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SoR > Lecture 11: Diagnostic Assessment of Human Sperm Parameters > Flashcards

Lecture 11: Diagnostic Assessment of Human Sperm Parameters Flashcards

1
Q

What is the first diagnostic step in male fertility investigation?

A
  • Semen analysis
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2
Q

What is the definition of semen analysis?

A
  • Analysis of seminal fluid & sperm parameters as an indicator of male fertility potential.
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3
Q

What do we use to determine whether the semen is normal or abnormal?

A
  • WHO criteria for normal semen parameters (2021)
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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
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5
Q

Which method of semen analysis is performed clinically mainly?

A
  • Manual semen analysis

(- CASA used in research settings mainly)

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6
Q

WHO reference values 2020 vs 2021

A
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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
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8
Q

What should you see in the appearance of a normal semen sample?

A
  • Grey-opalescent appearance
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9
Q

How do you determine which semen sample has normal liquefaction vs abnormal?

A
  • Normal liquefaction = 20-30 minutes post-production
  • Abnormal = <30mins
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10
Q

What is the name given to abnormal liquefaction?

A
  • Delayed liquefaction
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11
Q

What may delayed liquefaction indicate?

A
  • Infection e.g. bacterial prostatitis
  • Accessory glands infected -> secretions from these glands altered -> delayed liquefaction
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12
Q

How can we measure sperm volume?

A
  • Direct volume measurement (common in diagnostic setting)
  • Volume from weight
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13
Q

Describe direct volume measurement

A
  • Serological pipette attached to electronic pipette
  • Aspirate sample from container -> Volume (ml) measured using graduate scale
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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)
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15
Q

What can you use to calculate sperm concentration?

A
  • Haemocytometer

(can be used to calcite [RBC] & [sperm]

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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
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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
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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
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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

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20
Q

How do you count the sperm?

A
  • Many different ways, depends on sample & dilution factor (refer to WHO)
  • one method of counting:
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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)

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22
Q

Which sperm to count in a box and which to not?

A
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23
Q

Definition of [sperm]

A
  • quantity of sperm present in a sample
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24
Q

[sperm] units

A
  • millions/ml
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25
Q

How is [sperm] determined?

A
  • counting chamber
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26
Q

How many types of counting chambers?

A

2 types:
- Neubauer haemocytometer (recommended)
- Makler counting chamber

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27
Q

Reference values of [sperm]?

A

≥16million/ml

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28
Q

[seprm] below ref value is called?

A

-Oligozoospermia

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29
Q

If a male is oligospermic, is this clinically significant?

A
  • 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
Q

Sperm concentration is also known as?

A
  • Sperm density
  • Sperm count
31
Q

When is sperm motility assessed?

A
  • ASAP after sample liquefaction
  • within 1 hr following ejaculation,
  • to limit deleterious effects of dehydration, pH or changes in temperature on motility.
32
Q

What must you do in the lab when assessing sperm motility due to the time-sensitivity?

A
  • 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
Q

How to perform sperm motility analysis?

A

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
Q

What are the different categories of sperm motility?

A

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
Q

What are the WHO ref values for sperm motility?

A
  • a+b≥30%
  • a+b+c≥42%
36
Q

What is the term when sperm motility is below ref values?

A
  • Asthenozoospermia
    (sample is asthenbozoospermic)
37
Q

What do you use to keep count of sperm when assessing sperm motility?

A
  • Counter
38
Q

Which sperms to count and which sperms to include in your counts?

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
  • 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
Q

How is sperm morphology assessed?

A
  • Assessed directly on the wet preparation
  • Using stains
40
Q

Where are the different techniques of assessing morphology done?

A
  • 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
Q

What are the different type of stains?

A
42
Q

How do you use stains for morphology analysis?

A
  • Sperm smeared on slides
  • fixed & immobilised before staining protocol of choice is applied.
43
Q

Which stain gives better nuclear detail?

A
  • Papanicolaou
44
Q

What is the normal morphology of sperm head?

A
  • 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
Q

What is the normal morphology of sperm tail?

A
  • 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
Q

Where is the nuclear material contained in sperm?

A
  • Postacrosomal region
47
Q

How was normal morphology of sperm determined?

A
  • Sperm successfully pass the cervix of female tract, analysed.
48
Q

What is the term when morphology is below the reference values?

A
  • Teratozoospermia
49
Q

Types of different abnormal sperm morphology

A
50
Q

What is sperm vitality a measure of?

A
  • number of sperm alive
51
Q

How is sperm vitility determined?

A
  • By assessing membrane integrity of spermatozoa
  • Important for samples with low progressive motility.
52
Q

When is sperm vitality measured?

A
  • 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
Q

How is sperm vitality tested in labs?

A
  • 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
Q

What is the permeability status of dead and live cells?

A
  • Dead: Permeable
  • Alive: Impermeable
55
Q

How is vitality measured quantitatively?

A
  • Number of spermatozoa with intact membrane expressed as % live spermatozoa.
55
Q

How is vitality measured quantitatively?

A
  • Number of spermatozoa with intact membrane expressed as % live spermatozoa.
56
Q

What are the variations of hypo-osmotic swellings?

A
57
Q

What is vitility values below reference values called?

A
  • Necrozoospermia
58
Q

Types of necrozoospermia?

A
  • Incomplete Necrozoospermia = live cells <45% but >5%
  • Complete Necrozoospermia = all cells dead in sample
59
Q

What is HOS medium made up of?

A

0.74% sodium citrate + 1.35% fructose in double-distilled H2O

60
Q

How is sperm pH determined?

A
  • pH strips - dip strips in sample
61
Q

What is the normal pH range of sperm sample?

A
  • 7.2-8.0
62
Q

What is the clinical significance of pH levels in sperm?

A
  • 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
Q

When is pH assessed?

A
  • ASAP after liquefaction of semen sample, preferably at 30 minutes, but no later than 1 hour post-ejaculation
  • loss of CO2
64
Q

How can we test for presence of leukocytes in sperm sample?

A
  • Count round cells using counting chamber
65
Q

What is the value of round cells to be considered a concern?

A
  • 1million/ml
66
Q

When is immunocytochemical staining used when looking for leukocytes?

A
  • Round cell numbers are >1 million/ml after using counting chamber
67
Q

What is an example of immunocytochemical staining for CD45-bearing leucocytes?

A
  • Granulocyte peroxidase
68
Q

What do high numbers of leukocytes indicate?

A
  • Infection in male reproductive tract
69
Q

Are all round cells leukocytes?

A
  • No,
  • Some count be germ cells that are present due to incomplete spermatogenesis
69
Q

Are all round cells leukocytes?

A
  • No,
  • Some count be germ cells that are present due to incomplete spermatogenesis

SoR (12 decks)

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