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Embryo freezing
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.
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Embryo freezing
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- 2. EMBRYO FREEZING process ofpreserving 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 freezingrate should be:- • Slow enough to pass water outside cells. • Fast enough to prevent increasing salt concentration from damaging membranes.
- 6. WHY DO WECRYOPRESERVE 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 Methodsof 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 & evaluationof 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 qualityembryos 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 postthaw 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 adaptersuck 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 strawsto -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-ccstraw 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 forthawing:- 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 ofThawing 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 strawinto 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 embryoand 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 isthe 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:- Step1 -- 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.
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- 23. -35°C -50°C/min RT -6°C LN2 (196°C) Slow cooling Programmed Equipment (Planner) Equilibrationwith 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 NonPermeable 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 RATESSURVIVAL VITRIFICATION 45- 50 % 93% SLOW FREEZING 30-60% 70-90% FAST FREEZING 45-55% 80-90% SURVIVAL RATES
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Editor's Notes
- #24 One of the main difference concerns the concentartion of CP that is very high in the vitrificcation step as compared to the slow freezing procedure.