Semen Analysis

Practical Guide To
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A. INTRODUCTION
This information is being published to assist laboratory personnel determine the
general quality of semen.
The basic aim of semen analysis is to evaluate descriptive parameters of
spermatozoa and seminal fluid in other to define its general quality.
The parameters that are usually assessed include;
♣ Appearance
♣ Odour
♣ Liquefaction
♣ Viscosity
♣ pH
♣ Volume
♣ Sperm concentration
♣ Sperm motility
♣ Sperm morphology
♣ Sperm vitality
Normal values of semen parameters issued by the WHO in 1992 are generally
used as reference values (Table I).
Ideally, each laboratory should set its own normal values, reflecting the specific
population analyzed.
Depending on the specifications of the doctor, all the parameters above might
not be important in your findings.

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B. GENERAL OVERVIEW
1. Normal values
Table 1: Normal values of semen variables
Standard tests Expected Values
volume 2.0 – 5.0
pH 7.2-8.0
Colour Gray-white
Liquefaction 15 – 60 minute
Viscosity Normal
Sperm concentration ≥20x106
cells/ml (20 000cells/µl)
Total sperm count ≥40x106
cells/ejaculate (40 000cells/ejaculate)
Motility ≥50% with forward progression (categories “a” and “b”) or
≥25% with rapid progression (category “a”) within 60 minutes
of ejaculation
Morphology ≥30% with normal forms
Vitality ≥75% or more live, i.e.,excluding dye
White blood cells <1x106
/ml (1 000/µl)
Immunobead test <20% spermatozoa with adherent particles
MAR test <10% spermatozoa with adherent particles
Optional tests
αααα -Glucosidase(neutral) 20 mU or more per ejaculate
Zinc(total) 2.4 µ -mol or more per ejaculate
Citric acid(total) 52 µ -mol or more per ejaculate
Acid phosphatase(total) 200 U or more per ejaculate
Fructose(total) 13 µ -mol or more per ejaculate

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2. Normencleture
Table 2: Normenclature for semen variables
Variable Meaning
Hypospermia Semen volume <2.0ml
Hyperspermia Semen volume >5.0ml
Normozoospermia Normal ejaculate as defined above (No abnormality
detected).
Oligozoospermia Sperm concentration <20x106
/ml
Asthenozoospermia <50% spermatozoa with forward progression
(categories a and b) or
<25% spermatozoa with category a movement.
Teratozoospermia <30% spermatozoa with normal morphology
Oligoasthenoteratozoospermia Alteration in all three variables (combination of only
two prefixes can be used)
Azoospermia no spermatozoa in the ejaculate
Aspermia no ejaculate
C. SUMMARY OF PROCEDURE
1. Sample collection and delivery (WHO recommendation)
The subject should be provided with clearly written or oral instructions
concerning the collection and, if required, transport of the semen sample.
The sample should be collected after a minimum of 48 hours and no
longer than 7 days of sexual abstinence.
The sample should be obtained by masturbation and ejaculated into a
clean, wide-mouthed glass or plastic container. If plastic is used, it should
be checked for lack of toxic effects on spermatozoa. The container should
be warm to minimize the risk of cold shock.
Condoms and other methods like coitus interuptus are discouraged.
Incomplete samples should not be analyzed, particularly if the first
portion of the ejaculate is lost. Sample should be brought to the
laboratory maintained at 20 to 40o
C

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2. Macroscopic evaluation
Appearance
The semen sample is first evaluated by simple inspection. A normal sample has
a grey-opalescent appearance.
The sample may appear clear if the sperm concentration is too low. It may also
appear brown when red blood cells are present in the ejaculate
(haematospermia).
Samples which do not liquefy need additional treatment such as exposure to
bromelin, to make the sample amenable to analysis.
The sample should be well mixed in the original container. Incomplete mixing
is probably a major contributor to errors in determining sperm concentration.
Liquefaction
Semen usually liquefies within 60 minutes. Assessment of motility is
recommended only after complete liquefaction. If not see treatment of viscous
samples below.
Consistency (Viscosity)
The consistency, also called viscosity, of the liquefied sample can be estimated
by gentle aspiration into a 5-ml pipette and then allowing the semen to drop by
gravity and observing the length of the thread formed. A normal sample leaves
the needle as small discrete drops, while in cases of abnormal consistency the
drop will form a thread of >2 cm.
Increased consistency has the same clinical meaning as abnormal liquefaction,
and may be related to prostate dysfunction resulting from chronic inflammation.
Treatment Of Viscous Samples
High viscosity interferes with sperm motility and concentration. In such a
case viscosity can be reduced by adding a known volume of Saline,
phosphate buffer solution (PBS) or culture medium and careful mixing to
give a homogenous dilution for examination.
Volume
The volume of the ejaculate should be measured either with a graduated
cylinder or by aspirating the whole sample into a wide-mouthed pipette by
means of a mechanical device.

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pH
The pH is determined by acidic secretions of the prostate and alkaline secretions
of the seminal vesicles. It should normally be in the range of 7.2-8.0.
3. Initial microscopic investigation
This checks for sperm motility and concentration.
Motility (Using Wet preparation without adding any liquid)
10ul of completely liquefied semen is delivered onto a clean glass slide
and covered with a cover slip.
The freshly made, wet preparation is left to stabilize for approximately
one minute.
The microscopic field is scanned systematically and the motility of each
spermatozoon encountered is graded as A, B ,C or D depending on the
following observations:
CATEGORY INTERPRETATION
A Linear Rapid Progressive Motility
B Slow or Sluggish Profressive Motility
C Non-Progressive Motility
D Immotility
At least 100 spermatozoa are classified in this way. Visual field close to
the border of the coverslip should be avoided.
It is advisable to repeat the procedure at different intervals (over 24
hours) on different drop of semen processed in the same way.
Assessment Of appropriate Dilution
The wet preparation is used to estimate the concentration and select the most
accurate dilution. When Azoospermia is suspected, centrifugation may be
needed.
Dilution of the ejaculate
Spermatozoa/HPF Dilution Semen(µl) Diluent (µl)
Swim up 1+1 [1:2] 100 100
<15 1+4 [1:5] 100 400
15 - 40 1+9 [1:10] 50 450
40 - 200 1+19 [1:20] 50 950
>200 1+49 [1:50] 50 2450

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Spermatozoa Count Proper
The Neubauer Counting chamber is used in counting.
Prepare the chamber and load as you would do for RBC count
Determine the number of squares that should be counted. First count the
number of spermatozoa in the upper, left corner square.
<10 sperm Count all the grid (25 squares)
10 – 40 sperm Count 10 squares in each chamber
>40 sperm Count 5 squares (4 corners and 1 central) as for RBC.
Another way is to count the sperm as for WBC, i.e counting is done in the 4
WBC zone.
Calculation
Using the WBC: [Number Of sperm counted x 100 000]/ml
4. Morphology
Make a thin film by sliding one slide over the other containing a drop of
semen.
Stain with Giemsa and air-dry it.
Assess 100 – 200 sperm cells under oil immersion for the following
defects, scoring each defect as a percentage.
The following categories of defects should be scored.
Head shape/size defects, including large, small, tapering, pyriform,
amorphous, vacuolated (>20% of the head area occupied by unstained
vacuolar areas), or double heads, or any combination of these.
Neck and midpiece defects, including absent tail, non inserted or bent tail
(the tail forms an angle of about 90° to the long axis of the
head),distended/irregular/bent midpiece, abnormally thin midpiece or any
combination of these.
Tail defects, including short, multiple, hairpin, broken, irregular width, or
coiled tails, tails with terminal droplets, or any combination of these.
Cytoplasmic droplets greater than one-third of the area of a normal sperm
head.
For infertility assessment, 2 – 3 specimens collected at about 2 weeks interval
might be needed to draw final conclusion.

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This information is not meant to replace the knowledge of your
personal findings. Remember it is just to supplement, so do more
research. You might find it necessary to modify this article.
We Thank You For Using Our Article‼

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© July 2011. LaboHDS
Compiled by
Nkengacha Marcellous Agendia
Clinical Laboratory Scientist
District Laboratory, Sangmelima
E-mail: labo_hds@yahoo.com
Website: www.labohds.wordpress.com

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