Embryo Evaluation and Grading

Embryo searching

• Filter to rinse with 20 ml fluid using syringe and needle
• Bottom dish (below the filter) use to collect the fluid and for embryo searching

Handling abnormal cervix

• ‘Y’ shaped cervix

Single vaginal opening , however, two opening in the body

Such condition horn flushing and horn insemination advised

• Inverted ‘Y’ Shaped cervix

Two vaginal opening with single uterine opening – Do not hamper flushing or AI

• Double barrel cervix

Two separate vaginal and uterine opening – Separate flushing of both horns to be followed

Damages

• Brown blood clot in the first flush

Indicate trauma to uterine body by AI gun. Brown colour indicate old injury

May also reflect that oocytes were not fertilized

• Disappearance of infuse fluid

Injury by AI gun that do not heal (brown clot)

Fresh rupture by catheter over inflation (red clot)

• Leaky valve (UTJ)

Happens when many follicles remains unovulated (high E2)

More E2, leads to improper closer of UTJ

Fluid leaked from oviduct to abdomen

• Catheter patency

Squeezing the bifurcation area gently 2-3 times

To and fro movement of bubbles at the outflow end of the tubing reflect patency of catheter

Reposition catheter if no patent

• Catheter washing when mucous is trapped in the catheter

While passing through difficult cervix mucous get trapped in the catheter

Mucous is enemy to embryo

Cuff bifurcation area with thumb and four fingers (blocking)

Allow 15 ml fluid to enter the catheter   and Flush out and discard

Selection of recipient for transfer

• Size

Should be with the expected calf size to avoid dystocia  and Heifer should be over 350 kg

• Milk–Should have sound udder
• Prior history – Regular calving
• Lactation

Good body condition lactating animals (major source of recipient)havig  40-50 days post partum

Dry cows are excellent recipient, however avoid problematic animals& already weaned calf

• BCS – Not too fatty , ideally BCS 3 to 3.5
• Mineral deficiency – As per the area deficiency, incorporate area specific minerals
• Low stress

Adequate shelter in cold and heat and Do not allow to go them in mud

Bio security

Should be disease free

No such disease as BVD, Neospora canis, JD, Bovine leukosis

• Vaccination – Against BVD, IBR, Parainfluenza 3, Leptospira

Recipient synchronization

• Natural or induced heat do not have any impact on pregnancy • Fixed time ET (FTET) also work good, however, have low pregnancy rate but yield is more
• Explanation :
• If 100 animals synchronized and observed for heat• 85% will be observed in heat
• 75% will have good CL & will be selected for transfer
• If pregnancy rate is 60% the calf obtained will be 45
• For timed ET
• Out of 100 animals, good CL will be 90%
• If pregnancy rate considered as 55% than calf obtained will be 49.5

Acceptable synchrony of recipient

• Fresh embryo tolerate greater degree of asynchrony than frozen
• 24 h before to 48 h after donor heat , recipients have been used
• Day 7 recipients are always best
• For frozen embryo – 5.5 – 7.0 day heat used
• For fresh emrbyo – 5-8 days heat used
• Grade 1 embryos used for cryopreservation
• Grade 2 & 3 embryos used for transfer  but  Day 8 embryos are to be avoided

Recipient preparation

• Should be full rumen and not fasting
• Recipient in chute
• Epidural -3- 5 ml lignocaine
• Palpate ovary quickly
• Transfer embryos if good CL and normal uterus palpated
• Transfer should be in horn ipsilateral to ovary having CL
• Provide comfortable environment to recipient
• Transport to short distance do not cause any harm
• Site of transfer – Cranial 1/3rd of the horn (deep transfer)

Avoid any trauma during transfer, as it will lead to PG release

• Recipient restraining – Properly squeezed with Minimum movement
• Footing – Firm footing in chute (no metallic floor)
• Climate – Comfortable and cool but Avoid air blown through fan or plenty of air flow

Cooler part of the day should be used for transfer

• Embryo deposition

Time spent in passing the gun through cervix do not effect pregnancy rate

Time spent in passing through horn adversely affect pregnancy rate

Double sheath gun is used for transfer

• Frozen thawed ET

Transfer similar as fresh embryo transfer, however, frozen-thaw are transfer immediately while fresh can be retain for 6-8 h

• Thawing – Water thaw – Air thaw – Combination (water + air)
• Water thaw : straw directly transfer from LN2 into water bath or thawing unit

Chances of zona cracking

• Air thaw: straw after taking out from LN2 , kept in air till completely thawed
• Combination: after taking out from LN2, straw kept in air for 5-6 seconds and than transferred to water bath – Prevent zona cracking

Cracked zona embryo can not be used for export

Some time crack zona is helpful in easy hatching

For cryopreserved embryos, combination therapy is commonly used

• ET gun – IMV 52 cm ET gun with blue sheath
• Success rate

5% difference with fresh embryo (AETS: American Embryo Transfer Association)

Evaluation of embryos

• Morphological evaluation performed for three reasons

To differentiate between embryo & unfertilized ova (UFO)

Determining the developmental stage coinciding with the expected development

To unable technician to have sufficient information on which to base the decision to transfer or cryopreserve

Parameters considered for embryo evaluation

• Embryo size & shape • Presence of extruded / degenerated cells
• Colour characteristics, number and compactness of the cells (blastomeres)
• Integrity of zona pellucida • Presence or absence of vacuoles in cytoplasm of blastomeres

Grading and classification (IETS)

• IETS established in 1974 (Denver, Colorado, USA)
• Two digit coding system to describe embryo & their characteristics – 1st digit represent embryo stage – 2nd digit represent embryo quality – Both digit separated by hyphen ‘ – ’
• Embryo stage code ranges from 1- 9 (unfertilzed to expanded hatched blastocyst)
• Embryo quality code ranges from 1- 4 (excellent /good to dead/degenerated)

Microscope for embryo evaluation

• Good microscope having high resolution to be used
• Steriomicroscope with maximum magnification of 50x should be employed
• Embryos initially searched in 10-15x • For zona & cytoplasmic evaluation – 50x
• Microscope should have long working distance (distance b/w objective to stage)
• Stage plate should be clear (No frosted plate) • No clamp or clip on the stage (free stage)
• Illumination from beneath the stage • Bright field illumination as standard but should also have facility for dark field illumination (helpful when searching through cloudy fluid)

Normal development of embryo (in-vivo)

• Embryo diameter – 150-190 μm • Zona thickness – 12-15 μm
• The size remain unchanged till blastocyst stage.
• Zona function – Provide shape to embryo – Provide receptor for sperm

Block accessory sperm (cortical reaction) – Rupture of zona leads to hatching

Zona is one of the landmark in recognizing the embryos

Stage of embryos encountered on day 7 :

Morula – blastocyst  • Individual cells of embryos called blastomeres

• Morula – Blastomeres appears like cluster of grapes & individual blastomere could not be differentiated
• Compact Morula – Cells differentiated as ‘inside’ & ‘outside’ part of embryos
• Blastocyst

Two types of cells differentiated

Outer ring of trophoblast cells form the outermost layer of placenta

Inner clump of cells called inner cell mass (ICM) form entire foetus as well as most of the layer of Placenta

Posses cavity called blastocoele\

Stages of embryonic development recovered on D-7

• Stage code 1 : 1 cell

Getting a single cell between D 6-8 indicate unfertilized ovum (UFO)

Stage may confused with compact morula – All UFO many not have similar morphology

• Normal UFO

Perfectly spherical zona – Spherical vitelline membrane

Normal granular cytoplasm – Moderate pervitelline space

• Other UFO

Shows signs of degeneration (variable degree)  – Cytoplasmic fragmentation

May give illusion of blastocoele or cell division

Extreme condensation (resemble compact morula)

• Stage code 2: 2-cells to 12-cells

Any embryo representing these cells number between day 6 –8 is not coordinating with expected development and considered dead or degenerated

Developmental delay, not fit for transfer or cryopreservation

• Stage code 3 : Early morula (latin – mulbery)

Minimum 16 cells to considered morula – Some blastomere visible some not

• Stage code 4 : compact morula

Blastomere coalesed to form a compact-light ball of cells-Individual blastomere not visible

Cells can be allocated as ‘inside’ or ‘outside’ part of emrbyos

Occupy 60-70% of perivitelline space.

• Stage code 5 : early blastocyst – Fluid filled cavity appears

Cells start differentiating between zona & blastocoel (but no clear ICM)

No change in thickness of zona – Cells occupy 70-80% of perivitelline space

• Stage code 6 : blastocyst

Cells clearly differentiated – Trophoblast layer, ICM, Blastocoele

Zona thickness unchanged

No perivitelline space (except where cells are partially collapsed)

• Stage code 7: expanded blastocyst

Increase in overall diameter ( 1.2-1.5 times of original 150-190 µm)

Zona pellucida thining 1/3rd of original (12-15 µm)

No perivitelline space– Expanded blastocoele stage embryo frequently appears collapsed

• Stage code 8 : hatched blastocyst

Embryo can be seen undergoing hatching process or hatched

Embryo has blastocel cavity or may be collapsed

Difficult in identification of this stage as may confuse with endometrial debris

• Stage code 9 : expanded hatched blastocyst

Similar to stage 8, however has larger diameter (size) – Difficult to get this stage on D 7

Embryo quality grade

• By visual estimation of embryo morphological characteristics
• Assessment features

Uniformity of blastomeres (size, shape & color)  – Presence of extruded cells

Presence of dead/degenerated cells – Degree of cytoplasmic granulation

Presence of cytoplasmic vacoule – Presence of cytoplasmic fragmentation – Zona integrity

• Code 1 : excellent /good

Symmetrical and spherical embryo mass

Individual blastomere uniform in size, color , density

Developmental stage of embryo coinciding with the expected development age.

85% normal blastomere – Zona spherical and smooth (no concave / flat surface)

• Code 2 : Fair

Moderate irregularities – 50% blastomere normal and intact

Mild unevenness in cytoplasmic pigmentation with in individual blastomeres

• Code 3: Poor

Major irregularities in shape, size & color of individual blastomeres (25% cells intact)

Extreme unevenness of cytoplasmic pigmentation – Presence of vacuoles in the blastomeres

• Code 4: Dead or Degenerated

Extreme dark cytoplasm– If no embryo at this stage of collection reach to morula stage than embryo should be considered as dead/degenerated

Embryo Evaluation

• Recovered embryo transfer from filter to search Petridis
• Embryo searched in flushing / recovered media at 10 -15 x (steriomicroscope )
• Searched embryos transfer to holding media in another Petridis Evaluation for morphology at 50 x
• Commonly recovered embryo stage on day 7 : compact morula, early blstocyst & blastocyst
• Embryo recognized by zona (translucency & refract light)
• Two digit code : first representing stage than quality
• Example : 6 – 1 code means, blastocyst stage –excellent /good quality

Note : Why there is difference in embryo stage and quality : time of ovulation differ

Non-transferable embryos  • Key identification features

Normal embr U

• Degenerated ova appears similar to compact morulla
• Fragmentation: referred as cytoplasmic blebbing is a process during which some of

cytoplasm (which does not contain any chromosome or chromosomal DNA) segregate itself from ovum, resulting in a specimen that appears multicellular

• Can be checked by DNA staining • Resemble 2-4 cells stage embryos
• Getting a 2-4 cells stage embryo at this time period is illogical.

Dead / Degenerated embryos

Any embryo between 2 cell to 12 cells stage collected on day 7 is considered as dead /degenerated. – Indicate slow and retarded development which will not leads to pregnancy

Embryos whose blastomeres are not fused to one another (lack of tight junction formation) is also considered as dead /degenerated

 Transferable embryos

• Embryos despite having normal quality may have many deviations. These may includes

Irregular size of blastomeres – Large/ multiple vacuoles within the cytoplasm of blastomere

Degeneration of one or more blastomere

Extruded blastomeres (some cells not forming tight junction)

Damaged or mishapen zona pellucida

• Extruded cells

Adhered blastomere communicate with one another and participate in development of embryos

Non adhered cells considered as extruded

Size and number of extruded cells directly impact embryo grade and pregnancy rate

Extruded cells do no divide

Extruded cells pressed with zona layer (stage 6-7) difficult to identify

Rolling of embryo assist in identification

• Misshapen or flat zona pellucida

If zona is not spherical than considered as misshapen having flat or concave surface

Such zona interfere with rolling & allow embryos to stick

The defect is sufficient to lower the embryo 1 grade than what actually observed

• Lipid in cytoplasm

Abnormal accumulation of lipid droplet (vacuoles) in the cells is not good

Presence of more than few vacuoles in the cells cytoplasm is grade lower in quality

• Cracked zona pellucida

Cracking usually observed at the time of hatching

Cracking of zona may occur at early stage

Cracked zona embryo can not be washed properly

Embryos not fit for export, however can be frozen well

• Irregular shape of cells comprising the embryos

Commonly observed in embryos of blastocyst stage

Formation of blastocoele cavity leads to irregularity

Collapsing of blastocoele is considered normal physiology.

Not a cause for lowering the grade

• Materials adhering to zona pellucida

Some embryos may have adhered material on the outer surface of zona

• Adhered material may include – Mucus – Individual cells – Clumps of tissue
• Adhered material also reflects signs of endometritis in donor
• Cumulus cells / endometrial cells constitutes the adherent materials (do not separate during transport)
• Adherent materials can be washed successfully or even can be washed with 0.25% solution of trypsin • The quality of embryo is not affected by adherent materials , however, such embryos are not fit for commercial use or export

Challenges to accurate embryo grading & classification

• UFO with compact morula

Good microscope – Higher magnification help in accurate grading

• UFO with blastocyst

Happens when a portion of UFO degenerate and become lighter resembling blastocoel

Check trophoblastic layer surrounding the blastocoele – ICM cells in blastocoele

 All are absent in UFO

Embryo evaluation tips

• Most common disagreement amongst workers are between grades (excellent /good to fair to poor quality) rather than stage
• Rolling of embryo will help in evaluation of zona & embryo make up
• Each embryos carefully examined with zoom in and out way and rolled (to observe all sides as it’s a two dimensional figure)
• Effectiveness of morphological embryo evaluation

Photograph helps as a learning tool, however, actual evaluation needed

Video images further assist in the process

Reports based on video images showed agreement by experienced technician for

• Embryo stage – 89% • Embryo quality – 68.5% (Farin et al., 1995, Theriogenology
• The difference in agreement is because of lack of rolling as can be done in a petridish
• Success to embryo evaluation depends upon

Appropriate training (have seen all stage & grade of embryos)

Ample experience – Proper equipments (good microscope)

• Embryo stage has little influence on pregnancy rate when transfer between compact morulla to expanded blastocyst for in-vivo fresh embryo
• Similarly stage code 4,5 & 6 of in-vivo derived embryo survived freezing – thawing process well , however, grading is important