Cryobiology of Gametes and Embryos

Question 1

What are the 3 non-physiological conditions required of cells during cryopreservation?

Answer 1

1) Exposure to multi-molar concentrations of cryoprotective agents
2) cooling to subzero temperatures
3) removal of almost all cell water/conversion into solid state

Question 2

What factors characterize CPAs

Answer 2

1) low molecular weight
2) high solubility in water
3) cell permeability properties
4) non-toxic at high intracellular concentrations

Question 3

what is the relationship between CPA and freezing point?

Answer 3

CPA lowers freezing point of solutions which allows time for the cell to dehydrate during cooling

Question 4

Why are spermatozoa ideal cells to cryopreserve?

Answer 4

1) small volume and large surface area
2) little cytoplasm
3) contain less intracellular water
4) exist individually for efficient dehydration

Question 5

Why are oocytes difficult to cryopreserve?

Answer 5

1) lot of cytoplasm
2) smaller surface area to volume ratio than sperm
3) complex cytoskeleton, localization of organelles
4) they are more fragile

Question 6

What are the consequences of freezing sperm without CPA?

Answer 6

1. plasma membrane swelling as water expands
2. acrosomal leakage/ breakdown

Question 7

What are the functions of glycerol as a CPA?

Answer 7

1. Remove/reduce water content
2. Minizmize intracellular ice formation

Question 8

What happens to sperm after addition of glycerol?

Answer 8

1) sperm shrink initially due to increased extracellular osmolarity
2) osmotic equilibrium is reached as CPA penetrates teh cell and displaces water
3) sperm returns to almost original volume

Question 9

What is supercooling during sperm freezing?

Answer 9

After adding CPA, temperatures are reduced. When temperature reaches -5 to -15, extracellular ice formation occurs. Forms extracellular solid phase.

Inside of sperm is unfrozen but supercooled
supercooled intracellular watrer has higher chemical potential and diffuses out of the cell osmotically

freezing continues extracellularly resulting in hypertonicity and further dehydration

Question 10

Can tissues frozen slowly be warmed quickly and vice versa?

Answer 10

No. Cells frozen slowly should be warmed slowly. Cells frozen rapidly should be warmed rapidly.

Question 11

What concentration of glycerol is used for human spermatazoa?

Answer 11

5-10% v/v

Question 12

What is the function of a sperm extender in slow freezing solutions?

Answer 12

liquid diluent added to semen. buffer that protects sperm from their own toxic byproducts
protects from cold shock and osmotic shock during cryopreservation

Question 13

What are example buffer medium extenders in sperm cryopreservation?

Answer 13

Zwitterions (TES and TRIS) bind free hydrogen and hydroxyl ions in sorrounding medium to aid dehydration

sodium citrate

Egg yolk maintains viability and improves membrane fluidity

Question 14

What is TEST yolk bufer?

Answer 14

Commonly used for sperm cryo

contains 12% glycerol

1:1 v/v dilution

final glycerol 6%

Question 15

Why is vitrification not typically used for sperm?

Answer 15

1. Shows no superiority over conventional methods with normal sperm
2. Sensitive to high concentrations of CPAs (sucrose causes osmotic shock)
3. Seminal plasma can be protective (zinc and antioxidants) in conventional freezing but not vit

Question 16

What is the relationship between embryonic stage and prgenancy rates after cryopreservation?

Blastocyst vs cleavage stage rates

Answer 16

blastocyst stage associated with higher pregnancy rate per embryo transferred than cleavage stage embryos

similar to transfer with fresh embryos

Question 17

Name 4 common CPAs used in embryo cryopreservation

Answer 17

1) DMSO
2) glycerol
3) ethylene
4) propylene glycol

All of these have low molecular weights, high solubility in water, permeate embryos quickly and minimal/no toxicity to embryos even at high concentrations

Question 18

What are the most common CPAs for cleavage stage and blastocysts?

Answer 18

1.5M propylene glycol with 0.2M sucrose for cleavage stage

10% Glycerol with 0.2M sucrose for blastocyst

Question 19

How do CPAs affect the freezing point of solutions?

Answer 19

Low molecular weights and high solubility of CPAs mean they depress freezing point of solutions to temperatures of -40 to -50 or lower

During slow cooling, there is liquied present in the solution even at low subzero temperatures allowing time for embryo to dehydrate during cooling

Question 20

What is the impact to embryos of long exposure ( >15 minutes) to low toxicity CPAs?

Answer 20

CPAs are generally low toxicity even at multimolar concentrations. However expsoure >15 minutes at >20 degrees C will cause embryos to metabolize in the presence of non-physiological concentrations of cryoprotectants

evidence of embryo damage that may occur during continued develpment inot he presence of these CPAs

Question 21

What are the effects of adding CPA gradually on embryo survival versus radidly diluting out cryoprotectant after freezing when thawing?

Answer 21

Gradual versus abrupt exposure to CPAs is similar

In contrast, rapid dilution out after freeezing can cause osmotic shock

Question 22

What factors affect cryo-injury during slow freezing?

Answer 22

1) CPA used
2) temperature during dilution
3) rapidity of dilution

Question 23

What is osmotic shock and how does it occur?

Answer 23

When intracellular solution has higher osmotic pressure than extracellular solution water enters more rapidly than intracellular solute such as glycerol or propylene glycol can leave.

Depending on relative permeabilities of cell to water and to solute, cell volume may increase and lead to lysis/cell bursts.

Most embryos are relatively impermeable to glycerol, which makes thjem sensitive to osmotic shock when they are direclty diluted out of a glycerol solution into isotonic saline

Embryos are more permeable to propylene glycol than to glycerol

Question 24

What is the relationship between membrane permeability and osmotic shock?

Answer 24

Embryos are less sensitive to osmotic shock when they are more permeable to a CPA

Embryos are more permeable to propylene glycol than glycerol

This is why impermeable solutes such as sucrose are used during dilution of embryos after cryopreservation

Question 25

Why is sucrose commonly used during thawing?

Answer 25

Sucrose is impermeable. It reduces susceptibility of embryo to osmotic shock

Question 26

What is seeding during slow freezing?

Answer 26

Method by which solution that has been cooled below freezing point is induced to undergo crysstallization under controlled conditions

Aqueous solutions spontaneously freeze when colled below 0. The temperature at which this occurs is random and varies from -3 to -20 etc.

seeding produces a phase change of the aqueous solution that causes an increase in solute concentration. embryos then lose water.

small differences between seeding temperatures (-4 to -10) have little effect on embryo survival if sufficeint time after seeding is allowed for embryos to undergo osmotic contraction

spontaneous crystallization depends on the sample volume

embryos frozen in volumes of <0.1 will tend to supercool so deliberate seeding must be used. and in >0.5 will not supercool very much. in high volumes intentional crystallization may not be required since it will occur spontaneously at high subzero temperatures

SEEDING IS REQUIRED FOR HIGH SURVIVAL OF EMBRYOS THAT ARE COOLED AT LOW RATES

Question 27

During slow cooling at what temperature can the embryos be plunged?

Answer 27

Slow cooling can be terminated over broad subzero temperature range of about -25 to -40 with same results

Question 28

What are the most commonly used cryoprotectants in vitrifying solution?

Answer 28

• Ethylene glycol and dimethylsulfoxide (DMSO) at high concentrations
• Become viscous and form solid glass-like state without formation of ice crystals

Question 29

List the important factors that influence permeability and toxicity of cryoprotectant and results of freezing

Answer 29

1. volume of sample/concentration of cryoprotectants
2. technique by which CPA is added
3. time and temperature of equilibration
4. cooling rate
5. changes in volume

Question 30

Describe the two steps of CPA introduction and their concentrations during vitrification

Answer 30

1. Equilibration in relatively low concentrations of CPAs (7.5% DMSO and 7.5% Ethylene glycol) for 7-15 minutes
2. Short 30-90 second exposure to vitrification solution containing higher CPA concentrations (15% DMSO and 15% EG) as well as a dehydrating agent (sucrose/tetralose)

Done at room temperature or 37 degrees.

Cell size and water content influence rate of dehydration and dictate minimum CPA exposure time to achieve maximal CPA equilibration

Question 31

What is the glass transition temperature of water?

Answer 31

about -130 degrees celsius

Question 32

What is the minimum temperature that embryos must be stored at after vitrification

Answer 32

below -130 (glass transition temperature)

Question 33

What are the optimum warming rates for embryos that are slow frozen?

Answer 33

Depends on cooling rate
1. Embryos cooled slowly to high subzero temperatures of about -30 to -40 before being rapidly cooled to -196 require moderately rapid warming of about 200 to 350 degrees/minute for maximum survival
2. Embryos cooled slowly to -60 or below require rather slow warming of 25 degrees/minute or less

Slow frozen embryos are usually held in iar for 10 to 15 seconds etc before being placed in warm water bath.

Question 34

What is the relationship between sample volume and container on warming rates?

Answer 34

Sample volume and container influence warming rate

2. 1. Embryo frozen in volume of 0.5ml in glass ampoule will warm at 350 degrees/minute in warm water bath
3. embryo frozen in volume of 0.05ml in plastic straw will warm at 2,500 degrees/minute in water bath

Slow frozen embryos are usually held in iar for 10 to 15 seconds etc before being placed in warm water bath because of the fast warming rate if submerged right away

Question 35

What is the purpose of sucrose during thawing?

Answer 35

Functions as osmotic buffer since it is non-permeating
lessens the chance of osmotic shock when diluting out cryoprotectants

Question 36

Why is serum protein supplement increased to 20% in post-thaw culture?

Answer 36

1. provides enhanced membrane-stabilizing effects that improve cell survival

Note: higher serum content will cause a small decrease in pH of medium and alter medium substrate concentrations
these can be toelrated for 2-3 hours but if longer culture is required, the oocytes or embryos must be moved to medium containing 10%v/v protein