Diagnostic assessment of sperm parameters Flashcards
1
Q
What is semen analysis?
A
- Defined as analysis of seminal fluid and sperm parameters as an indicator of male fertility potential.
- Usually the first diagnostic step in male fertility investigations. When a couple have been trying to conceive for at least a year and a GP is looking to carry out investigations on both sides, the first port of call for male fertility investigations is semen analysis.
- Remains the gold standard.
- WHO criteria for normal semen parameters (2021). The semen analysis test is standardized by the WHO via their publication (known as the semen analysis manual = “WHO laboratory manual for the examination and processing of human semen”). This is the most recent publication which was just released a couple of months ago (in 2021).
- Basic Semen Analysis is what is routinely done. Further diagnostic parameters may be introduced depending on the testing centre/patient. Parameters include anti-sperm antibodies, hypo-osmotic swelling test, sperm DNA frag testing.
2
Q
What are the two different ways of carrying out semen analysis?
A
- There are mainly two different ways of carrying out semen analysis = manual (performed by a lab practitioner using a microscope and a cell counter) and computer-assisted semen analysis (makes use of light microscopy and assisted by computer software)
- The device shown is one of the most compact and neatest systems existing; the box houses a microscope and heated stage. In terms of operating it, the button ejects the heated stage is ejected (like a CD player). Slide/slide chamber can then be inserted, button pressed again and the heated stage returns into the device. It goes straight underneath the view of the microscope objective that has been housed inside, focus can be adjusted using the knob, the other buttons allow movement from one field of view to another.
- This software allows analysis of sperm count, sperm motility, sperm morphology and more, e.g. can track sperm and different movements, can set algorithms to look at hyperactivation.
- In the clinical setting, when a GP requests/refers semen analysis for example, those labs that perform the analysis usually carry it out the manual way. CASA is found more in the research setting (in research labs). There are some clinical labs that use CASA, but this is not commonplace.
3
Q
How is computer-assisted semen analysis (CASA) carried out?
A
- The device shown is one of the most compact and neatest systems existing; the box houses a microscope and heated stage. In terms of operating it, the button ejects the heated stage is ejected (like a CD player). Slide/slide chamber can then be inserted, button pressed again and the heated stage returns into the device. It goes straight underneath the view of the microscope objective that has been housed inside, focus can be adjusted using the knob, the other buttons allow movement from one field of view to another.
- This software allows analysis of sperm count, sperm motility, sperm morphology and more, e.g. can track sperm and different movements, can set algorithms to look at hyperactivation.
- In the clinical setting, when a GP requests/refers semen analysis for example, those labs that perform the analysis usually carry it out the manual way. CASA is found more in the research setting (in research labs). There are some clinical labs that use CASA, but this is not commonplace.
4
Q
What are the WHO reference values (2021)?
A
- Volume = 1.4 - 6.0 ml
- Appearance = Grey-opalescent appearance
- Liquefaction = <30 minutes
- Sperm concentration = ≥16million/ml
- Motility = ≥42%
- Progressive motility = ≥30%
- Morphology (normal forms) = ≥4%
- Vitality (live) = ≥54%
- pH = 7.2 - 8.0
- Leucocytes = <1 million/ml
- The key changes from the previous 2010 criteria and the current 2021 criteria are in the motility values and also the switching from counting three categories of motility to four categories of motility.
5
Q
What is the normal appearance and liquefaction time of a sample?
A
- When a normal sample is produced and collected by the lab, a grey-opalescent appearance is expected to be seen in line with WHO criteria.
- Normal liquefaction should take place within 20-30 minutes post-production. 30+ minutes is considered to be delayed liquefaction.
- An abnormally long liquefaction time (1h+) may be indicative of an infection in the male reproductive tract, e.g. bacterial prostatitis
- When these accessory glands are infected, e.g. the prostate, the seminal vesicle, the bulbourethral gland, the secretions from these glands are altered and this is responsible for the delayed liquefaction of the semen sample.
6
Q
What are the two methods used to measure sperm volume?
A
1) The direct volume measurement is the most commonly used in a diagnostic setting. A serological pipette is attached to an electronical pipette. The entire sample is aspirated from the container and the volume is measured using the graduated scale on the serological pipette.
2) The second method is not as commonly used, but is also very accurate = volume from weight. Can weigh sample pots before and after sample production. The difference between them = sample volume. This can be carried out because studies on human semen have shown weight to be an accurate index of volume (1g = 1 ml sample).
7
Q
How are haemocytometers used to calculate sperm concentration?
A
- Although they were originally developed for counting red blood cells, haemocytometers can also be used to calculate sperm concentration. This makes them a valuable tool in reproductive biology.
- Haemocytometers consist of two counting chambers, each inscribed with a microscopic grid. Two raised pillars sit either side of the counting chambers. These pillars hold the cover slip in place exactly 1/10th of a mm above the chambers so the precise volume of sample above each grid is known. Prior to loading the sample into a haemocytometer, the sperm need to be immobilised. A diluent, such as 3% saline, not only kills sperm but preserves them if the counts need to be conducted at a later date. The sample must be dilute enough to allow individual sperm to be counted (a factor of 400 is usually sufficient). Sample should be mixed thoroughly to evenly distribute sperm before counting.
- Once loaded, the sample needs to be allowed to settle (for about 3-5 minutes). Can be placed in a humid chamber to prevent it from drying out in this time. The haemocytometer needs to be viewed under a phase-contrast microscope. The counting chamber is located at the centre of this field of view. It consists of 25 largest squares, each containing 16 smaller squares (usually only need to count sperm in 5 of the larger squares; use distributed squares as shown for an unbiased count).
- Calculating sperm concentration in sample per ml = average of total counts from the two counting chambers x 5 to make up the other 20 squares) x dilution factor (usually 400) x 10,000 (volume of haemocytometer)
8
Q
How is sperm concentration (density) measured?
A
- Sperm concentration/sperm density/sperm count = quantity of sperm present in a semen sample.
- There are two main types of counting chamber used = the Neubauer haemocytometer and the Makler counting chamber (depends on the choice of the clinic or lab). The Neubauer is usually what is recommended by the WHO in terms of standardising counts.
- In terms of the clinical terminology, sperm samples or semen samples in which the sperm count is below the normal WHO reference values are known as samples that are oligozoospermic/the individual has oligozoospermia. The journey in the female tract always begins with high numbers of spermatozoa (hundreds of million) and ends at the site of fertilisation with about 10 to 100. Therefore, it is important to have a very good starting number in order to attain successful fertilisation and having good numbers of sperm getting to the site of fertilisation.
- Important to highlight that the method for counting sperm on the previous slide (four corner boxes and the one in the middle) is just one way of performing sperm count. There are different ways of going about it - the choice of method really depends on the sample; looking at the sample under the microscope for the first time to look at density informs the choice of counting method. Also depends on the dilution factor, because a small aliquot needs to be diluted with water to immobilise sperm cells (can’ t count while they are swimming). The dilution factor will determine how many boxes should be counted in the grid, e.g. the video used a 1 in 20 dilution factor (standard), but the WHO manual shows a table of different possible dilution factors and the corresponding number of boxes to count.
- THE VIDEO IS JUST ONE WAY OF GOING ABOUT IT WITH THE NEUBAUER SAMPLE! DEPENDS ON THE SAMPLE AND DILUTION FACTOR APPLIED BEFORE CARRYING OUT THE COUNT!
9
Q
How is sperm motility assessed within semen?
A
- Sperm motility within semen should be assessed as soon as possible after liquefaction of the sample (after the sample has liquefied), within 1 hour following ejaculation, to limit the deleterious effects of dehydration, pH or changes in temperature on motility (especially the longer it stays outside of the body and is exposed to these elements) .
- Time sensitive aspect of semen analysis.
- Due to this time sensitivity, it is important that your workflow is such in the lab that it does not compromise the time sensitivity. As soon as liquefaction, volume and appearance have been assessed, the first thing that is done is a small aliquot is taken from the sample and diluted for carrying out the sperm concentration. That dilution is kept aside and then the next thing is sperm motility, which is performed immediately.
- The volume assessment begins by mixing with the serological pipette to draw the sample up and down a few times. A positive displacement pipette (the ones with pipette tips) is used to remove aliquot (usually 10 microlitres each) and prepare replicate preparations on a slide (allows duplicate counts). Covered with cover slips. Prepared slides can be observed with a microscope. Expected to assess at least 200 sperm per replicate.
WHO manual has a table that shows the accepted limits between replicates. If the replicate counts aren’t acceptably close according to that, the motility count has to be repeated using a fresh slide. - 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 (x20 or x40 objective) 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.
- WHO manual has a table that shows the accepted limits between replicates. If the replicate counts aren’t acceptably close according to that, the motility count has to be repeated using a fresh slide.
10
Q
What are the four categories of sperm motility based on current WHO (2021) criteria?
A
- Sperm motility is classed into 4 categories based on current WHO (2021) criteria:
1) 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.
2) 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
3) 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.
4) Immotility (d): no movement. - Can sometimes fit an accessory, called a reticule, to the microscope eyepiece that has graduated measurements. This can be used to assist you in determining whether something is rapid (%a) or slow progressive (%b).
- Slowly progressive motility satisfies the same progression criteria as rapidly progressive motility, but it moves at a slower speed.
- WHO reference values =
1) a+b≥30%
2) a+b+c≥42% - Asthenozoospermia = motility below WHO ref values
- Example:
Total no. of spermatozoa assessed = 210
a=80, b=32, c=20, d=78
a=38%, b=15%, c=10%, d=37%
11
Q
What were the three categories of sperm motility based on WHO (2010) criteria?
A
- Sperm motility was classed into 3 categories based on previous WHO (2010) criteria:
1) Progressive motility (a): spermatozoa moving actively, either linearly or in a large circle, regardless of speed.
2) 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.
3) Immotility (c): no movement. - Looking at the previous WHO semen analysis criteria in the 2010 manual
- The change is that progressive motility has been further classified as rapidly progressive and slowly progressive
- WHO (2010) Ref Values =
1) a≥32%
2) a+b≥40%
12
Q
What is a sperm counter?
A
- Sperm counter was used to record different categories of sperm motility
- Each button is assigned a category (one for A, B etc.). With practice, fingers align. As you look in a field of view, it is most practical to focus on A and B, since they are likely to move out of the field. The figures tally and the total is shown on the end screen circles. There is usually an alert when a count of 200 is reached.
- The right picture is amore modern version with the same principle. The only difference is that it is more programmable, e.g. can save counts and calculate percentages (instead of manual calculations).
13
Q
How is sperm motility measured?
A
- 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.
- When performing a semen analysis, it is important to only count sperm with an intact head and tail (don’t have to be normal sperm).
- Move slides systematically in a way that once a field of view has been counted, you do not come back to it; need a starting point and then to move away from it.
- How to determine different motility categories? Create a visual segment and count A, B, C and D in this region only before moving to another field of view and focusing on the exact same region until reaching 200; important to keep this region consistent! When the sample distribution is more sparse, you can be more generous with your visual segment.
- When using a graduated reticule with a micron scale in the eyepiece, it usually divides the field into quadrants already and then one can be chosen to count (have to be consistent!).
- In some cases when the sperm count is very low (sparsely distributed) and probably not swimming very fast, the entire field of view can comfortably be counted before moving to the next field. WHATEVER IS DONE IN ONE FIELD SHOULD BE DONE IN ALL FIELDS OF VIEW UNTIL 200 IS REACHED!!!
- Recommended to finish counting the field of view even after 200 is reached (usually tends to go over)
14
Q
What are the two ways in which sperm morphology is assessed?
A
- Sperm morphology can be assessed in two ways in the lab.
1) Assessed directly on the wet preparation
2) Using stains - It can either be assessed directly on the wet preparation (same as the slides used for motility). Assign morphology categories the same way (abnormal or abnormal). Requires a skilled eye for picking out normal morphology from freely swimming sperm; can be acquired upon semen analysis training in the lab.
- The second method uses stains. Sperm cells need to be smeared, fixed and stained. Once immobilized, morphology can be carefully assessed in the sample.
- In terms of the staining techniques, there are three stains that are commonly used depending on the lab = Papanicolaou, diff-Quik (based on H&E) and Shorr. Each of these three staining methods have individual protocols. Sperm is smeared on slides and fixed before the staining protocol of choice is applied.
- Important to note that labs and diagnostic (treatment) centres tend to use the first method (assess directly on the wet preparation). This is because a lot of these stains can be toxic; don’t want them near sperm/eggs/embryos in the IVF lab. In IVF labs that provide both diagnostic and treatment, they tend to just go with wet preparation. Centres that are strictly diagnostic and centres where the andrology lab is completely separated to the IVF lab may use stains.
15
Q
What is normal sperm morphology (WHO, 2021)?
A
- In terms of sperm morphology criteria, the 4% reference value was decided based on data over the years of individuals with certain morphology counts who have been able to father children. However, in terms of the actual morphological features (how normal sperm should appear), it was determined by extracting sperm from the female tract (sperm that had made it past the cervix to different points upstream of the female tract) for analysis. They profiled sperm cells from the tubes, sperm that had made it past the cervix etc. and were able to come up with a consensus on what normal sperm should look like; only physiologically and functionally normal sperm that would be capable of making it past the cervix and cervical mucus and further into the female reproductive tract.
- Excess cytoplasm (that wasn’t removed during spermiogenesis) is seen continuing from the sperm head and usually surrounding the midpiece.
- Teratozoospermia = normal morphology is less than 4% of the sample
- Sperm head
1) Smooth, regularly contoured and generally oval in shape.
2) Well-defined acrosomal region comprising 40–70% of the head area.
3) 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.
4) The post-acrosomal region (where the nuclear material is found) should not contain any vacuoles.
