Diagnosis of human sperm parameters Flashcards
What is semen analysis needed for?
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.
WHO criteria for normal semen parameters (2010).
What are the 2 methods of doing semen analysis
Manual semen analysis (through microscope)
Computer assisted semen analysis
What are the WHO reference values for semen
Volume 1.5 – 6.0 ml Appearance/Colour Grey-opalescent appearance Liquefaction <30 minutes Sperm concentration ≥15million/ml Motility ≥40% Progressive motility ≥32% Morphology (normal forms) ≥4% Vitality (live) ≥58% pH 7.2 - 8.0 Leucocytes <1 million/ml
What should the appearance and liquefaction be like in normal semen
It should have a grey-opalescent appearance
Normal liquefaction should take place within 20-30 minutes post-production.
An abnormally long liquefaction time may be indicative of an infection e.g. bacterial prostatitis
What are the 2 methods for measuring semen volume
Direct volume measurement using a serological pipette
calculating 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.
How to measure concentration or density of sperm
Sperm concentration = quantity of sperm present in a sample.
Millions per ml.
Determined using a counting chamber.
Low concentration of sperm is known as OLIGOZOOSPERMIA
Neubauer haemocytometer and makler counting chamber are used to measure the concentration
How to examine sperm motility
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.
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 3 categories of motility for sperm
Sperm motility is classed into 3 categories based on current WHO (2010) criteria:
Progressive motility (a): spermatozoa moving actively, either linearly or in a large circle, regardless of speed.
Non-progressive motility (b): 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 (c): no movement.
In any sample, to find motility:
Count only 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
What are the 2 methods for examining sperm morphology (shape)
Assessed directly on the wet preparation
Using stains
The 3 types of stains that can be used are:
Papanicolaou staining
Diff quik
Shor staining
Describe the normal morphology of the sperm cell
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.
The structure is tail, post acrosomal region, acrosomal region
Describe the normal morphology of the sperm tail
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.
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.
If the morphology of the tail is below the referenced level it is called tetrazoospermia
How to assess sperm vitality
Determined by assessing the membrane integrity of the spermatozoa - especially important for samples with low progressive motility.
Assessed as soon as possible after liquefaction of the semen sample, preferably at 30 minutes, but no later than 1 hour post-ejaculation.
Number of spermatozoa with intact membrane expressed as % live spermatozoa.
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 solutions.
How to assess sperm pH
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. pH is assessed immediately, after half an hour but no longer than an hour post ejaculation due to the loss of CO2
How to assess sperm leucocytes
High number of leucocytes could indicate infection
A counting chamber is used to assess round cells
Immunocytochemical staining is also used
Summary
A semen analysis is usually the first diagnostic step in male fertility investigations. It is the analysis of seminal fluid and sperm parameters as an indicator of male fertility potential.
A standard semen analysis is carried out using the WHO (2010) parameters for the laboratory assessment of human semen.
sperm parameters analysed include: volume, liquefaction, appearance, concentration, motility, morphology, vitality, pH, leucocytes. All of these parameters are of clinical significance to male infertility investigations.
