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
What is Asthenozoospermia?
Motility before ref values
Example- motility
Example:
Total no. of spermatozoa assessed = 210
a=80, b=32, c=20, d=78
a=38%, b=15%, c=10%, d=37% = normal progressive motility
Sperm motility (WHO, 2010) OLD CRITERIA
Only contained three categories: Progressive motility, Non-progressive motility and Immobility.
NOW they have further classified progressive motility as rapidly progressive and slowly progressive.
How is sperm motility measured?
As different categories of sperm motility are observed you register it using a counter.
Before you count you assign a button for each category.
It is important that you 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
Make sure that the total count of at least 200 is derived from at least 5 fields of view. It is important that you don’t keep viewing the same field, you need to move the slide systematically.
It is important to track the sperm, so it is important to create a visual segment to focus on. It is important to keep this region consistent:
It is recommended to finish up the count in the segment even if you have reached 200.
How many ways are there to assess sperm morphology and what are?
2
Assessed directly on the wet preparation
Like what is used for motility. Requires a trained eye
Used by centres that provide diagnostics as well as treatments as many of the stains are toxic. So do not want this in IFV labs
Using stains
Sperm is smeared on slides and fixed before the staining protocol of choice is applied
Used in centers that are strictly diagnostic
What was the normal morphology for the sperm head like according to WHO 2021?
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 (where you have the nuclear material) should not contain any vacuoles.
What was the normal morphology for the sperm tail like according to WHO 2021?
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 in the center.
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.
What does Teratozoospermia mean?
Morphology below ref value
What are the abnormal morphologies classified into?
- head defects
- neck & midpiece defects
- tail defects
- excess residual cytoplasm
