Diagnostic assessment of Human Sperm Parameters Flashcards
What is semen analysis?
It is the analysis of seminal fluid and sperm parameters as an indicator of male fertility potential.
Usually the first diagnostic step in male fertility investigations.
Remains the gold standard for male fertility investigation
WHO criteria for normal semen parameters (2021).
What are the two ways to carry out semen analysis?
Manual semen analysis – is usually performed by a lab practitioner using a microscope and a cell counter
Computer assisted semen analysis (CASA) – Makes use of light microscopy and assisted by computer software. Uses a microscope and a heated stage.
What method is normally carried out in the clinical settings?
In clinical settings analysis is normally manual. CASA is used in research.
Key WHO values
Key changes are in motility values and the switch from count 3->4 categories of motility.
What factors are assessed in semen analysis?
- appearance and liquefaction
- sperm volume
- concentration & moility
Apperance & Liquefaction
When the sample is produced and collected you expect to see a Grey-opalescent appearance.
Normal liquefaction should take place within 20-30 minutes post-production.
Anything above 30 mins is considered to be delayed liquefaction.
An abnormally long liquefaction time may be indicative of an infection e.g. bacterial prostatitis
When the glands are infected, the secretions from the glands are altered, which results in delayed liquefaction.
Sperm Volume
There are two methods:
Direct volume measurement (most commonly used in diagnostics):
Volume (ml) measured directly using serological pipette.
Volume from weight (not commonly used):
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.
Assessment of concentration & motility
Haemocytometer can be used to calculate sperm concentration.
Haemocytometer have two counting chambers with a microscopic grid
First need to insure that the sperm are immobilised using 3% saline.
Sample needs to be diluted to count the sperm, normally by 400
The Haemocytometer needs to be viewed under a phase contrast microscope
You need to count sperm in 5 of the larger squares, 4 corners and the centre square.
To calculate concentration of sample:
Sperm conc per ml = Average of sperm count * 5 *dillution factor (400) *10,000 (volume of Haemocytometer)
Assessment of concentration & motility
Haemocytometer can be used to calculate sperm concentration.
Haemocytometer have two counting chambers with a microscopic grid
First need to insure that the sperm are immobilised using 3% saline.
Sample needs to be diluted to count the sperm, normally by 400
The Haemocytometer needs to be viewed under a phase contrast microscope
You need to count sperm in 5 of the larger squares, 4 corners and the centre square.
To calculate concentration of sample:
Sperm conc per ml = Average of sperm count * 5 *dillution factor (400) *10,000 (volume of Haemocytometer)
Sperm concentration
Sperm concentration = quantity of sperm present in a sample.
Measured in millions per ml.
Determined using a counting chamber. There are usually two main types:
Neubauer haemocytometer (recommended by WHO)
Makler counting chamber
What does Oligozoospermia mean?
Concentration below ref value
It is important to have a good starting number as at the site of fertilisation there is only 10-100.
It is important to immobilise sperm first as it would hard to measure whilst the hard moving. Dilution factor is often 1:20.
What is sperm motility
This is a time sensitive aspect of semen analysis
Sperm motility within semen should be assessed as soon as possible after liquefaction of the sample, within 1 hour following ejaculation, to limit the deleterious effects of dehydration, pH or changes in temperature on motility.
Method for testing sperm motility
Mix the semen sample well.
Remove aliquots of semen immediately after mixing (~10µl each), allowing no time for the spermatozoa to settle out of suspension.
Make a wet preparation approximately 20µm deep (2 replicates). Wait for the sample to stop drifting (within 60 seconds).
Examine the slide with phase-contrast optics at ×200 or ×400 magnification. Assess approximately 200 spermatozoa per replicate for the percentage of different motile categories.
Compare the replicate values to check if they are acceptably close. If so, proceed with calculations; if not, prepare new samples.
What are the 4 categories of sperm motility according to WHO?
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 a rate of 25 μm (or ½ tail length) in one second.
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
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.
Immotility (d):
no movement.
WHO Ref Values
a+b≥30% for a normal sample
a+b+c≥42% for a normal sample
