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WHO 5TH EDITION STANDARD for SEMEN ANALYSIS

BY : WAFA ALAHMED

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AIM

To standardize semen analysis method

To implement a good practice for the entire analytical process based on the WHO 5th standard.

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Learning OutcomesRecognize 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.

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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 daysColor : opaque greyVolume : 0.5 mlPH: 8.0Liquefaction time: 45 min There was no sperms seen on wet preparation.

How you will report the result?

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Sample collection for microbiologyPass urine Clean the area with soap Rinse with waterBlot dry with disposable towelCollect sample in sterile containerProcess the sample within 3 hours. Why?

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Make sure to maintain Sample Temperature through the whole analysis.

Why?

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Appearance of Semen SampleNormal:

Homogenous, Opaque Grey colorAbnormal:

*Less Opaque Why? Very Low Sperm Concentration *Red-brown Why? RBCs *Yellow Why? Vitamins, Drugs, or icteric *Creamy Why? WBCs

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Liquefaction timeWatery like R.R < 60 minutesIf > 60 minutes How to overcome this problem?

Mechanical MixingJelly-like granules

Has no significanceMucus strands Interferes with Semen

Analysis

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Semen ViscosityEstimation of ViscosityWhen?

After LiquefactionHow?

Aspirate sample by wide-bore plastic pipette

Normal Viscosity: Small discrete drops

Abnormal Viscosity: Drop form thread > 2 cm

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Semen VolumeMeasures by Weight Why?Low Semen Volume Why? 1.Obstruction of ejaculatory duct 2.Congenital absence of vas deference 3.Collection problemHigh Semen Volume Why? Active exudation due to inflammation

of accessory organs.R.R 1.5 - 6.5 ml

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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 daysColor : opaque greyVolume : 12 mlPH: 8.0Liquefaction time: 45 min

How you will report the result?

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Semen PHMeasured 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

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Role Play

How to instruct a patient for semen sample collection for routine analysis?

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Microscopic investigations at x100 total magnification

Mucus strand formationSperm aggregation & agglutination

Cells other than spermatozoa

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Sperm Aggregation

Adherence of immotile sperms to each other or motile sperms to mucus strands, non-sperm cells or debris.

a. b. c. d. Views of spermatozoa aggregated with an epithelial cell (a), debris (b) or spermatozoa (c, d).

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Sperm Agglutination

Motile sperms sticking to each other by their heads, tails or mid-pieces.

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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

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Microscopic investigations at x400 total magnification

Assessment of sperm motilityDetermination of dilution required for accurate assessment of sperm number

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Important Notes

Thorough mixing

Avoid bubble formation

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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

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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

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Wet PreparationMix the semen sample wellTransfer standard volume 10 μl on clean slideCover with 22mm x 22mm coverslipTo 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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Wet PreparationExamine at x400 Look for sperms in area 5mm from the edgeScan slide randomly & score PR, then NP,

then IMCount sperms of each category using counterEvaluate 200 sperms in each replicateHigh sperm concentration -- score random

fieldsLow sperm concentration ---score entire

slideCalculate average & difference between two

percentageDetermine acceptability difference

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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.

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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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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 (%)

*Acceptable

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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Learning OutcomesRecognize 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.

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?

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Thank you