Lecture 11: Diagnostic Assessment of Human Sperm Parameters Flashcards
(71 cards)
1
Q
What is the first diagnostic step in male fertility investigation?
A
- Semen analysis
2
Q
What is the definition of semen analysis?
A
- Analysis of seminal fluid & sperm parameters as an indicator of male fertility potential.
3
Q
What do we use to determine whether the semen is normal or abnormal?
A
- WHO criteria for normal semen parameters (2021)
4
Q
How do we analyse semen?
A
- Manual semen analysis: performed by lab practitioner using microscope & cell counter
- Computer-assisted semen analysis (CASA): light microscopy + computer software
5
Q
Which method of semen analysis is performed clinically mainly?
A
- Manual semen analysis
(- CASA used in research settings mainly)
6
Q
WHO reference values 2020 vs 2021
A
7
Q
What other parameters other than sperm analysis can be used to test for male fertility?
A
- Anti-sperm antibodies
- hypo-osmotic swelling test
- sperm DNA frag testing
8
Q
What should you see in the appearance of a normal semen sample?
A
- Grey-opalescent appearance
9
Q
How do you determine which semen sample has normal liquefaction vs abnormal?
A
- Normal liquefaction = 20-30 minutes post-production
- Abnormal = <30mins
10
Q
What is the name given to abnormal liquefaction?
A
- Delayed liquefaction
11
Q
What may delayed liquefaction indicate?
A
- Infection e.g. bacterial prostatitis
- Accessory glands infected -> secretions from these glands altered -> delayed liquefaction
12
Q
How can we measure sperm volume?
A
- Direct volume measurement (common in diagnostic setting)
- Volume from weight
13
Q
Describe direct volume measurement
A
- Serological pipette attached to electronic pipette
- Aspirate sample from container -> Volume (ml) measured using graduate scale
14
Q
Describe the method of measuring sperm volume via volume from weight
A
- 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)
15
Q
What can you use to calculate sperm concentration?
A
- Haemocytometer
(can be used to calcite [RBC] & [sperm]
16
Q
Describe the structures present in a haemocytometer?
A
- 2 counting chambers, each with microscopic grid
- 2 raised pillars on either side of chambers
17
Q
What is the significance of the raised pillars in haemocytometers?
A
- to hold slip in place 1/10mm above chambers thus PRECISE volume above each grid is known
18
Q
How do you prepare the sperm prior to haemocytometer?
A
- 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
19
Q
How do you use a haemocytometer for [sperm]?
A
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
20
Q
How do you count the sperm?
A
- Many different ways, depends on sample & dilution factor (refer to WHO)
- one method of counting:
21
Q
How to calculate [sperm]?
A
sperm/ml= average of total counts from 2 counting chambers x 5 (other boxes) x dilution factor (usually 400) x volume of haemocytometer (10000)
22
Q
Which sperm to count in a box and which to not?
A
23
Q
Definition of [sperm]
A
- quantity of sperm present in a sample
24
Q
[sperm] units
A
- millions/ml
25
How is [sperm] determined?
- counting chamber
26
How many types of counting chambers?
2 types:
- Neubauer haemocytometer (recommended)
- Makler counting chamber
27
Reference values of [sperm]?
≥16million/ml
28
[seprm] below ref value is called?
-Oligozoospermia
29
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
30
Sperm concentration is also known as?
- Sperm density
- Sperm count
31
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.
32
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...
33
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.
34
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.
35
What are the WHO ref values for sperm motility?
- a+b≥30%
- a+b+c≥42%
36
What is the term when sperm motility is below ref values?
- Asthenozoospermia
(sample is asthenbozoospermic)
37
What do you use to keep count of sperm when assessing sperm motility?
- Counter
38
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
39
How is sperm morphology assessed?
- Assessed directly on the wet preparation
- Using stains
40
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
41
What are the different type of stains?
42
How do you use stains for morphology analysis?
- Sperm smeared on slides
- fixed & immobilised before staining protocol of choice is applied.
43
Which stain gives better nuclear detail?
- Papanicolaou
44
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.
45
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.
46
Where is the nuclear material contained in sperm?
- Postacrosomal region
47
How was normal morphology of sperm determined?
- Sperm successfully pass the cervix of female tract, analysed.
48
What is the term when morphology is below the reference values?
- Teratozoospermia
49
Types of different abnormal sperm morphology
50
What is sperm vitality a measure of?
- number of sperm alive
51
How is sperm vitility determined?
- By assessing membrane integrity of spermatozoa
- Important for samples with low progressive motility.
52
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))
53
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.
54
What is the permeability status of dead and live cells?
- Dead: Permeable
- Alive: Impermeable
55
How is vitality measured quantitatively?
- Number of spermatozoa with intact membrane expressed as % live spermatozoa.
55
How is vitality measured quantitatively?
- Number of spermatozoa with intact membrane expressed as % live spermatozoa.
56
What are the variations of hypo-osmotic swellings?
57
What is vitility values below reference values called?
- Necrozoospermia
58
Types of necrozoospermia?
- Incomplete Necrozoospermia = live cells <45% but >5%
- Complete Necrozoospermia = all cells dead in sample
59
What is HOS medium made up of?
0.74% sodium citrate + 1.35% fructose in double-distilled H2O
60
How is sperm pH determined?
- pH strips - dip strips in sample
61
What is the normal pH range of sperm sample?
- 7.2-8.0
62
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
63
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
64
How can we test for presence of leukocytes in sperm sample?
- Count round cells using counting chamber
65
What is the value of round cells to be considered a concern?
- 1million/ml
66
When is immunocytochemical staining used when looking for leukocytes?
- Round cell numbers are >1 million/ml after using counting chamber
67
What is an example of immunocytochemical staining for CD45-bearing leucocytes?
- Granulocyte peroxidase
68
What do high numbers of leukocytes indicate?
- Infection in male reproductive tract
69
Are all round cells leukocytes?
- No,
- Some count be germ cells that are present due to incomplete spermatogenesis
69
Are all round cells leukocytes?
- No,
- Some count be germ cells that are present due to incomplete spermatogenesis
