Lecture 11: Diagnostic Assessment of Human Sperm Parameters Flashcards
What is the first diagnostic step in male fertility investigation?
- Semen analysis
What is the definition of semen analysis?
- Analysis of seminal fluid & sperm parameters as an indicator of male fertility potential.
What do we use to determine whether the semen is normal or abnormal?
- WHO criteria for normal semen parameters (2021)
How do we analyse semen?
- Manual semen analysis: performed by lab practitioner using microscope & cell counter
- Computer-assisted semen analysis (CASA): light microscopy + computer software
Which method of semen analysis is performed clinically mainly?
- Manual semen analysis
(- CASA used in research settings mainly)
WHO reference values 2020 vs 2021
What other parameters other than sperm analysis can be used to test for male fertility?
- Anti-sperm antibodies
- hypo-osmotic swelling test
- sperm DNA frag testing
What should you see in the appearance of a normal semen sample?
- Grey-opalescent appearance
How do you determine which semen sample has normal liquefaction vs abnormal?
- Normal liquefaction = 20-30 minutes post-production
- Abnormal = <30mins
What is the name given to abnormal liquefaction?
- Delayed liquefaction
What may delayed liquefaction indicate?
- Infection e.g. bacterial prostatitis
- Accessory glands infected -> secretions from these glands altered -> delayed liquefaction
How can we measure sperm volume?
- Direct volume measurement (common in diagnostic setting)
- Volume from weight
Describe direct volume measurement
- Serological pipette attached to electronic pipette
- Aspirate sample from container -> Volume (ml) measured using graduate scale
Describe the method of measuring sperm volume via volume from weight
- Weighing sample pots before & after sample production
- Difference = sample volume.
- Studies on human semen have shown weight to be an accurate index of volume (1g=1ml of semen sample)
What can you use to calculate sperm concentration?
- Haemocytometer
(can be used to calcite [RBC] & [sperm]
Describe the structures present in a haemocytometer?
- 2 counting chambers, each with microscopic grid
- 2 raised pillars on either side of chambers
What is the significance of the raised pillars in haemocytometers?
- to hold slip in place 1/10mm above chambers thus PRECISE volume above each grid is known
How do you prepare the sperm prior to haemocytometer?
- IMMOBILISE: Add dilute, 3% saline - kills sperm & preserves sperm for later count use
- DILUTE: factor of 400 (1/20 dilution) to allow counting of individual sperm
- VORTEX: evenly distribute sperm
How do you use a haemocytometer for [sperm]?
1- moisten pillars (prevent coverslip from moving)
2- Add cover slip
3- Aspirate 10uL sample at edge of coverslip (sample drawn into counting chambers by capillary action)
4- Place haemocytometer in humid chamber (prevents drying out & allows sample to settle)
5- View under phase contrast microscope & count
6- Use cell counter to keep counts
How do you count the sperm?
- Many different ways, depends on sample & dilution factor (refer to WHO)
- one method of counting:
How to calculate [sperm]?
sperm/ml= average of total counts from 2 counting chambers x 5 (other boxes) x dilution factor (usually 400) x volume of haemocytometer (10000)
Which sperm to count in a box and which to not?
Definition of [sperm]
- quantity of sperm present in a sample
[sperm] units
- millions/ml
How is [sperm] determined?
- counting chamber
How many types of counting chambers?
2 types:
- Neubauer haemocytometer (recommended)
- Makler counting chamber
Reference values of [sperm]?
≥16million/ml
[seprm] below ref value is called?
-Oligozoospermia
If a male is oligospermic, is this clinically significant?
- Yes,
- race to fertilisation in female tract begins with high no. of sperm (10-100 million) and at site of fertilisation (10-100)
- Higher no. of starting sperm = higher chance of fertilisation
Sperm concentration is also known as?
- Sperm density
- Sperm count
When is sperm motility assessed?
- ASAP after sample liquefaction
- within 1 hr following ejaculation,
- to limit deleterious effects of dehydration, pH or changes in temperature on motility.
What must you do in the lab when assessing sperm motility due to the time-sensitivity?
- Work flow in lab must not compromise time sensitivity, thus must check:
1- liquefaction
2- volume
3- Appearance
4- THEN sperm motility right after - small aliquot taken from sample & diluted for [sperm] to be done later i.e. after sperm motility
5- [sperm] etc…
How to perform sperm motility analysis?
1- Mix semen sample using serological pipette (draw up & down)
2- Remove aliquots of semen using positive displacement pipette immediately after mixing (~10µl each), allowing no time for spermatozoa to settle out of suspension.
3- Make a wet preparation approximately 20µm deep (2 replicates). Wait for sample to stop drifting (within 60 secs)**.
4- Examine the slide with phase-contrast optics at ×200 (x10 x20) or ×400 magnification. Assess approximately 200 spermatozoa per replicate for the percentage of different motile categories.
5- Compare replicate values to check if they are acceptably close. If so, proceed with calculations; if not, prepare new samples.
What are the different categories of sperm motility?
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.
What are the WHO ref values for sperm motility?
- a+b≥30%
- a+b+c≥42%
What is the term when sperm motility is below ref values?
- Asthenozoospermia
(sample is asthenbozoospermic)
What do you use to keep count of sperm when assessing sperm motility?
- Counter
Which sperms to count and which sperms to include in your counts?
- 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
- must stick to one field of view i.e. if only counting the round area in centre, then each view you count sperms in the centre
How is sperm morphology assessed?
- Assessed directly on the wet preparation
- Using stains
Where are the different techniques of assessing morphology done?
- wet preparation (free-moving sperm- skilled eye required): labs/clinics where treatment occurs
- stains: diagnostic labs/andrology separate from IVF lab due to toxicity of stains
What are the different type of stains?
How do you use stains for morphology analysis?
- Sperm smeared on slides
- fixed & immobilised before staining protocol of choice is applied.
Which stain gives better nuclear detail?
- Papanicolaou
What is the normal morphology of sperm head?
- Smooth, regularly contoured & generally oval in shape.
- Well-defined acrosomal region comprising 40–70% of head area.
- Acrosomal region = no large vacuoles & no more than 2 small vacuoles (not occupy > 20% of sperm head).
- Post-acrosomal region = no vacuoles.
What is the normal morphology of sperm tail?
- Slender, regular midpiece about same length as sperm head.
- Major axis of midpiece = aligned with major axis of sperm head.
- Residual cytoplasm = considered anomaly only when in excess i.e. exceeds 1/3 of sperm head size.
- Principal piece = thinner than midpiece & ~10 times the head length. Can be looped back on itself provided there is no sharp angle indicative of a flagellar break.
Where is the nuclear material contained in sperm?
- Postacrosomal region
How was normal morphology of sperm determined?
- Sperm successfully pass the cervix of female tract, analysed.
What is the term when morphology is below the reference values?
- Teratozoospermia
Types of different abnormal sperm morphology
What is sperm vitality a measure of?
- number of sperm alive
How is sperm vitility determined?
- By assessing membrane integrity of spermatozoa
- Important for samples with low progressive motility.
When is sperm vitality measured?
- ASAP after liquefaction of semen sample,
- preferably at 30 mins - no later than 1 hr post-ejaculation
- (prolonged exposure to external conditions affect vitality (same as motility))
How is sperm vitality tested in labs?
- Dye exclusion (Eosin-Nigrosin) - Damaged plasma membranes (found in dead cells) allow entry of membrane-impermeant stains.
- Hypo-osmotic swelling (HOS) test - Only spermatozoa with intact membranes (live cells) will swell in hypotonic solutions/culture.
What is the permeability status of dead and live cells?
- Dead: Permeable
- Alive: Impermeable
How is vitality measured quantitatively?
- Number of spermatozoa with intact membrane expressed as % live spermatozoa.
How is vitality measured quantitatively?
- Number of spermatozoa with intact membrane expressed as % live spermatozoa.
What are the variations of hypo-osmotic swellings?
What is vitility values below reference values called?
- Necrozoospermia
Types of necrozoospermia?
- Incomplete Necrozoospermia = live cells <45% but >5%
- Complete Necrozoospermia = all cells dead in sample
What is HOS medium made up of?
0.74% sodium citrate + 1.35% fructose in double-distilled H2O
How is sperm pH determined?
- pH strips - dip strips in sample
What is the normal pH range of sperm sample?
- 7.2-8.0
What is the clinical significance of pH levels in sperm?
- pH of semen reflects balance between pH values of different accessory gland secretions:
- Mainly alkaline seminal vesicular secretion & acidic prostatic secretion => come together = neutral to alkaline pH
When is pH assessed?
- ASAP after liquefaction of semen sample, preferably at 30 minutes, but no later than 1 hour post-ejaculation
- loss of CO2
How can we test for presence of leukocytes in sperm sample?
- Count round cells using counting chamber
What is the value of round cells to be considered a concern?
- 1million/ml
When is immunocytochemical staining used when looking for leukocytes?
- Round cell numbers are >1 million/ml after using counting chamber
What is an example of immunocytochemical staining for CD45-bearing leucocytes?
- Granulocyte peroxidase
What do high numbers of leukocytes indicate?
- Infection in male reproductive tract
Are all round cells leukocytes?
- No,
- Some count be germ cells that are present due to incomplete spermatogenesis
Are all round cells leukocytes?
- No,
- Some count be germ cells that are present due to incomplete spermatogenesis
