Cryopreservation

Question 1

What is GIFT, ZIFT and ET

Answer 1

GIFT:
sperm and egg both placed in tube hoped proximity = fert

ZIFT:
zygote placed back in tube (2 cell)

ET:
embryo transfer

Question 2

What is the basics of cryopreservation?

Answer 2

cooling to sub zero temperatures

removed intracellular water and replaces with cryoprotectants to stop cells being damaged/killed with ice crystals

allows for storage at -196 degrees in liquid nitrogen (LN2) tank

does not age or decay cells

Question 3

What temp do ice crystals form and and why do we not want them?

Answer 3

-5 to -15 degrees

they are extremely detrimental in large cells like sperm, eggs and embryos and ince formation grow exponentially

Question 4

what is super cooling?

Answer 4

term used for cells that do not have ice crystal formation in temps they should exist

Question 5

What are cryoprotectants and what are the two types?

Answer 5

factors that replace water inside the cells

aims to curb the growth/eliminate crystal formation

two types:
- permeable
- non-permeable

Question 6

How do non permeable cryoprotectants work and what are some examples?

Answer 6

• do not enter cell
• work through osmotic pressure by moving water out of cell
• control cell dehydration

examples:
- sugars (sucrose)
- proteins (albumin)

Question 7

What do permeable cryoprotectants work and what are some examples?

Answer 7

• enters through the cells membrane to replace water
• stablises intracellular proteins
• reduce the temperature at which ice crystals form at

examples:
- dimethyl sulfoxide (DMSO)
- ethylene glycol (EG)
- glycerol
- propanediol (PROH)

Question 8

How has cryopreservation radically changed the way we use and control ART?

Answer 8

1. fertility preservation
   - cancer/ chemo patients
   - single women
2. donor gametes
   - can fly sperm/ eggs around the world
   - can allow for better sperm screening
   - cycles do not have to linked
3. Avoid OHSS
   - in cycles where egg number are high the risk of OHSS increases
   - ET = risky for further OHSS due to pregnancy
4. Allows family planning
   - individuals can plan when they would like to be pregnant
   - multiple children can be born from one fresh OPU cycle

Question 9

What is slow freezing?

Answer 9

slow cooling machine gently drops temp between -40 to -70
- drops temp at less than 1 degree per minute

slowly cools the embryo wile cryoprotectants draw out the water dehydrating the cell
- relies on drawing water out so only small (non-lethal) crystals can form

Uses low amounts of cryoprotectants (10%)
- less toxic for the cells

Requires specialised equipment (machine) and LN2

Question 10

What cells is slow freezing commonly used for and what are its successful thaw rates?

Answer 10

commonly used on cleavage stage embryos

success rates are variable (about less than 60%)

Question 11

What are common errors in slow freezing?

Answer 11

cell can pop when being thawed
- occurs from cell lysis
- when water rapidly enters the cell the membrane become damaged and can pop
seen under microscope by dark blobs with non membrane

takes out human errors but can have machine error

Question 12

How are the embryos thawed after slow freezing?

Answer 12

slowly warmed
- gentle reintroduction of H20 ad removal of cryoprotectant

steps:
1. cut straw and drian liquid into dish
2. search for embryo
3. move embryo through thawing protocol
4. culture overnight to assess survival and resumption of development

Question 13

What is embryo vitrification and what is the process?

Answer 13

rapidly cooling cells using 30-40% cryoprotectant (high toxic levels but occurs very quickly)
- aims to achieve glass like state (no ice crystals)

cools at 25000 degrees per minute

1. handling media
2. equilibrium solution (low conc cryoprotectant)
3. vitrification solution (high conc of cryoprotectant)
4. embryo is then quickly exposed to LN2 to snap freeze

Question 14

What can go wrong during vitrification?

Answer 14

embryo collapsing in ES is essential for freezing process

minimal media on device is best
- more media = more crystals

exposure timing to cryoprotectant is critical

touching the frozen block as quickly as possible is key
- too slow and embryo does not snap freeze

avoid taking freezing device too far out of tank to avoid warming

Question 15

How does embryo warming occur after vitrification?

Answer 15

rapidly warm the embryos
- completed on heat stage
- draws out cryoprotectant and H20 is added
1. high sucrose solution
2. lower sucrose solution
3. handling media

Question 16

What are the success rates of embryo warming

Answer 16

90+
- measured by implantation rate and clinical pregnancies

great FET pregnancy rates

Question 17

How are sperm samples frozen and warmed?

Answer 17

ejaculate sperm samples are robust and easy to freeze
- add cryoprotectant slowly into raw ejaculate before loading into straws/vials
- frozen using LN2

warmed by:
- removing from LN2 and leaving to thaw on bench

Not all sperm will survive the freeze approximately 50%

Question 18

What is the survival rate of oocyte freezing and how can you identify damage to oocyte by this process?

Answer 18

survival rates:
- approx 85% vitrified
- 57% slow frozen

easy to tell if there is external damage to oocyte

difficult to tell if there is internal damage:
- zona hardening
- spindle effects
- organelle damage
- cell membrane damage

Question 18

Vitrification has been the most effective way to freeze eggs why is this?

Answer 18

oocytes are very sensitive to freeze/thaw damage
- cooling rate and speed of freezing
- dehydration (they form ice crystals faster)
- surface/volume ratio of egg
- use of cryoprotectants

several eggs can also fit on one vitrification device optimising storage

Question 18

Cryopreservation is great for fertility preservation of sperm as they can be frozen in small amounts for future use. How can testicular biopsy samples be obtained?

Answer 18

TESA
- sperm taken directly from testis

TESE
- tissue sample taken from testis

PESA
- sperm taken from epididymis

Post vasectomy samples

Post chemo samples (when count is low)

Question 19

How is the outcome of oocyte freezing measured?

Answer 19

not just measured by survival but also:
- fert rate
- blastulation rate
- implantation rate

Question 20

How many frozen oocytes are need to make a baby?

Answer 20

most clinic say at least 20 (VARTA says at least 30)
- however depends on many factors (AGE is a big one)

average number of eggs collected at OPU is 8

Question 21

Ovarian and testicular tissue can be frozen for 10+ years potential regrafting later on. What type of patients is this mostly used for?

Answer 21

done in cancer patients where:
- egg collection not possible
- chemo will eradicate egg reserve/ spermatogenesis
- prepubescent patients

low return rate for regrafting (MIVF) due to short life span of patients

low occurrence of cancer following grafting

Question 22

gametes/ embryos can be moved in dry transport shippers. What are these containers?

Answer 22

1. contains no free LN2
   - absorbent panel keeps sample cold up to a week
2. complies with shipping regs
3. tanks have vacuum insulated panels
4. charged with LN2 prior to transport
5. maintains low temp of -150 to -190 for several days
6. usually fitted with temp tracker for piece of mind

Question 23

How has the transport of embryos and gamets changed donor access internationally?

Answer 23

huge international donor bases (australia is low)

gives many individuals/couples options

can order sperm in mail