Embryo freezing involves preserving embryos at sub-zero temperatures, usually before implantation. There are two main methods - controlled rate freezing and vitrification. Controlled rate freezing involves slow cooling and seeding to prevent ice crystal formation, while vitrification solidifies the solution without any ice crystals using high concentrations of cryoprotectants. Both aim to minimize damage to embryos from freezing and thawing. Cryopreservation allows storage of surplus embryos and avoids multiple pregnancies from single retrievals. It also enables disease screening before transfer.

1. SUBMITTED BY:-
ROHIT KUMAR
(V-11-3-43)
EMBRYO FREEZING

2. EMBRYO FREEZING
process of preserving an embryo at sub-zero
temperatures, generally at an embryogenesis stage
corresponding to pre-implantation.

3. BASICS OF EMBRYO
CRYOPRESERVATION
• Use of Cryoprotectant:-
*lower the freezing point and permit embryo freezing.
* no intracellular ice crystals.
• Seeding:- is the process of inducing freezing of extracellular
medium, thereby precluding the potentially damaging effects
of ice crystals formation.
• Slow freezing at a programmed rate:- controls the rate of
freezing.
• Storage of frozen embryos is sustained in liquid nitrogen at
-196°C.
• Thawing

4. cryoprotactantscryoprotactants
Intracellular
(permeating/ invasive)
Intracellular
(permeating/ invasive)
Extracellular (non-
permeating /non-invasive)
Extracellular (non-
permeating /non-invasive)
LOW MOLECULAR WEIGHT
•1,2 propanediol
•Dimethyl sulfoxide DMSO
•ethylene glycol
•glycerol
•methanol
LOW MOLECULAR WEIGHT
•1,2 propanediol
•Dimethyl sulfoxide DMSO
•ethylene glycol
•glycerol
•methanol
LOW MOLECULAR
WEIGHT
•Glucose
•Sucrose
•Trehalose
•Lactose
•Proteins
•Raffinose
•Lipoproteins—egg yolk,
milk, blood serum
HIGH MOLECULAR
WEIGHT
•Polyvinyl alcohol
•Polyvinylpyrrolidone
LOW MOLECULAR
WEIGHT
•Glucose
•Sucrose
•Trehalose
•Lactose
•Proteins
•Raffinose
•Lipoproteins—egg yolk,
milk, blood serum
HIGH MOLECULAR
WEIGHT
•Polyvinyl alcohol
•Polyvinylpyrrolidone
Functions of
cryoprotactants
•Protect cells from ice
crystal damage.
•Reduce amount of ice
formed at any given
temperature as they lower
freezing point.
•Penetrate into cells
and should have low
toxicity.
Functions of
cryoprotactants
•Protect cells from ice
crystal damage.
•Reduce amount of ice
formed at any given
temperature as they lower
freezing point.
•Penetrate into cells
and should have low
toxicity.

5. PRINCIPLE OF CRYOPRESERVATION
freezing rate should be:-
• Slow enough to pass water outside cells.
• Fast enough to prevent increasing salt concentration from
damaging membranes.

6. WHY DO WE CRYOPRESERVE EMBRYOS?
• To avoid multiple pregnancy.
• for storage of good quality surplus
embryos for later use..
• provides chance to detect infectious
disease and genetic abnormalities, by
offering extended time for proper
screening and analysis.

7. Methods of embryo
freezing
Methods of embryo
freezing
Controlled
rate freezing
Controlled
rate freezing Rapid freezingRapid freezing vitrificationvitrification

8. controlled rate freezing
Steps:-
1. collection and Evaluation of embryos
2. Washing Embryos
3. Equilibration
4. Loading Embryos
5. Cooling of embryos
6. Thawing
7. Evaluate embryo and transfer

9. 1.)collection & evaluation of embryos
Embryos from donor
female
(surgically or non-surgically
removed)
Place them into an
isotonic embryo holding
medium (10 minutes)
Place them into an
isotonic embryo holding
medium (10 minutes)
Under low magnification (≤ 10X) in stereomicroscope
Grade the embryos
Grade 1
grade2
Grade 3:-
can be
transferred
fresh only
•Grade 1 and Grade 2 embryos are at compact morula through expanded blastocyst
stages of development and are suitable for cryopreservation.
•Inspect the grade 1 and 2 at magnification ≥ 50X for intact zona pellucida and any
adherent material.
•Segregate or Discard the embryo with a cracked or missing zona pellucida or with a
zona pellucida having adherent material.

10. Wash good quality embryos in Dulbecco phosphate buffered saline+ 4% BSA.
Merit:-
• Washing Reduces Likelihood of Disease Transmission.
Precautions:-
• Keep embryos from each donor female separate.
• Do not wash more than 10 embryos at one time.
• If transmission of viral diseases is of concern:-
1.) wash embryos 2 times in a 0.25% trypsin solution.
2.) Total combined trypsin exposure time should not exceed 90 seconds.
• After 2 wash of trypsin, wash embryos 5 times more in embryo washing medium.
• After the final wash, place embryos into isotonic embryo holding medium.
2.)Washing Embryos

11. For better post thaw survivability embryos
stored :-
• at low temperature
• for few hours in some suitable medium.
Mediums for storage:-
• ( PBS + 0.4 % BSA + 5 % glycerol for 5 min)
5% glycerol- 1 ml PBS serum+ 1 ml glycerol
• ( PBS + 0.4 % BSA + 10 % glycerol for 10-30 minutes)
10% glycerol- 9 ml PBS serum+ 1 ml glycerol
3.)EQUILIBRATION

12. with straw adapter suck 1.3 cm of CPA solution.with straw adapter suck 1.3 cm of CPA solution.
Now aspirate air to create an air bubble (0.15 cm)Now aspirate air to create an air bubble (0.15 cm)
aspirate single embryo in hypertonic CPA solution (0.8 cm)aspirate single embryo in hypertonic CPA solution (0.8 cm)
Again aspirate 0.15 cm air to create another air bubbleAgain aspirate 0.15 cm air to create another air bubble
Aspirate 9.1 cm CPA solution to completely fill the strawAspirate 9.1 cm CPA solution to completely fill the straw
Seal the end with heat or PVCSeal the end with heat or PVC
4.)Loading Embryos
• place straws in freezing machine.
•Purpose of air bubbles are to physically isolate the embryo.
Working length of straw is 11.5 cm capacity (0.25 ml)Working length of straw is 11.5 cm capacity (0.25 ml)
Embryo in CPA solution
Air bubbles
CPA SOLUTION
COTTON PLUG
COTTON PLUG
/HEAT SEALED END

13. • Cool straws to -7°C for 5 minutes.
• cooling rate during this step can be slow or rapid.
Seeding of straws:- supercooling can occurs so seeding is an
important step.
Seeding done by touching the side of the embryo container with
forceps dipped into liquid nitrogen.
• keep straws at -7°C for an additional 10 minutes.
• Be sure that they remain seeded.
• Cool straws from -7°C to -30°C at 0.5°C/minute.
• Temperature reach -30°C, plunge them into liquid nitrogen (within 2
to 3 minutes)for storage.
• The cooling rate should average 0.5°C/minute
5.)Cooling of embryos

14. • hold 0.5-cc straw quietly in the air for exactly 20 seconds.
• followed by 20 seconds in a 37°C water bath;
• 0.25-cc straws should be thawed for 15 seconds in the air plus 15 seconds in
37°C water.
• For removal of cryoprotactant pass embryos from serial dilutions of thawing
media.
The standard method is to dilute in six steps:
• PBS plus 0.4 percent BSA plus 8.3 percent glycerol,
• 6.7 percent,
• 5 percent,
• 3.3 percent,
• 1.7 percent and then
• 0 percent glycerol,
6.)Thawing
6 minutes
/step at
ambient
temperature

15. alternative method for thawing:-
This method have 4 steps of dilution in:-
(1) 6 percent glycerol plus 10 percent sucrose;
(2) 3 percent glycerol plus 10 percent sucrose;
(3) 10 percent sucrose (all in PBS plus 0.4 percent BSA);
(4) PBS plus 0.4 percent BSA with no sucrose or glycerol.
• Instead of 0.4 percent BSA, 10 percent serum can be used in step 4.
• Both procedures lead to similar results, but the four-step method is faster.
Each step 6
minute at normal
temperature in
warm conditions 5
minutes.

16. New method of Thawing straws
step 1:- Take straw out of goblet
and Hold the straw in air for 3-5
second.
step 2:- submerge into a 37°C water bath
for an additional 25-30 seconds.
Step 3:- Remove the straw from water bath and Wipe the straw
carefully.
Step 4:- Cut the sealed end of the straw.

17. load the straw into an embryo transfer device and
transfer as quickly as possible to a synchronous
embryo recipient.(only if ethylene glycol is used
as CPA)
In Step 5 either:-
• Contents of the straw should let freely
flow into the sucrose solution
Dish containing 1 M
sucrose solution.
•Allow embryos to remain in sucrose
solution for 10 minutes
•Transfer to an isotonic embryo
holding medium for 10 minutes
•Load into a new straw and now embryo
ready to transfer into a suitable recipient.

18. • Evaluate embryo and transfer within few minutes of removing the
cryoprotectant.
• Discard degenerate embryos
• If recipients are available, transfer the degenerate embryos anyway (non-
surgically).
7.)Evaluate embryo and transfer
Disadvantages of slow freezing:-
Expensive equipment
Higher incidence of intracellular ice
formation
Advantage:-
Low concentration of cryopropectant, less
toxic

19. RAPID FREEZING
steps:-
Step 1 -- Embryo collection and evaluation.
Step 2 -- Washing of good quality embryos.
Step 3 -- Equilibration
BEST COMBINATION OF CRYOPROTECTANT:-
Total 10 ml of media- .85 g of sucrose +2.34 ml of DMSO and the quantity of
basic media -7.66 ml
Step 4 -- Loading of embryos in mini straw.
Step 5 -- Direct plunging into liquid nitrogen.
Step 6 -- Thawing
THAWING MEDIA
– 0.33 M sucrose + 0.95 M DMSO
– 0.33 M sucrose + 0.47 M DMSO
– 0.33 M sucrose in holding media
– Holding media for 10 minutes

20. vitrification
• Vitrification is the solidification of a solution at low
temperature without ice crystal formation
Liquid Phase Solid phase
Amorphous state/
Vitrified state
/Glassy state
No ice crystals
formation
-Absence of mechanical
injuriy
-osmotic gradient
Dewar container

21. steps of vitrification:-
Step 1 -- Embryo collection
Step 2 -- Washing of good quality embryos
Step 3 -- Equilibration
(First in intracellular vitrification media at room temp for 7-10
min)
(Then in extra cellular vitrification media at 0-4 c for 10-15
seconds)
Step 4 -- Loading of embryos in mini straw
Step 5 -- Direct progressive plunging into liquid nitrogen
Step 6 – Thawing
Mixing of embryos with 1 m sucrose solution and
keep it for 10 min for removal of cryoprotectants.

22. SLOW FREEZING
VERSUS
VITRIFICATION

23. -35°C
-50°C/min
RT
-6°C
LN2
(196°C)
Slow cooling
Programmed Equipment
(Planner)
Equilibration with cryoprotectants
Drop in LN2
Time
Seeding
-2°C/min
- 0.3 °C/min
1-3hr Time
Vitrification
Dewar container
Drop in LN2
5-10min

24. 24
Concentration of
cryoprotectants
Permeable
Low MW
Non Permeable
Low MW
High MW
Vitrification
Ethylene
glycol
DMSO
Erythritol
Intercellular
cryoprotectant
Sucrose
Trehalos
e
Dehydration
Ficoll,
PEG
Extracellular
cryoprotectant
10-20 M
0.5-0.75 M
10 mg/ml
Slow cooling
1,2
propanediol
DMSO
Glycerol
Sucrose
Trehalose
0.2-1 M
1- 1.5 M

25. PROCEDURE PREGNANCY RATES SURVIVAL
VITRIFICATION 45- 50 % 93%
SLOW FREEZING 30-60% 70-90%
FAST FREEZING 45-55% 80-90%
SURVIVAL RATES

26. thanks