Multiple Ovulation Embryo Transfer

Multiple Ovulation Embryo Transfer

Sanjay K. Mishra¹ and Atul Saxena²

• 1.PhD. Scholar 2. Professor and Head,

Department of Obstetrics & Gynaecology, College of Veterinary Science & A.H,

U.P. Pt Deen Dayal Upadhyay Pashuchiktsa, Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura (U.P) INDIA

• Commercial advancement in late 1970s

Objectives

– Acceleration and proliferation of genetic material (complete genome male +female, in AI only male)

– Fertilized embryos transported around the world

– Disease control

– Bio security programme

– Genetic salvage of valuable individuals

– Development of new lines / breeds

Donors selection

• Genetic merit
• Reproductive soundness

– Superior performance (individual or herd)

– Good BCS (preferably growing BCS)

– Free from diseases

– 50-60 days post partum

– Per rectal examination : cervix to ovary free with no adhesion

– Patency of cervical canal (esp Boss indicus) – should be patent

– Should have regular cyclicity

• Blood typing of both sires to further confirm offsprings (especially in cases of import of embryos)
• Donor Management\

– Selected from naturally cyclic group or those used for superovulation

– 2-4 donors for optimum efficacy

– 8-10 recipient for each donor

 

Superovulation (bovine)

• Aim: maximize the number of fertilized & transferable embryos
• Wide variation in superovulatory response
• Variability noticed for superovulatory response and embryo quality
• Dairy cows:

– Variability similar to beef

• Variability in ovarian response due to

– Difference in superovulatory treatments

– Gonadotrophin preparation use

– Batch of gonadotrophin

– Dose of gonadotrophin

– Duration and timing of treatments

– Use of additional hormones

• Animals and environmental factors

– Nutritional status

– Reproductive history

– Age

– Season

– Breed

– Effect of repeated stimulation

– Ovarian status at the time of treatment

– Environmental temperature /stresses

Gonadotrophins

• Three preparations are used

– Gonadotrophins (porcine or other animal pituitary) – eCG  – hMG

• Pituitary extracts contain FSH
• Biological half life of FSH in cow is 5 h or less
• Two injections daily (morning & evening) induces superovulatory response
• All injection by intramuscular route for 4-5 days
• Doses

– Crude pituitary : 28-50 mg

– Partially purified (NIH-FSH-PI) : 260-400 mg (folltropin)

– Purified pituitary : 450 ug (porcine pituitary) 

• ECG

– Contain both FSH & LH has half life in cow is 40 h , persist up to 10 days

– Used as single injection I/M – PG after 48 h of eCG

• Long half life resulted in

– Continued ovarian stimulation and Unovulated follicles

– Abnormal endocrine profiles  and  Reduce embryo quality

• Total dose (eCG) : 1500 -3000 IU (2500 IU commonly use)
• Intravenous administration of antibody to eCG 12-18h after onset of oestrus (time of AI) overcome some of the ovulatory problems
• Endocrine studies suggest that eCG treated animals had more frequently abnormal profile of LH and progesterone (reason for low production of transferable embryos)
• eCG produced from same mares had variations (FSH to LH ratio)
• Experimental proof

– Three groups exposed to pure FSH & different LH concentration

– Results for better embryo quality & superovulatory response was with

group having lowest LH

• It was concluded that no additional LH is required for superovulation
• Endogenous LH is sufficient • Exogenous LH is detrimental to superovulation
• What is the best time for superovulation to optimize superovulatory response

– Concept of follicular wave utilized – FSH used before the selection of DF

– Experiment done using recombinant bFSH, treatment starts 1 day before, on the day, 1 or 2 day after wave emergence (based on the fact that endogenous FSH surge starts 1 day before emergence of wave)

– Significantly more follicles were recruited when FSH treatments initiated on the day / day before wave emergence.

• Traditional approach (followed for many years in ET)
• FSH treatment in mid cycle • Treatment of 4 or 5 days (no difference in ovulation)
• FSH used in decreasing or constant dose •

PG added on D 3 or D 4 of FSH treatment

• Mid cycle coincide with the period of second wave emergence
• Drawback :
• Skill person employed for oestrus detection (before FSH treatment and after FSH for insemination)
• All donors to undergo superovulation be in oestrus at the same time (need synchronization)
• Time wasted in initiating the superovulatory treatment (mid cycle waiting period) 

Synchronizing wave emergence

• Use of E2 and progestins (1990s)

– E2 suppresses FSH release & follicle atresia

– After E2 metabolization, FSH surge & new wave emergence occurs

– On an average, wave emergence takes place 4 days after the E2 – progestin treatment

Oestradiol -17 β – 5 mg Or EB (oestradiol benzoate) – 2.5 mg (I/M)+P4 100 or 50 mg (I/M) & Progesterone implant (intra-vaginally) D 0 FSH treatments on D 4 (morning & evening)

PG on D 6 (M & E) – Removal of implant (D 6 evening)

Estrus on D 8 (48 h of PG) – AI (12 & 24 h after estrus)

• Disadvantage – Oestradiol banned in many countries
• Ablation of follicle

– Used to suppress the effect of E2 on FSH and Procedure initiate new wave

– Ultrasound guided oocyte aspiration used for ablation

– 5 mm or more diameter follicle aspirated

• Research further suggest to aspirate two large follicle (instead of all) to initiate wave
• Drawback

– Required USG with pick-up facilities and trained person but Field application not possible

• Gonadotrophin releasing hormone (GnRH)

– Used to induce ovulation of the DF & emergence of new wave 1-2 days later

– Induction of ovulation is < 60% in random stage of estrus cycle

– Lower superovulatory response compare to > follicle ablation

• How to improve ovulatory response

– Pre-synchronization and – Synchronization of ovulation

Progestin insertion + PG administration (D 0) GnRH (7 days later for inducing ovulation) FSH treatment to start 36 h after GnRH

FSH treatment (4 or 5 days, if 5 days, remove progestin device a day later)

Remove progestin device (on D 4 FSH treatment )

• Note : • >95% animals ovulated to first GnRH
• Embryo number & quality were similar to oestradiol response (Reprodu Ferti Dev , 2010)
• Fixed time AI

Oestradiol + Progesteron device

-FSH treatment (D 4) –PG ( M & E, D6)

Removal of progesterone (D 7,morning)

– GnRH or LH (D 8 morning , 24 h after removal)

FTAI (12 h & 24 h later , i.e D 8 evening & D 9 morning)

• Note:
• Changes made by other workers – removal D 7 evening (additional 12 h) & GnRH or LH 24 h later (i.e D 8 evening)

How to avoid multiple use of FSH in superovulation

• Drawback (multiple injection)

– Needs multiple handling of animals

– Painful to animals & some showed variations in preovulatory LH surge concentration

– Non-compliance of FSH schedule happens in certain cases

• Single subcutaneous FSH injection

– Used in higher BCS beef cattle (> 3.0 on 5 scale) – Results were not repeatable in less BCS

– Superovulatory response similar to traditional

• Mixing FSH with slow releasing polymers (hyaluronan)

– Tagged substance biodegradable & non-irritant to body tissue

– 2% hyaluronan solution used with FSH – Single injection FSH with 2% solution produces similar number of ova/embryo as of traditional method

• Drawback

– 2% hyaluronan solution is much viscous and pose difficulties in FSH to dissolve

– Less viscous (diluted) concentration not effective as single injection

– Efficacy with less viscous solution was improved by splitting the dose (75% & 25%)

– All inj. given intramuscularly. Less concentration solution allows easy solubility of FSH

• In HF superovulatory response improved by splitting dose into two (75% & 25%)

– 75% S/C on first day – 25% 48 h (day of PG administration)

Lyophilized FSH dissolved in 10 ml of 1% or 0.5% hyaluronan solution

75% dose on D 1     – 25% dose on D 3 (day of PG administration)

Embryo collection

• First successful non-surgical bovine embryo collection – 1976 by

– Dr Robert Rowe – Dr Peter Elsden– Dr Martin Drost

• Time of Collection

– Calculation based on standing heat of donor

– Collection follows 6.5 day – 7.0 day

– Before or on D 6, embryo posses poor cryotolerance but D 7.5 – 8.0 emrbyos in hatched stage and becomes less useable.

If multiple donors are used, flush the one who comes in heat first.

– Usually variation of 12-24 h in heat observed in multiple donor Superovulation

Collection media

– Commercially ready to use is best (not cost effective)

– Ringer lactate or D-PBS + 0.1% BSA, cheap and good for short term holding of emrbyos

– Embryos meant for export use polyvinyl alcohol (non animal resource)

• Donor preparation

– Anteriorly elevated ramp (30-35 cm) to be placed in the chute (allow access to uterus easily as abdominal viscera shift posteriorly) – Donor should be full stomach (no need for fasting)

– Full stomach push uterus posteriorly, access to uterus easy.

– Empty stomach makes negative abdominal pressure, which causes air to sucked around arms and into the rectum and colon, making handling of uterus difficult

– When donor is in chute , give epidural anaesthesia (3-5 ml with 18 G needle)

– Tranquilization (10 mg xylazine) before epidural can be used for excitable animals

– Avoid using disinfectant at the site of epidural or perineal region cleaning.

– Simply wipe with water or clean towel the desired areas. Donor is ready for collection

Equipments

• Catheter : 52 cm, silicon, French foleys 16 /18G with 5 ml cuffs
• Stylet : 60 cm stainless steel
• ‘Y’ tubing : 150 cm plastic tubing, one end with syring tip and other for filter attachment
• Disposable three way plastic valves • 50-60 ml capacity air tight syringe
• Embryo filter 75µm • Jelly (non-spermicidal)
• Cervical dilator (ET / AI gun will be helpful)
• Flushing medium D-PBS +0.1% BSA • 10 ml syringe to inflate cuffs
• Sterilization of equipments
• Manufacturer supplied materials are gamma radiated sterilized
• Ethylene dioxide is embryotoxic
• Disposable items even can be reused (cost saving) by simply cleaning with lab reagent followed by multiple washing with distilled water 

Embryo collection

• 52 cm silicon, 16/18 G French Foleys catheter is preferred even human Foley’s catheter 45 cm long , 16/18 G has been used
• Silicon catheter preferred as it causing minimum tissue irritation as compared to latex.
• The catheter is made rigid by inserting stylet.
• Stylet tip should be lubricate with jelly, which allow easy withdrawal
• Stylet should always be longer in length compare to catheter (52 cm vs 60 cm)
• After passing the stylet, catheter should be stretched to fit the stylet
• This procedure will prevent stylet to accidently pass into the fluid port
• In difficult cervix (‘s’ or 90º bending), cervical dilators (AI gun /ET gun) is helpful.
• Pass catheter immediately after dilator is removed otherwise cervix will collapse to its original shape • Squeezing cervical ring in front of the catheter will help in passing the catheter through tough cervix
• Opening of all cervical rings are not aligned and as such dilators is helpful

Body flushing

• Uterine body anatomy should be clear • Uterine body space varies 1.25 -5.0 cm
• Heifers body space 1.25 – 1.9 cm, aged animal 2.5 -5.0 cm
• Anterior to cuff, catheter tip length 2.5-3.75 cm
• Inflation of cuff will protrude the tip into the horn
• 2-3 ml inflation – 1.25 cm space occupied in the body
• 6-10 ml inflation – 2.5-3.75 cm space occupied
• Sufficient inflation required to place the catheter
• Inflation of cuff to be made with flushing media (any leakage can be detected at the valve , not so with air)

Placement of catheter into the body

• Placed catheter in horn (5.0 cm deep)
• Inflate with 1-2 ml fluid or till sensation of inflation perceived
• Retract stylet 5-6 cm into the catheter and than retract the catheter until cuff is in the body
• Continue inflating the cuff, tug catheter caudally to check sufficient inflation achieved
• Once inflation is sufficient, remove stylet
• Underinflated cuff pull back into the last cervical ring as the cervix relaxed
• Cervical pressure on the cuff block lumen of the catheter

 Solution

– Pinch behind the cuff in the cervix will squirt it back into the body. If this technique does not work, deflate, remove stylet, take out catheter and repositioned again

• Over inflation : the tip of the catheter will flush one horn
• After right placement of catheter, flush with media (gravity, syringe or pump) •
• Syringe infusion is preferred
• 400 ml media is required to flush both horns (50 ml x 8 = 400 ml)
• Before first flushing, the ‘Y’ tubing should be filled with media to remove trapped air.
• After each infusion, retrieved the maximum amount of infused fluid
• Half of the infused fluid come out by gravity, however, further retrieval required milking of the horns • Horn can be milked by grasping the tip of the horn between second and third finger and thumb used to pull the fingers (worm like movement)
• Gentle tug at the catheter on the body area will help in retrieval of fluid.
• With the last flush , infuse 25 mg PG into the uterine horn
• Before the use of embryo filters, separate evaluation in dish follows, which is time consuming. However, a beginner must use it.
• 400 ml fluid in 8 flush can be examined separately
• If embryos are detected in the lash dish (7-8) it indicates some embryos are left inside and need re-flush
• Also, after flushing the search in different dish, pass the fluid through embryo filter (75 μm ) in order to get any embryo which can not be searched
• Embryos of day 7 were present at the tip (Utero-tubal side) of the horn
• Heifers do not have a dilated apex (no pregnancy settled earlier) hence require more fluid to dilate and thus require more flushing media 

Horn flushing

– Take less time , Less media and Blockage of the oviduct can be detected

• Placement of catheter near 1/3 of apex (deep inside the horn)
• Cuff should not be overinflated (shape should be elongated and not round)
• Over inflation leads to rupture of endometrial wall and fluid leaked into uterine wall and broad ligament (crepitating sound)
• If rupture occurs, reposition the catheter ahead of the rupture site
• If under inflation, the catheter will slip
• Under such condition, re-inflate to go for body flushing

 

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

Normal Embryo	UFO
Vitelline membrane smooth & spherical	Membrane fragmented /degenerated
Cellular cytoplasm	Granular cytoplasm
No perivitelline space	Moderate perivitelline space

 

• 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