2. Topic: SEMEN ANALYSIS | By: M.danish | Date: 22/04/2016
• Semen consists of spermatozoa suspended in plasma like fluid and is
formed at ejaculation.
• Only the spermatozoa and a small amount of secretions are produced
in the testis and the epididymis (5% of total volume)
• Bulk of the semen consists of the secretions of seminal vesicles (46-
80%) and prostate (13-33%).Bulbourethral and urethral glands
contribute about 2-5% of total volume.
• Initially the sperms are not motile but while passing through the
epididymis they acquire motility by virtue of carnitine that is added to
the seminal fluid in epididymis.
SEMEN ANALYSIS
3. SEMEN ANALYSIS
• Seminal vesicles provide an important energy source in the form of
fructose that enhances motility of spermatozoa.
• Prostaglandins and fibrinogen like substances are also added to
seminal fluid by seminal vesicles.
• Prostatic fluid provides a number of
• enzymes, spermine (a bacteriostatic substance), citrates, calcium and
zinc.
• The bulbourethral and urethral glands contribute mucoproteins and
IgA to semen
Topic: ABC | By: EFG | Date:
4.
5. INDICATIONS FOR SEMEN ANALYSIS
• These include
• Infertility,
• hypogonadism,
• Follow up after vasectomy,
• prior to donations for artificial insemination and storage of semen
before radiotherapy
6. SAMPLE COLLECTION
• A period of abstinence is important as it affects both the quantity and
motility of spermatozoa.
• Ordinarily abstinence for 3-5 days is adequate.
• The specimen should be collected in the morning to allow sufficient
time for its analysis.
• Masturbation is the ideal method for producing the semen specimen.
• Condoms must never be used for collection of semen by intercourse
• Note the volume, colour, appearance and the pH.
7. Continue
• A clean, dry, wide-mouth glass or plastic jar should be used as semen
container.
• Its lid must not be rubber lined. Detergents, water and rubber are
injurious to sperms.
• Specimen should be transported to the laboratory at a temperature
as close to 37°C as possible and delivered to the laboratory in less
than 2 hours.
8. PHYSICAL EXAMINATION
• Transfer the semen into a clean graduated small cylinder.
• Note the volume, colour, appearance and the pH.
• Normally, the human semen, soon after ejaculation, forms a gel-like
clot that liquefies in 5-20 min.
• Absence of liquefaction in a semen sample must be noted.
9. Viscosity
• Viscosity of semen should be assessed.
• It can be measured by dropping a drop of semen from a 10 cm long
capillary tube containing 0.1 ml semen.
• Time taken by the drop to form and leave the capillary tube is a
measure of its viscosity.
10. SPERM COUNTING
• Visual assessment
• Place a drop of semen on a clean glass slide
• place a cover slip over it.
• Examine the slide under the high power objective of a microscope
• make a visual assessment of the sperm count and to determine the need
for any dilution.
• Dilution
• The diluent used is 3.5% buffered formal saline
• Normally 1 in 20 dilution is made by adding 50 μl of well-mixed and
liquefied semen to 950 μl of diluent .
11. Counting procedure
• Improved Neubauer chamber (Haemocytometer) is used for counting
• The diluted semen is carefully mixed and the chamber is charged
using a Pasteur pipette.
• The chamber is then examined by using x10 objective of microscope.
• Sperms are counted in the four large corners and one large central
square.
12. Continue
• It is important that loose tails and germinal cells are not counted. At least
200 spermatozoa must be counted If these are not available in these
5squares, more squares must be counted.
• Calculation:-
• Where
• C = Count in 5 large squares
• D = Dilution factor
• V = Vvolule of 5 large s1ures
• 1000 = To convert mm3 into ml
13. ASSESSMENT OF SPERM MOTILITY
• Assessment of motility must be performed soon after production of
sample, 3 and 6 hours later.
• Both motile and immotile sperms are counted at least in 5 fields with
a minimum count of 200.
• The count should be performed in duplicate and the average
recorded.
• Only forward movement of the sperms is taken as positive
• Percentage motility is then calculated.
14. Continue
• The sperm count can be calculated using the
• formula:
• Motile sperm count= Sperm count/ml % motility semen vol/100.
• More objective results can be obtained by following procedure.
• About 30 min after collection transfer the semen in a capped tube.
Gently mix by inverting the tube several times.
• Pipette one drop of semen onto a clean glass slide; place a clean
cover slip over it.
15. Continue
• Observe with a x40 objective and estimate the percentage of
spermatozoa moving at
• following speeds:
• Grade 0: No movement at all
• Grade 1: Moving with no forward progression.
• Grade 2: Moving with slow and wandering movement.
• Grade 3: Moving rapidly in almost straight line
• Grade 4: Moving with high speed in straight line
16. • Calculate a motility score by adding up the product motility grade and
percentage of spermatozoa in that grade. Example is as under
17. • Normal motility score for spermatozoa is ≥150.
• Motility depends upon temperature. At 37°C only 50% sperms are
motile after 3 hours.
• Exposure to cold: Return of sperm motility after placing the semen
sample for 30 min in the incubator is diagnostic of reduced motility
due to cold.
• Infection: Manifested by the presence of excess white cells or
bacteria. Bacterial culture will help.
18. continue
• Fructose deficiency:
• Addition of an equal volume of warm Bakers buffer (3.0g glucose,
• 0.46g Na2HPO4 7H2O, 0.2g NaCl, 0.01g KH2PO4 and distilled water
up to 100 ml) to an aliquot of semen on a glass slide will produce a
resumption of sperm motility if due to fructose deficiency
19. REPORTING
• Some of the special terms used for reporting the results of semen analysis
are:
• Aspermia: No ejaculate.
• Oligospermia/Hypospermia: Reduction in volume of ejaculate.
• Hyperspermia: Increase in volume of ejaculate.
• Oligozoospermia: Low sperm count (<30million/ml).
• Polyzoospermia: High sperm count (>300million/ml)
• Asthenozoospermia: Absence or marked reduction in sperm motility
(Motility score<150)
• Necrospermia: Dead sperm
20. ASSESSMENT OF SPERM MORPHOLOGY
• A normal sperm consists of a head and a tail joined together by a
short neck.
• The head is oval in shape and measures 4.5x3x1.5 μm while the tail is
about 50 μm long.
• Assessment of morphology can be made in a wet preparation or in a
stained smear of semen.
• The slides are then examined under oil immersion objective of the
microscope.
• Abnormalities of the head including small, large, tapering, pyriform,
amorphous and double heads
21. Continue
• the tail including double, coiled or short tails and of the mid-pieces
should be noted.
• REPORTING:
• Some of the special terms used for reporting the results of semen
analysis are:
• Aspermia: No ejaculate.
• Oligospermia/Hypospermia: Reduction in volume of ejaculate.
• Hyperspermia: Increase in volume of ejaculate.
22. continue
• Oligozoospermia: Low sperm count (<30million/ml).
• Polyzoospermia: High sperm count (>300million/ml)
• Asthenozoospermia: Absence or marked reduction in sperm motility
(Motility score<150).
• Oligoasthenozoospermia: Low count with low motility.
• Necrospermia: Dead sperm
24. REFERENCE RANGE
• Volume 2-6 ml (1-10 ml)
• Colour Grey-yellow
• Appearance Opalescent
• Viscosity Viscous
• pH 7.2-8.9
• Sperm count 60-150 million/ml (Extreme range 30-300 million/ml)
• Motility >70% at 1 hour and 50% at 3 hours after ejaculation
• Motility score ≥150
• Morphology >70% should be morphologically normal
25. TESTING FOR ANTI-SPERM ANTIBODIES
• Testing for anti-sperm antibodies is as important in the evaluation of
infertile male.
• Agglutination tests, sperm immobilising antibody tests, testing for
cytotoxic antibodies are the various methods for demonstrating
sperm antibodies.
26. FRUCTOSE TEST
• Fructose is absent from the semen of patients with bilateral aplasia
and also absent in bilateral obstruction of the ejaculatory ducts.
• Procedure:
• Place 0.1 ml of semen in a test tube.
• Add to it 1 ml of resorcinol reagent.
• Boil for 5-10 min.
• The solution turns reddish brown in the presence of fructose.
• No change in colour indicates absence of fructose from the semen.
27. IMPORTANT NOTES
• An important cause of aspermia is retrograde ejaculation (ejaculation
backwards into urinary bladder). In all cases when ejaculate is not
obtained, a urine specimen should be immediately collected and
examined for spermatozoa.
• An immotile sperm does not necessarily mean a dead sperm. It is
important to distinguish between asthenozoospermia and
necrospermia.
• For evaluating infertility, semen analysis should be performed on
three occasions with a gap of 2-3 weeks between any two analyses.
