: ESHRE Atlas of Embryology

 Day 1 Oocyte quality Sperm quality

Oocyte collection Sperm preparation

Fertilisation assessment timing:
17 ± 1 HPI
Creation of human embryos
Insemination Fertilisation Embryo culture Embryo selection

Non-viable embryos Viable embryos

Discard Embryo transfer

Biohazard waste Embryo biopsy

Cryopreservation

“surplus to needs”

Dispose Donate

Allow to succumb For treatment

Biohazard waste ‘Other’ patient request For research

For training / lab QC

Rienzi, L., Ubaldi, F., Iacobelli, M., Romano, S., Minasi, M.G., Ferrero, S., Sapienza, F., Baroni, E. and Greco, E., 2005. Significance of morphological
attributes of the early embryo. Reproductive biomedicine online, 10(5), pp.669-681.
 Day 1 Fertilisation assessment timing: Fertilised oocyte
17 ± 1 HPI
▪ 2PN and 2PB

The ‘strictness’ of fert grading

depends on the clinic’s protocols,
from the presence of 2PN to
example criteria listed below.

Example assessment criteria

- Spherical
- 2 polar bodies
- 2 centrally located, juxtaposed PN
- Even PN with distinct membranes
- Equivalent numbers and size of
nucleoli that are ideally equatorially
aligned at the region of membrane
juxtaposition
- Angle between the PB <50°

Image from: ESHRE Atlas of Embryology

 Day 1 Fertilisation assessment timing: Fertilised oocyte
17 ± 1 HPI
▪ 2PN and 2PB

The ‘strictness’ of fert grading

depends on the clinic’s protocols,
from the presence of 2PN to
example criteria listed below.

Example assessment criteria

- Spherical
- 2 polar bodies
- 2 centrally located, juxtaposed PN
- Even PN with distinct membranes
- Equivalent numbers and size of
nucleoli that are ideally equatorially
aligned at the region of membrane
juxtaposition
- Angle between the PB <50°

Image from: ESHRE Atlas of Embryology

 Day 1 Syngamy assessment timing: Syngamy check
23 ± 1 HPI
▪ 0PN and 2PB
(provided 2PN were observed at 17 HPI)

Most useful as a quality control tool

for the laboratory. Syngamy rates
indicate the quality of the oocytes
coming into the system.

Clinics may elect to do an early

cleavage check instead at 26-28
HPI (looking for 2-cell embryos),

Note- it’s syngamy or early

cleavage, not both

Image from: ESHRE Atlas of Embryology

 Day 2 Day 2 assessment timing: Cleavage grading
44 ± 1 HPI
▪ To identify viable cleavage embryos,
multiple factors must be considered

1. Cell number
Correlation between slow/fast cleavage and
chromosomal abnormalities

2. Even or uneven cells

Uneven cells negatively affects pregnancy
rates. Thought to be due to uneven
distribution of proteins, mRNA,
mitochondria and organelles in the sister
cells.

3. Fragmentation
Fragmentation <20% has not been shown
to affect PR

4. Multinucleation
Associated with very low implantation rate

Image from: ESHRE Atlas of Embryology

 Cleavage grading
Fragmentation
▪ To identify viable cleavage embryos,
Fragmentation multiple factors must be considered
Anuclear, membrane-bound extra cellular cytoplasmic structures
1. Cell number
• Consider % of the space within the zona that are fragments Correlation between slow/fast cleavage and
• Fragments may or may not be re-absorbed (usually lower %) chromosomal abnormalities

2. Even or uneven cells

Uneven cells negatively affects pregnancy
rates. Thought to be due to uneven
distribution of proteins, mRNA,
mitochondria and organelles in the sister
cells.

3. Fragmentation
Fragmentation <20% has not been shown
to affect PR

4. Multinucleation
Associated with very low implantation rate

Image from: ESHRE Atlas of Embryology

 Cleavage grading
Fragmentation
▪ To identify viable cleavage embryos,
Fragmentation multiple factors must be considered
Anuclear, membrane-bound extra cellular cytoplasmic structures
1. Cell number
• Consider % of the space within the zona that are fragments Correlation between slow/fast cleavage and
• Fragments may or may not be re-absorbed (usually lower %) chromosomal abnormalities

2. Even or uneven cells

Uneven cells negatively affects pregnancy
rates. Thought to be due to uneven
distribution of proteins, mRNA,
mitochondria and organelles in the sister
cells.

3. Fragmentation
Fragmentation <20% has not been shown
to affect PR

4. Multinucleation
Associated with very low implantation rate

Image from: ESHRE Atlas of Embryology

 Cleavage grading
Multinucleation
▪ To identify viable cleavage embryos,
Multinucleation multiple factors must be considered
A blastomere containing more than one nucleus
1. Cell number
• Correlations with culture media, improper temperature control Correlation between slow/fast cleavage and
(particularly during OPU) or inherent uncontrolled cell cycle chromosomal abnormalities
• Highly correlated with chromosomal abnormalities
• Can make very nice blastocysts! 2. Even or uneven cells
Uneven cells negatively affects pregnancy
rates. Thought to be due to uneven
distribution of proteins, mRNA,
mitochondria and organelles in the sister
cells.

3. Fragmentation
Fragmentation <20% has not been shown
to affect PR

4. Multinucleation
Associated with very low implantation rate
https://fertilitysolutions.com.au/multinucleated-embryos/
 Day 3 Cleavage grading
▪ To identify viable cleavage embryos,
multiple factors must be considered

1. Cell number
Correlation between slow/fast cleavage and
chromosomal abnormalities

2. Even or uneven cells

Uneven cells negatively affects pregnancy
rates. Thought to be due to uneven
distribution of proteins, mRNA,
mitochondria and organelles in the sister
cells.

3. Fragmentation
Fragmentation <20% has not been shown
to affect PR

4. Multinucleation
Associated with very low implantation rate

Rienzi, L., Ubaldi, F., Iacobelli, M., Romano, S., Minasi, M.G., Ferrero, S., Sapienza, F., Baroni, E. and Greco, E., 2005. Significance of morphological attributes of the early
embryo. Reproductive biomedicine online, 10(5), pp.669-681.
Day 4 Day 4 assessment timing: Morula grading
92 ± 2 HPI
▪ Morula (Latin: mulberry)

EGA → gene expression of adhesion

proteins changes → cell junctions change
to permit cell-to-cell adherence

1. Indistinguishable mass
In a fully formed morula individual cells can
not be seen or counted (although there
would be between 16-32 cells). Compaction
should begin at the 8-cell stage.

2. ‘All in’
All cells should be included in the morula
and involved in compaction

3. Delayed compaction/exclusions
Associated with lower implantation rates
Day 4 Day 4 assessment timing: Morula grading
92 ± 2 HPI
▪ Morula (Latin: mulberry)

EGA → gene expression of adhesion

proteins changes → cell junctions change
to permit cell-to-cell adherence

1. Indistinguishable mass
In a fully formed morula individual cells can
not be seen or counted (although there
would be between 16-32 cells). Compaction
should begin at the 8-cell stage.

2. ‘All in’
All cells should be included in the morula
and involved in compaction

3. Delayed compaction/exclusions
Associated with lower implantation rates
Day 5 Day 5 assessment timing: Blastocyst transition
116 ± 2 HPI
▪ Blastocyst (Latin origin: cyst)

Compacted cells experience pressure

differences from other cell contact, i.e a cell
can tell if full surrounded or on the edge.
For the first time, the embryo can detect
‘outside’ and ‘inside’
Day 5 Day 5 assessment timing: Blastocyst transition
116 ± 2 HPI
▪ Blastocyst (Latin origin: cyst)

Compacted cells experience pressure

differences from other cell contact, i.e a cell
can tell if full surrounded or on the edge.
For the first time, the embryo can detect
‘outside’ and ‘inside’

1. Cavity formation
Fluid is transported by the outer cells into
the embryo.

‘Cavitating morula’ can be used to

describe these embryos (the point
where cell differentiation has not
started, but a cavity can be seen)
Day 5 Day 5 assessment timing: Blastocyst transition
116 ± 2 HPI
▪ Blastocyst (Latin origin: cyst)

Compacted cells experience pressure

differences from other cell contact, i.e a cell
can tell if full surrounded or on the edge.
For the first time, the embryo can detect
‘outside’ and ‘inside’

1. Cavity formation
Fluid is transported by the outer cells into
the embryo.

‘Cavitating morula’ can be used to

describe these embryos (the point
where cell differentiation has not
started, but a cavity can be seen)

‘Blastocoel’ is the fluid filled cavity

of the blastocyst
Day 5 Day 5 assessment timing: Blastocyst grading
116 ± 2 HPI
Dynamic process where the embryo,
• Increases cell number
• Volume of the cavity increases
• Zona thins

1. Degree of expansion

1 Blastocoel is <50% of the

embryo’s volume
2 Blastocoel is >50%
3 Full blastocoel cavity
4 Blastocoel is greater than the
original volume of the embryo
5 TE is herniating out of zona
6 Blastocyst entirely out of zona
Day 5 Day 5 assessment timing: Blastocyst grading
116 ± 2 HPI
After an expansion level of 3, it is
possible to easily distinguish and
grade the two cell populations

We are looking for the

cohesiveness of these cell
populations

2. ICM morphology

A Clear ICM, tight compaction

B Loose ICM, some compaction
C Few cells and loose
D (extra) absent/deg
Day 5 Day 5 assessment timing: Blastocyst grading
116 ± 2 HPI
After an expansion level of 3, it is
possible to easily distinguish and
grade the two cell populations

We are looking for the

cohesiveness of these cell
populations

3. TE morphology

A Cohesive barrier “cobblestones”

B Loose barrier
C Few/large cells, poor barrier
D (extra) absent/deg
Focus on expansion
Focus on expansion
3AA
Focus on expansion
4AA 3AA
 Focus on ICMs
A A A

Grading and Image from: ESHRE Atlas of Embryology

 Focus on ICMs
B A C

Grading and Image from: ESHRE Atlas of Embryology

 Focus on ICMs
B D C

Grading and Image from: ESHRE Atlas of Embryology

 Focus on TE
A B C

Grading and Image from: ESHRE Atlas of Embryology

 Focus on Hatching
5BB 5AA

Grading and Image from: ESHRE Atlas of Embryology

 Focus on Hatching
5AA 6AA

Grading and Image from: ESHRE Atlas of Embryology

 Collapsing; need to be familiar with vit/warming

Image from: ESHRE Atlas of Embryology

Collapsed blast: https://es-la.facebook.com/www.ivfexplained.org/posts/post-thaw-embryotoday-we-have-a-beautiful-and-enormous-collapsed-blast-post-thaw/1230134867130653/
Tutorial activity
Static vs TL embryo assessments
You work in a clinic that does single blastocyst transfer. ASSESSMENT TIMES
Fert check 17 HPI
Task 1: Use the static images provided to grade embryos 1-3
Syngamy check 23 HPI
Task 2: Use the timelapse videos provided on Moodle to grade
embryos 4-5
Day 2 44 HPI
Day 3 68 HPI
The clinic has a strict protocol that blastocysts suitable for vitrification must
be grad A/B only (eg 4AC 3BC, 5CA would not be suitable) Day 4 92 HPI
Task 3: Rank the 5 embryos Day 5 112 HPI

Indicate which embryo you would ET (#1) and which embryos you would vitrify
in the ‘Outcome’ column.
Tutorial activity
Discuss the pros and cons of static vs TL grading

STATIC ASSESSMENT TIMELAPSE

PROS

CONS