Lab Practical: Cryopreservation

Question 1

At what temperature are cryopresered gametes and embryos kept

Answer 1

-196 degrees Celsius

Question 2

what is the purpose of cryopreservation

Answer 2

to maintain the viability of sperm, oocytes and embryos for manyyears

Question 3

when was the first pregnancy resulting from cryopreserved sperm

Answer 3

1953

Question 4

What are the main risks of cryopreservation to gametes/embryos

Answer 4

Internal ice crystal formation

Osmotic stress

Question 5

Which cells are more likely to experience ice crystal formation and why

Answer 5

Oocytes as they are larger cells

Question 6

What is used to help prevent the sperm being compromised during freezing/thawing

Answer 6

cryoprotectant agents

- evidence suggests that this helps to enhance sprerm cryosurvival rate

Question 7

What are the main uses of cryopreserved sperm

Answer 7

Sperm donation for IVF treatment

Fertility preservation e.g. before cancer treatment

Question 8

Describe how you would carry out the freeze process in the lab

Answer 8

1. Measure the volume of the liquefied sample
2. Make a wet-preparation of the liquefied semen sample to estimate the concentration and motility
3. Slowly add the Sperm Freezing solution (Origio; add 1:1 ratio; pre-warm the freeze solution at room temperature for at least 15 mins prior to starting) to the liquefied semen. This should take a minimum of 5 minutes. Mix the sample by swirling the pot in hand through-out the procedure
4. Label a cryovial and add semen to a maximum volume of 1 mL and put into the liquid nitrogen tank immediately

Question 9

Describe how you would carry out the thaw process in the lab

Answer 9

1. Take the cryovial out of the liquid nitrogen and immediately put in the 30 °C water bath for 2 minutes
2. Over a minimum period of 5 minutes, add 4 mL of sperm wash solution to the semen in a 50 mL Falcon tube. Shake the sample through-out
3. Perform density gradient centrifugation (DGC) using to separate gradients (2.5 mL thawed sample on each). Centrifuge 0.3 RCF for 20 minutes
4. Pool 80% pellets into 4 mL sperm wash and centrifuge for 5 minutes at 0.3 RCF
5. Aspirate the supernatant leaving 0.5 mL of sperm wash
6. Resuspend the pellet

Question 10

What are the two main methods of sperm cryopreservation and what is typically used now as it is shown to give superior results

Answer 10

slow programmable freezing

vitrification (often preferred these days)

Question 11

Why does ice formation occur during cryopreservation

Answer 11

When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing
Some cells can tolerate extra cellular ice formation but any intracellular ice is usually fatal

Question 12

how can osmotic stress cause injury to the sperm during freezing

Answer 12

Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The associated stresses on the cell can cause damage directly