Lab Practical Exam

Question 1

Describe how you carry out a wet preparation for semen analysis?

Answer 1

• Keep sperm incubated
• Examine colour, pH, viscosity and volume.
• Take 10uml and examine under a phase contrast microscope
• Count the numbers of motile sperm (200 in 4 fields of vision)
• Examine for the presence of other cells and debris

Question 2

What counts as a type A (Rapidly progressively motile) sperm?

Answer 2

Velocity of more than 25 um/s

Question 3

What counts as a type B (slow progressive cell)

Answer 3

Velocity of 5 - 24 um/s

Question 4

What speed in a class c moving at?

Answer 4

Velocity of less than 5 um/s

Question 5

What is the normal pH of semen?

Answer 5

7.2 - 7.8

Question 6

How many days abstinence do you need to get a good quality sample?

Answer 6

Between 3 and 7

Question 7

If the semen analysis seems abnormal how long should you wait before taking a new one and why?

Answer 7

3 months. This is how long the entire process of spermatogenesis takes. A cold/bing drink 3 months before the sample can affect it.

Question 8

What is the density of a mature, morphologically normal sperm?

Answer 8

1.12 g/ml

Question 9

What is the density of an abnormal sperm?

Answer 9

1.06 - 1.09 g/ml

Question 10

What is the density of the 40% solution used in density graded centrifugation?

Answer 10

1.06g/ml

Question 11

What is the density of the 80% solution used in density graded centrifugation?

Answer 11

1.10g/ml

Question 12

Describe how you carry out a density graded centrifugation sperm preparation

Answer 12

1. Add 1ml 80% solution to the bottom of the test tube
2. Add 1ml 40% solution
3. Add semen on top slowly
4. Centrifuge at 300 xg for 20 mins
5. Aspirate the upper raft (contains the seminal plasma)
6. remove the 40% layer and the lower interface raft whilst leaving most of the lower 80% layer in place.
7. Transfer the pellet (75ul) into a single clean 15ml centrifuge tube and wash with 4ml sperm wash.
8. Centrifuge at 300xg for 5 minutes
9. Aspirate most of the upper supernatant and leave around 1ml to examine and use.

Question 13

Why are normal sperm more dense?

Answer 13

Their DNA is more condensed

Also abnormal cells eg those with round heads are much less dense so don’t make it down to the bottom.

Question 14

List some drawbacks of density graded centrifugation

Answer 14

1. Overcentrifugation can cause a build up of reactive oxygen species.
2. Overloading the gradient can block the interface layer and prevent the passage of functional sperm.
3. Large amount of debris also collected (not the same in swim up where you get a small number of highly motile cells)

Question 15

What are some of the advantages of density graded centrifugation?

Answer 15

1. Suitable for all samples including very viscous samples
2. Get a higher number of cells.
3. Faster than swim up

Question 16

What temperature are sperm cryopreserved at?

Answer 16

• 196 degrees

Question 17

What are the three main risk to cells during cryopreservation?

Answer 17

Mechanical damage from ice crystal formation
Osmotic stress
Cold shock injury

Question 18

Why do sperm freeze better than eggs?

Answer 18

Eggs are larger and are more prone to ice crystal formation

Question 19

Why do sperm cryopreserve relatively well?

Answer 19

1. They are small
2. Large surface area to volume ratio
3. Plasma membrane is highly permeable to water which facilitates dehydration when a cryoprotectant is added.
4. The sperm head is mostly condensed DNA and contains little cytoplasm making them less susceptible to ice crystal formation.

Question 20

What is the cryoprotectant added to sperm and how does it work?

Answer 20

Glycerol (egg yolk or soy have also been used)
Cell permeable so enters the cell and lowers the freezing point of intracellular water to induce greater dehydration at lower temperatures and reduce ice crystal formation.

Question 21

Roughly what % of motile sperm are lost during cryopreservation?

Answer 21

60 - 90%

Question 22

Why, in the clinic re samples stored in vapour rather than liquid nitrogen?

Answer 22

To reduce the risk of infection spread

Question 23

How is sperm prepared following its removal from liquid nitrogen?

Answer 23

Thawed rapidly in a room temperature water bath
Slow dilution with sperm wash
Density graded centrifugation

Question 24

Describe what is done before the catheter is passed through in an embryo transfer procedure

Answer 24

1. Ensure the bladder is appropriately full
2. Optimise the transabdominal scan of the uterine cavity
3. Remove cervical mucus with cotton buds

Question 25

Above what grading is classed as a top quality embryo?

Answer 25

3AA

Question 26

What day is an embryo transferred at?

Answer 26

Day 5 blastocyst stage

Question 27

What do the three figures mean in an embryo quality score?

Answer 27

Number: Expansion grade of the embryos (scored from 1 - 6)
First letter: Inner cell mass grade (scored from A to c)
Second letter: Trophectoderm quality (Scored from A to C)

Question 28

If the length of the cervix is 5cm what do you set the catheter length to?

Answer 28

5cm. You want the outer catheter to sit just at the internal os.

Question 29

Where is the ideal place to put the embryo back?

Answer 29

At the junction between the lower and middle third of the uterine cavity

Question 30

Name two types of catheters and the differences between them?

Answer 30

Cook catheter is the normla catheter and it has a round tip so as not to damage the endometrium.
Wallace catheter is used if the cervix is unable to tolerate this widened tip.

Question 31

How long after ET do you take a pregnancy test?

Answer 31

2 weeks

Question 32

What is the ideal number of eggs to get at an oocyte collection?

Answer 32

8 - 15