Cryopreservation Flashcards
What is GIFT, ZIFT and ET
GIFT:
sperm and egg both placed in tube hoped proximity = fert
ZIFT:
zygote placed back in tube (2 cell)
ET:
embryo transfer
What is the basics of cryopreservation?
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
What temp do ice crystals form and and why do we not want them?
-5 to -15 degrees
they are extremely detrimental in large cells like sperm, eggs and embryos and ince formation grow exponentially
what is super cooling?
term used for cells that do not have ice crystal formation in temps they should exist
What are cryoprotectants and what are the two types?
factors that replace water inside the cells
aims to curb the growth/eliminate crystal formation
two types:
- permeable
- non-permeable
How do non permeable cryoprotectants work and what are some examples?
- do not enter cell
- work through osmotic pressure by moving water out of cell
- control cell dehydration
examples:
- sugars (sucrose)
- proteins (albumin)
What do permeable cryoprotectants work and what are some examples?
- 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)
How has cryopreservation radically changed the way we use and control ART?
- fertility preservation
- cancer/ chemo patients
- single women - donor gametes
- can fly sperm/ eggs around the world
- can allow for better sperm screening
- cycles do not have to linked - Avoid OHSS
- in cycles where egg number are high the risk of OHSS increases
- ET = risky for further OHSS due to pregnancy - Allows family planning
- individuals can plan when they would like to be pregnant
- multiple children can be born from one fresh OPU cycle
What is slow freezing?
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
What cells is slow freezing commonly used for and what are its successful thaw rates?
commonly used on cleavage stage embryos
success rates are variable (about less than 60%)
What are common errors in slow freezing?
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
How are the embryos thawed after slow freezing?
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
What is embryo vitrification and what is the process?
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
- handling media
- equilibrium solution (low conc cryoprotectant)
- vitrification solution (high conc of cryoprotectant)
- embryo is then quickly exposed to LN2 to snap freeze
What can go wrong during vitrification?
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
How does embryo warming occur after vitrification?
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
What are the success rates of embryo warming
90+
- measured by implantation rate and clinical pregnancies
great FET pregnancy rates
How are sperm samples frozen and warmed?
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%
What is the survival rate of oocyte freezing and how can you identify damage to oocyte by this process?
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
Vitrification has been the most effective way to freeze eggs why is this?
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
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?
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)
How is the outcome of oocyte freezing measured?
not just measured by survival but also:
- fert rate
- blastulation rate
- implantation rate
How many frozen oocytes are need to make a baby?
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
Ovarian and testicular tissue can be frozen for 10+ years potential regrafting later on. What type of patients is this mostly used for?
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
gametes/ embryos can be moved in dry transport shippers. What are these containers?
- contains no free LN2
- absorbent panel keeps sample cold up to a week - complies with shipping regs
- tanks have vacuum insulated panels
- charged with LN2 prior to transport
- maintains low temp of -150 to -190 for several days
- usually fitted with temp tracker for piece of mind
How has the transport of embryos and gamets changed donor access internationally?
huge international donor bases (australia is low)
gives many individuals/couples options
can order sperm in mail
