Table 5 Methodological differences between the three versions of WHO guidelines

WHO guildline of 1992

(Third Edition) [26]

The volume of the ejaculate should be measured either in a graduated cylinder with a conical base or by aspirating the whole sample into a wide mouthed pipette by means of a mechanical device

The concentration of spermatozoa should be determined using the haemocytometer method, containing more than 100 × 10⁶ spermatozoa/ml, a 1:50 dilution may be appropriate. White-blood-cell pipettes and automatic pipettes relying upon air displacement are not accurate enough for making volumetric dilutions of such viscous material as semen. White-blood-cell pipettes and automatic pipettes relying upon air displacement are not accurate enough for making volumetric dilutions of semen. A positive-displacement type of pipette should be used

Each spermatozoa is graded 'a', 'b', 'c', or 'd'. Usually four to six fields have to be scanned to classily 100 successive spermatozoa, yielding percentage for each motility category. The count of 100 spermatozoa repeated and the average values calculated for each category

At least 100, and preferably 200, spermatozoa are counted. With stained preparations, a 100 × oil-immersion bright-field objective without a phase ring should be used

WHO guildline of 1999

(Fourth Edition) [11]

The volume of the ejaculate may be measured using a graduated cylinder with a conical base or by weighing standard containers with and without semen

The concentration of spermatozoa should be determined using the haemocytometer method on two separate preparations of the semen sample, one for each side of the counting chamber. The dilution is determined (1:5, 1:10, 1:20, 1:50) from the preliminary estimation of sperm concentration. White-blood-cell pipettes and automatic pipettes relying upon air displacement are not accurate enough for making volumetric dilutions of semen. A positive-displacement type of pipette should be used scanning the slide and estimating the number of spermatozoa per field or part of a field equivalent to 1 nl gives an approximate sperm concentration in 10⁶/ml. This estimate is used to decide the dilution for determining the sperm concentration by haemocytometry: < 15 spermatozoa, dilution 1:5;15–40 spermatozoa, dilution 1:10;40–200 spermatozoa, dilution 1:20; > 200 spermatozoa, dilution 1: 50

At least five microscopic fields are assessed in a systematic way to classify 200 spermatozoa. The motility of each spermatozoa is graded 'a', 'b', 'c', or 'd', according to whether it shows

With stained preparations, a 100X oil-immersion bright-field objective and at least a 10X ocular should be used. At least 200 consecutive spermatozoa are counted (assessing 200 once is better than l00 twice). Although it is preferable to count 200 spermatozoa twice to reduce counting error and variability

WHO guildline of 2010

(Fifth Edition) [12]

The volume is best measured by weighing the sample in the vessel in which it is collected. Alternatively, the volume can be measured directly.

1.Collect the sample directly into a modified graduated glass measuring cylinder with a wide mouth. These can be obtained commercially

2. Read the volume directly from the graduations

The concentration of spermatozoa in semen is their number (N) divided by the volume in which they were found, i.e. the volume of the total number (n) of rows examined for the replicates.

Sperm count > 101 per 400 × field of view,

 > 404 per 200 × field of view, dilution 1:20;

sperm count 16–100 per 400 × field of view;

64–400 per 200 × field of view, dilution 1:5;

sperm count 2–15 per 400 × field of view;

8–60 per 200 × field of view, dilution 1:2;

sperm count < 2 per 400 × field of view Dilution 1:5;

 < 8 per 200 × field of view

Examine the slide with phase-contrast optics at × 200 or × 400 magnification. Assess approximately 200 spermatozoa per replicate for the percentage of different motile categories. A simple system for grading motility is recommended that distinguishes spermatozoa with progressive or non-progressive motility from those that are immotile. The motility of each spermatozoa is graded as follows:

Progressive motility (PR); Non-progressive motility (NP) and Immotility (IM)

Examine the slide using bright field optics at × 1000 magnification with oil immersion. Evaluate at least 200 spermatozoa in each replicate, in order to achieve an acceptably low sampling error. Repeat the assessment of at least 200 spermatozoa, preferably on the replicate slide, but alternatively on the same slide