2. AIM
To standardize semen analysis method
To implement a good practice for the entire
analytical process based on the WHO 5th standard.
2
3. Learning Outcomes
Recognize the importance of WHO 5th edition for
standardizing the semen analysis.
Recall basic knowledge for Sample Collection & handling.
Assess liquefaction time, viscosity & PH.
Implement the new concept for measuring sample volume.
Identify the categories of sperm movement according to
this standard.
Determine the average motility using 95% CI table.
Apply the new technique for assessing sperm motility on
patient & External QC proficiency testing samples.
3
4. Case Study:
a 35 years old male brought a semen sample
within 30 min of collection to the laboratory
for routine semen analysis.
Abstinence: 3 days
Color : opaque grey
Volume : 0.5 ml
PH: 8.0
Liquefaction time: 45 min
There was no sperms seen on wet preparation.
How you will report the result?
4
5. Sample collection for microbiology
Pass urine
Clean the area with soap
Rinse with water
Blot dry with disposable towel
Collect sample in sterile container
Process the sample within 3 hours.
Why?
5
6. Make sure to maintain Sample
Temperature through the whole
analysis.
Why?
6
7. Appearance of Semen Sample
Normal:
Homogenous, Opaque Grey color
Abnormal:
*Less Opaque Why?
Very Low Sperm Concentration
*Red-brown Why?
RBCs
*Yellow Why?
Vitamins, Drugs, or icteric
*Creamy Why?
WBCs
7
8. Liquefaction time
Watery like
R.R < 60 minutes
If > 60 minutes
How to overcome this problem?
Mechanical Mixing
Jelly-like granules
Has no significance
Mucus strands
Interferes with Semen Analysis
8
9. Semen Viscosity
Estimation of Viscosity
When?
After Liquefaction
How?
Aspirate sample by wide-bore plastic pipette
Normal Viscosity:
Small discrete drops
Abnormal Viscosity:
Drop form thread > 2 cm
9
10. Semen Volume
Measures by Weight Why?
Low Semen Volume Why?
1.Obstruction of ejaculatory duct
2.Congenital absence of vas deference
3.Collection problem
High Semen Volume Why?
Active exudation due to inflammation of
accessory organs.
R.R 1.5 - 6.5 ml
10
11. Case Study:
a 28 years old male brought a semen sample within 30
min of collection to the laboratory for routine semen
analysis.
Abstinence: 3 days
Color : opaque grey
Volume : 12 ml
PH: 8.0
Liquefaction time: 45 min
How you will report the result? 11
12. Semen PH
Measured after 30 min of sample collection
How ?
1. Mix sample
2. Spread drop on PH paper
3. Wait < 30 sec
R.R 7.2 - 8.0
12
13. Role Play
How to instruct a patient for semen
sample collection for routine analysis?
13
14. Microscopic investigations
at x100 total magnification
Mucus strand formation
Sperm aggregation & agglutination
Cells other than spermatozoa
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15. Sperm Aggregation
Adherence of immotile sperms to each other or
motile sperms to mucus strands, non-sperm cells
or debris.
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a. b. c. d.
Views of spermatozoaaggregatedwithanepithelialcell(a),debris (b)orspermatozoa(c,d).
17. 17
Parts
involved
Degree of agglutination
1. Isolated
(<10 sperm/
agglutinate,
many free
sperm)
2. Moderate
(10–50 sperm/
agglutinate,
free sperm)
3. Large
(agglutinates
>50 sperm,
some sperm still
free)
4. Gross (all
sperm agglutinated,
and
agglutinates
interconnected)
A. Head-to-
head
B.Tail-to-tail
(heads
are seen to be
free and
move clear of
agglutinates)
B. Tail-tip-to-
tail-tip
D. Mixed (clear
headto-
head and tail-
to-tail
agglutinations)
E. Tangle
(heads and
tails enmeshed.
Heads
are not clear of
agglutinates
as they are in
tailto-
tail
agglutination)
Schematic diagram of different extents of sperm agglutination
18. Microscopic investigations
at x400 total magnification
Assessment of sperm motility
Determination of dilution required for
accurate assessment of sperm number
18
20. Categories of sperm motility:
Progressive (PR)
Active movement regardless of speed
- Linear
- In large circle
Non-progressive (NP)
Motility without progression.
Immotility (IM)
No movement
20
21. Categories of sperm motility:
Progressive (PR)
Active movement regardless of speed
- Linear
- In large circle
Non-progressive (NP)
Motility without progression.
Immotility (IM)
No movement
21
22. Wet Preparation
Mix the semen sample well
Transfer standard volume 10 μl on clean slide
Cover with 22mm x 22mm coverslip
To provide chamber depth of 20 μm (why?)
depth < 20 μm
constrains sperms movement
depth > 20 μm
sperms move in & out of focus
Assess when sample stop drifting
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23. Wet Preparation
Examine at x400
Look for sperms in area 5mm from the edge
Scan slide randomly & score PR, then NP, then IM
Count sperms of each category using counter
Evaluate 200 sperms in each replicate
High sperm concentration -- score random fields
Low sperm concentration ---score entire slide
Calculate average & difference between two percentage
Determine acceptability difference
23
24. Important Notes
Assess only intact sperms
Score three categories in the same
field until 200 sperms.
On rare in-homogenous samples—
unacceptable difference among replicates
Calculate mean and note it in the report.
24
25. Example:
Sperm motility in replicate of 200 sperms are:
PR : 60% & 68%
NP: 10% & 5%
IM: 30% & 27%
• Common category PR, average: 64%, difference 8%
• From table: acceptable difference for this average is 10
• Since diff. is 8% < acceptable diff. then result is
accepted
• Report the average: PR 64%, NP 8%, IM 28%
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26. Table: Acceptable differences between two percentages for a given average,
determined from replicate counts of 200 spermatozoa (total 400 counted)
*Based on the rounded 95% confidence interval (CI).
Average
(%)
*Acceptabl
e difference
Average
(%)
*Acceptable
difference
0 1 66-76 9
1 2 77-83 8
2 3 84-88 7
3-4 4 89-92 6
5-7 5 93-95 5
8-11 6 96-97 4
12-16 7 98 3
17-23 8 99 2
24-34 9 100 1
35-65 10
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27. Learning Outcomes
Recognize the importance of WHO 5th edition for
standardizing the semen analysis.
Recall basic knowledge for Sample Collection & handling.
Assess liquefaction time, viscosity & PH.
Implement the new concept for measuring sample volume.
Identify the categories of sperm movement according to
this standard.
Determine the average motility using 95% CI table.
Apply the new technique for assessing sperm motility on
patient & External QC proficiency testing samples.
27
