Semen
• Normal semen is an admixture of
spermatozoa suspended in secretions
(SEMINAL PLASMA ) from the glandular tissue
of the male genital system.
• Semen analysis is an evaluation of
spermatogenesis and spermiogenesis
INDICATIONS FOR SEMEN ANALYSIS
• Infertility
• Hypogonadism
• Follow up after vasectomy
• storage of semen before radiotherapy
Sample Collection
• The container has to be sterile and known NOT to be spermotoxic
(i.e. provided by the lab)
• The man must have had 3 – 5 days of abstinence
• The man must have washed his hands before collection
(particularly if microbiological analysis is requested)
• The sample must be kept at 37°C until analysis, which begins
ideally within 30 min, but absolutely within 60 min, of ejaculation
Macroscopic Evaluation-Appearance
• Colour (normal = white to grayish-yellow) – if
there is blood present, it may range from pink
to brown
• Opacity / translucence (normal = tends to
opaque)
• Whether mucus streaks or cell clumps are
present
Liquefaction
• Liquefaction is the breakdown of the gel portion
of the seminal plasma – the enzymes for this
are in the prostatic fluid
• At room temperature ,normal semen gets
liquefied within 30 minutes after collection.
• Presence of mucous streaks indicate incomplete
liquefaction
Viscosity
• After liquefaction aspirate sample by wide-
bore plastic pipette
• Normal Viscosity: Small discrete drops
• Abnormal Viscosity: Drop form thread > 2 cm
Volume
• The volume of sample should be measured in a graduated tube or a
small cylinder to the nearest 0.1 ml.
• Normal volume of ejaculate semen is 1.5 to 4.5 ml.
• If the volume of semen is below 1.5 ml , it is LOW VOLUME.
• Conditions which can lead to hypospermia or decreased volume are
• 1)DISORDERS OF PROSTATE GLAND & SEMINAL VESICLES.
2)RETROGRADE EJACULATION
• 3)CONGENITAL ABSENCE OF PROSTATE OR SEMINAL VESICLES.
4)OCCLUSION OF THE EJACULATORY DUCT
• 5)REPEATED SAMPLE COLLECTION.
PH
• The pH of the semen can be measured with pH paper ,
indicator dyes or pH mater. It depends upon sperms
conc.
• The pH should be measured within an hour of
ejaculation & should be in range of 7.2 – 8.0.
• If pH is less than 7.0 , dysgenesis of vas deferens,
seminal vesicles or epididymis may be present.
• Bacterial contaminated semen & semen with dead
sperms may produce ammonia which increases the pH
to make it alkaline.
SPERM COUNTING
• Visual assessment:
Examine semen slide under the high power objective of
a microscope to determine the need for any dilution.
• The diluent used is 3.5% buffered formal saline.
Gentian violet can be added to this fluid to stain the
sperms.
• Improved Neubauer chamber (Haemocytometer)is
used for counting.
• At least 200 spermatozoa must be counted.
• Sperm count (million/ml) = CxDx1000/V
• Sperm count 60-150 million/ml
ASSESSMENT OF SPERM MOTILITY
• 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.
• Only forward movement of the sperms is taken
as positive.
Motile sperm count=Sperm count/ml×%motility×semen vol/100
• Motility >70% at 1 hour and 50% at 3 hours after ejaculation
• 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
• Grade x Percentage =
• 0 x 30 0
• 1 x 10 10
• 2 x 15 30
• 3 x 30 90
• 4 x 15 60
• Total score 190
Normal motility score for spermatozoa is ≥150.
ASSESSMENT OF SPERM MORPHOLOGY
• A normal sperm consists of a head and a tail
joined together by a short neck.
• Assessment can be made in a wet preparation
or in a stained smear of semen.
• Stained by haematoxylin and eosin or
Papanicolaou or Giemsa.
• Abnormalities of the head including small,
large, tapering, pyriform, amorphous and
double heads.
• Abnormalities of the tail including double,
coiled or short tails
• >70% should be morphologically normal
• Also make a note of the presence of white
blood cells, epithelial cells, red blood cells,
lymphocytes, extraneous particles, protozoa
and bacteria
AGGLUTINATION
• Agglutination means motile spermatozoa stick to one another.
• Adherence of immotile sperms to one another or motile sperms to mucous
threads should be considered NON SPECIFIC AGGREGATION.
• The presence of AGGLUTINATION is suggestive of immunological cause of
infertility.
• Agglutination should be assessed in 20 random fields & avg. %age of
sperms clumped together is estimated .
• Type of agglutination is also noted i.e. HEAD to HEAD, TAIL to TAIL ,HEAD to
TAIL & MIDPIECE to TAIL.
• All types of agglutinations could be due to ANTI SPERM ANTIBODIES &
should be further subjected to IMMUNOLOGICAL TESTS
IMMUNOLOGICAL TESTS
• ANTI SPERM ANTIBODY ( ELISA ) :
• PRINCIPLE : The ELISA antisperm antibody kit uses sperm antigen which are
extracted from pool of treated sperms attached to wells of microlitre plates.
• Serial dilutions of sera are attached to the wells & after incubation , the
amt. of bound antibodies is measured by appropriate antisperm ab’s . This
test require serum.
• INTERPRETATION : a positive test is indicated by o.d of at least 0.3 at 405
nm.
• The dilution of 1/32 or more as antisperm ab’s is positive , while dilution of
1/16 is considered borderline
FRUCTOSE TEST
• FRUCTOLYSIS :
• Fructose is main sugar present in seminal plasma & is imp. nutrient for
the sperms.
• The quality of semen can be assessed by measuring the rate of
utilization of fructose.
• FRUCTOLYTIC INDEX is the amount of fructose used or lactic acid formed
by spermatozoa per hour at 37 deg.
• The semen sample should be well buffered otherwise the fructolysis will
stop at certain stage & result will be erroneous.
FRUCTOSE TEST
• Fructose is absent from the semen of patients with
bilateral aplasia of the vasa differentia and seminal
vesicles. It is also absent in bilateral obstruction of
the ejaculatory ducts.
• Place 0.1 ml of semen in a test tube. Add to it 1ml
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
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 (<30 million/ml).
Polyzoospermia: High sperm count (>300 million/ml)
Asthenozoospermia: Absence or marked reduction in sperm
motility (Motility score<150)
Oligoasthenozoospermia: Low count with low motility.
Necrospermia: Dead sperm
