Diagnostic Assessment of Sperm Parameters

Question 1

What does semen analysis achieve and what does it include ?

Answer 1

Analysis of seminal fluid and sperm parameters as an indicator of male fertility potential

Usually done as step 1 in male fertility investigation.

Couple trying to concieve for a long time with no success

Gold standard for male fertility test

Question 2

How is semen analysis carried out ?

Answer 2

1.Manual
2.Computer-assisted semen analysis
(CASA)

Question 3

How does CASA work?

Answer 3

It used light microscopy.

Press it the heater stage is ejected and insert slide/slide chamber and this will go under microscope objective.

Question 4

What is assessed in the appearance/liquefaction of sperm?

Answer 4

The normal appearance of sperm is grey-opalescent appearance

Normal liquefaction should take place within 20-320 minutes post-production

An abnormally long liquefaction time may indicate infection

e.g bacterial prostatitis

Accessory glands e.g.prostate become infected,secretions will become altered

Question 5

How can we measure the sperm volume?

Answer 5

Direct volume measurement
(Volume ml measured directly using a serological pipette)

Volume from weight
(Weighing sample pots before and after sample production)
Difference=Sample volume

Studies on human semen have shown weight to be an accurate index of volume

Question 6

How can we calculate the sperm concentration in the laborator?

Answer 6

This can be done using haemocytometers which contain two counting chambers.
These contain microscopic grids.
The sperm are immobilised using 3% saline which will kill them and preserve them.

The sample is diluted by a factor of 400.

Mixed to evenly distribute the sperm and then added to haemocytometer.

The sample is the settled and this is added to a moist chamber.

Question 7

What are we trying to do when working out the sperm concentration?

Answer 7

This is used in order to calculate the sperm concentration which is the quantity of sperm present in a sample.

This is measured in millions per ml.

This can be determined using a counting chamber

Question 8

What are the types of counting chambers used in determining sperm concentration?

Answer 8

1. Haemocytometer

2. Makler counting chamber

Question 9

What are the sperm concentrations which are below the reference value ?
What is the clinical relevance of this?

Answer 9

Oligozoospermia

The clinical relevance is that sperm concentration should start of high so that when the sperm is going through female reproductive tract less is lost.

This is very important for successful fertilisation

Question 10

What are some important factors to consider when carrying out a sperm motility count ?

Answer 10

Sperm motility within semen should be assessed as soon as possible after liquefaction of the sample within 1 hour following ejaculation.

This is to limit the deleterious effects of dehydration ,pH changes or temperature changes in motility.

Question 11

What are the steps to carrying out a sperm motility count?

Answer 11

1. Mix the semen sample well
2. Remove aliquots of semen immediately after mixing ,allowing no time for the spermatozoa to settle out of suspension

Make a wet preparation approximately 20 micrometetre deep,Wait for the sample to stop drifting.

Examine the slide with phase contract optics at x200 or x400 magnification.(x20/x40 objective)
Assess approximately 200 spermatozoa per replicate for the percentage of different motile categories

Compare the replicate values to check if they are acceptably close

Then carry out calculations.

Question 12

How is sperm motility classed ?What are the four catgeories

Answer 12

1. Rapidly progressive motility
2. Slowly progressive motility
3. Non-progressive motility
4. Immotility

Question 13

What is rapidly progressive motility?

Answer 13

Rapidly progressive motility (a): spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of at least 25 μm (or ½ tail length) in one second.

Question 14

What is Slowly progressive motility?

Answer 14

Slowly progressive motility (b): spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of 5 to < 25 μm (or at least one head length to less than ½ tail length) in one second

Question 15

What is non-progressive motility?

Answer 15

Non-progressive motility (c): all other patterns of motility with an absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed.

Question 16

What is immotility?

Answer 16

no movement.

Question 17

What is motility below reference value be called?

Answer 17

Asthenozoospermia

Question 18

When counting sperm motility what do we look for ?

Answer 18

We only count spermatozoa with intact head and tail

Evaluate at least 200 spermatozoa in a total of at least 5 fields per replicate.

Avoid repeatedly viewing the same field

Question 19

How is sperm morphology assessed?

Answer 19

It is assessed on the wet preparation when assessing motility.

Using stains:
Papanicolaou staining
Diff-Duik staining
Shorr stain

Prior to this the sperm will be smeared on slides and fixed before the staining protocol of choice is applied

Question 20

Give a description of a normal head morphology of the sperm

Answer 20

Sperm head
Smooth, regularly contoured and generally oval in shape.

Well-defined acrosomal region comprising 40–70% of the head area.

Acrosomal region should contain no large vacuoles, and not more than two small vacuoles, which should not occupy more than 20% of the sperm head.

The post-acrosomal region should not contain any vacuoles.

Question 21

Give a description of the normal tail morphology of a sperm

Answer 21

Slender, regular midpiece about the same length as the sperm head.

The major axis of the midpiece should be aligned with the major axis of the sperm head.
(Aligned at the centre )

Residual cytoplasm is considered an anomaly only when in excess i.e. when it exceeds one third of the sperm head size.

The principal piece should be thinner than the midpiece and around 10 times the head length. It may be looped back on itself provided there is no sharp angle indicative of a flagellar break.

Question 22

What are the different head defects which can arise ?

Answer 22

Check the slide

Question 23

What is sperm vitality ?

Answer 23

This is a measurement of how many sperms are alive.
This can be determined by assessing the membrane integrity of the spermatozoa -especially important for samples with low progressive motility.

Question 23

What is sperm vitality ?

Answer 23

This is a measurement of how many sperms are alive.
This can be determined by assessing the membrane integrity of the spermatozoa -especially important for samples with low progressive motility.

Number of spermatozoa with intact membrane expressed as % live spermatozoa

Question 24

How quickly does the vitality need to be assessed?

Answer 24

This is assessed soon following liquefaction of the semen sample, preferably at 30 minutes but not later than 1 hour post-ejaculation

Question 25

What are the methods that can be used to measure sperm vitality?

Answer 25

Dye exclusion (Eosin-Nigrosin)
Damaged plasma membranes such as those found in dead cells ,allow entry of membrane-impermeant stains 

Hypo-osmotic swelling test-
Only spermatozoa with intact membranes
(live cells ) will swell in hypotonic solution

Dead cells won’t be able to respond to a hypotonic environment

hypotonic = lower concentration of solutes than another solution

Question 26

What is a below ref value vitality ?

Answer 26

Necrozoospermia

Question 27

How can we check the pH of the sperm ?

Answer 27

We can measure pH by using strips.

The pH of semen reflects the balance between the pH values of the different accessory gland secretions, mainly the alkaline seminal vesicular secretion and the acidic prostatic secretion.

Question 28

What is the normal pH of a sperm and how does this make it better adapted

Answer 28

Normal semen is neutral to slightly alkaline. Serves to protect spermatozoa form the slightly acidic vaginal environment

Question 29

What are some important things to consider when wanting to measure pH ?

Answer 29

Assessed as soon as possible after liquefaction of the semen sample, preferably at 30 minutes, but no later than 1 hour post-ejaculation (loss of CO2).

Question 30

Why do we check the leucocytes ?

Answer 30

The presence of leucocytes could be indicative of an infection.

Can see round cells and if this exceed more than 1 million per ml.

This will raise a concern.

Round cells could be germ cells which haven’t completed spermatogenesis.

Question 31

How can we assess leucocyte/round cell count?

Answer 31

This can be done using counting chamber whilst doing concentration and density count.

This can then be sent for culture where it is assessed using immunocytochemical staining.
Granulocyte peroxidase
Staining for CD45-bearing leucocytes

Can indicate an infection of the male reproductive tract