Moderator :- Dr.

Shailendra Singh Thakur

Semen is body fluid that is ejaculated
at the time of orgasm, contain sperm
& secretion of seminal vesicle,
prostate, urethral gland &
bulbourethral gland..
Source Volume SEMEN Characteristics

Urethral and 0.1-0.2cc Viscous, clear

bulbourethral glands

Testes, 0.1-0.2cc Sperm present

epididymides,vasa
deferentia

Prostate 0.5-1.0cc Acidic,watery

Seminal vesicles 1.0-3.0cc Gelatinous, fructose

positive

Complete ejaculate 1.5-5.0cc Liquefies in 20-25min

Semen is viscous, neutral or slightly
alkaline & whitish opaque.

60 % semen volume is derived from

seminal vesicle which is also a major
source of high FRUCTOSE content of
semen.
Seminal vesicle secretion also provide the
substrate for the coagulation of the semen
following ejaculation.
About 20 % of the volume of semen is
contributed by the prostate gland.

It is milky in appearance & also rich in

proteolytic enzymes are responsible for the
liquefaction of semen.
About 10 -15 % of semen volume is also
contributed by EPIDIDYMIS,
VASDEFERENS, COWPER’S GLAND &
URETHERAL GLAND.

Less than 5 % of semen volume is

contributed by Spermatozoa.
The process of ejaculation result in the
mixing of these distinct fraction of
semen.

These enter the urethra individually in the rapid

succession.
The function of first clear fluid fraction may be
to cleanse & lubricate the urethra in
preparation for the bulk of following ejaculate
- It originate from urethral & cowper’s
gland.
The second fraction consist of small amount
of secretion from epididymis & vasdeferns
& large proportion of prostatic
secretion which contain spermatozoa.
The third & final fraction consist of mucoid
secretion resulting from emptying of seminal
vesicle.

The semen specimen collected for routine

examination should contain all above mention
fraction.
Sample should be collected in wide mouth
clean & dry bottle.
1. Semen collected following 3 days of
abstinence.
2.The specimen should be collected by
MASTURBATION in the clinical pathology
laboratory.
This allow a complete examination of
semen particularly liquefication time.
The specimen should be deliverd within 30 min
to the laboratory.( This specimen may be not
satisfactory).
1. Specimen should not be collected in
ordinary condoms since the powder or
lubricant applied to the condom may be
spermicidal.

The container in which the semen sample is

collected should be free from detergent.
The semen specimen should be examined
immediately after collection.

It should be kept at room temperature.

To determine the fertility of the man.
After a male has undergone vasectomy to
check the completeness of the procedure.
In medicolegal situations such as disputes
about the paternity of a child.
After reversal of vasectomy to confirm the
success of the procedure.
HISTORY TO BE NOTED:
•Name
•Date and time of collection
•Length of abstinence
•Interval between collection and analysis
•Drug intake
•Fever
•alcohal
NOTE:
Prolonged abstinence will lead to
increased volume, but reduced
motility.
The patient should evacuate his
bladder before specimen collection. If
retrograde ejaculation is suspected,
a post-ejaculate urine sample is
collected and examined for presence
of sperms.
ASSESSMENT OF SEMEN:(according to WHO)
Standard tests:
1. Volume
2. pH
3. Sperm concentration
4. Total sperm count
5. Motility
6. Morphology
7. Vitality
8. White blood cells
9. Immunobead test
10. MAR test
VOLUME:
Measured by aspirating into a pipette or by using a
syringe(non-toxic 1,2 or 5ml)
Normal-1.5ml or more.
Low volume<1.5ml
•B/l ejaculatory duct
obstruction
•B/l congenital
vasal aplasia
•Inadequate
erection &
improper mood at
collection
•Incomplete
collection
High volume>10ml:
Dilutional
COLOUR:
Homogenous grey opalescent appearance.
After prolonged abstinence, slightly yellow.
Deep yellow- Pyospermia.
Rust colour- Small bleedings in seminal vesicles.
Red or brown indicates presence of blood.
•Trauma to the genital tract.
•Inflammation.
•Tumour of the genital tract.

Increased turbidity indicates inflammatory process in some part

of the reproductive tract.
Liquefaction
■ Immediately after ejaculation  semen is typically a semisolid

■ coagulated mass 
due to enzyme PROTEIN KINASE from seminal vesicles.
■ Liquefaction is initiated by enzymes of prostatic origin.
■ Protein fragments degraded into free amino acids and ammonia
■ Failure to liquefy indicates inadequate prostatic secretion
■ Within a few minutes  the semen begins to liquefy (become
thinner), at which time a heterogeneous mixture of lumps will be
seen in the fluid.
■ As liquefaction continues, the semen becomes more homogeneous
and quite watery,
■ In the final stages only small areas of coagulation remain.

lll
Liquefaction
■ Normal – 15- 30 minutes after collection

■ The complete sample usually liquefies within 15 minutes at room

temperature
■ If the semen does not liquefy within 30 minutes, do not proceed
with semen analysis
but wait for another 30 minutes.
■ Rarely it may take up to 60 minutes or more.

■ If complete liquefaction does not occur within 60 minutes, this

should be recorded.

■ Semen samples collected at home or by condom will normally

have liquefied by the time they arrive in the laboratory.
■ ABSENCE OF INITIAL COAGULATION :
■ Congenital absence of vas deferens, seminal vesicles or obstruction
of the ejaculatory duct

■ INCOMPLETE LIQUEFACTION:
■ Dysfunctional accessory reproductive organs like prostate 
decreased production of
prostatic enzymes.
 VISCOSITY:
■ Freshly ejaculated semen  highly viscous due to substrate produced
by
seminal vesicles.
■ The coagulum liquefies spontaneously to form a
translucent, viscous fluid by the action of enzymes of
prostatic origin.
■ Viscosity of the sample estimated  After
liquefaction
■ Procedure : gently aspirate sample into a wide-bore
5ml plastic disposable pipette  allow
the semen to drop by gravity  observe the length of
any thread.

■ A normal sample leaves the pipette in small discrete

drops.
■ High viscosity  interfere with determination of sperm
motility, sperm concentration, detection of antibody-coated
spermatozoa and measurement of biochemical markers.

■ Absence of viscosity points to reduced cell content.

■ The semen from males with b/l congenital absence of vas deferens &
seminal vesicles fails to coagulate due to absence of substrate
pH:
Measured by using a pH meter or pH paper.
Normal:7.2-8
Semen is the strongest buffer in the body.
Seminal vesicle & vas deference
secretions alkaline
Prostatic secretion acidic(due to citric acid, proteolytic
enzymes, acid phosphatase)
Motility is reduced in acidic medium.
pH<7 is associated with largely prostatic secretions due to
congenital aplasia of vas & seminal vesicles and when
contaminated with urine.
pH>8 is associated with acute infection of prostate, seminal
vesicles or epididymis.
SPERM CONCENTRATION:
■ TOTAL SPERM NUMBER” and “SPERM CONCENTRATION”
are not synonymous.

■ Sperm concentration  number of spermatozoa per unit

volume of semen
■ Normal - ≥ 15 million/ml

■ Total sperm number  total number of spermatozoa in

the entire ejaculate and is obtained by multiplying the
sperm concentration by the semen volume.
■ Normal - ≥ 40 million per ejaculate
Counting chamber

■ 100 µm deep haemocytometer chamber recommended

■ Most commonly used – Improved Neubauer chamber
■ Disposable chambers also available
■ Shallow chambers not recommended
Normal count – 15 to 150 million/ml.
Oligozoospermia - < 15million/ml.
Causes:
• Mumps orchitis
• Prostatitis
•Hypopituitarism
•Hypogonadotropic hypogonadism
•Estrogen secreting tumours
•Hypo/Hyperthyroidism
•Drugs – Sulfasalazine, Cimetidine, Estrogen,
Nitrofurantoin, Caffeine, Alcohol, Cocaine, Smoking
tobacco, Herbal medications, Chemotherapeutic
agents
MOTILITY:
Routinely used technique:
•Place a drop of liquefied semen on a glass slide.
•Cover with coverslip and rim its edges with vaseline.
•Examine under 40X with reduced illumination.
•Count the number actively motile sperms out of 200.
•Calculate the percentage.

For accuracy,
Coverslip- 22mm x 22mm
Semen- 10ul , depth of 20um.
Phase contrast microscopy –
400x/600x at 37deg C.
Grading:
Rapid progressive motility
Slow/Sluggish progressive motility
Non - progressive motility
Immotility
Normal Motility:
After liquefaction or
If less than this, asthenozoospermia. within 60mins after
ejaculation.
Causes:

Cold
Radiation
Spermicides, Pesticides
Prolonged heat exposures
Prolonged abstinence
Autoimmunity
Progressive loss of motility by 5%/hr after 3hrs.
VIABILITY:
■ Mandatory - if the percentage of motile cells is low
■ Estimated by assessing the membrane integrity of the
cells
■ Assesed after liquefaction , at 30 mins
■ Can be done routinely on all samples

■ Normal - ≥ 58% live forms

■ high percentage of immotile and non-viable cells 

necrozoospermia  may indicate
epididymal pathology
■ Immotile live sperms  immotile cilia syndrome
(structural defects in the flagellum )
■ 2 methods :

1. The dye exclusion method - (using eosin-nigrosin)

• Most commonly used
• Principle: Damaged plasma membranes of non-vital (dead)
cells allow entry of
membrane-impermeant stains.

2. The hypo-osmotic swelling test (HOS)

• Alternative method
• Principle : only cells with intact membranes (live cells) will
swell in hypotonic
solutions
SPERM MORPHOLOGY:
•Thin smears similar to blood smears are mad e  feathering
technique.
•Before staining, mucoid material is removed by gentle washing
with semen dilution fluid. Then wash gently with buffered
distilled water.
•Several staining techniques are used 1)Pap is the best.
2) Haematoxylene technique.
3) Giemsa.
4)Leishman/basic fuchsin(0.25% acqeous).
5)Crystal violet.
6)Diff-Quick stain
•On basic fuchsin,
Sperm head caps: light blue.
Nuclear post: dark blue.
Body &tails: red/pink.
Sperm morphology: Ideal spermatozoon
Menkveld et al. 1991, WHO 1999, ESHRE 2002

Head oval shaped regular contour

Length: 4-5.5 micron
Width 2.5-3.5 micron
Darker posterior region
Base of head should be broad
Single tail symmetrically attached

BORDERLINE FORMS =
ABNORMAL
Morphology defects
COMPUTER AIDED SPERM ANALYSIS:
Automation seeks to establish standard methods of
analysis and set acceptable levels of accuracy in
measuring various parameters to promote
interlaboratory comparisons.
Advantages:
Subjective errors avoided
Motility can be assessed quantitatively
Capable of handling many samples without undergoing
fatigue
Morphological defects can be made out
Anti sperm antibodies
■ Immune response can be generated against sperm antigens
■ Since spermatogenesis begins during puberty
■ Normally the tight Sertoli-cell junctions provide the testis with a
barrier that prevents the immune system from coming in contact
with the post-meiotic germ cells.
■ This unique barrier can be violated,
■ In Testicular torsion, Vasectomy, Testicular trauma, testicular
surgeries
■ Sperms are exposed to immune system  l/t formation of anti
sperm antibodies
Anti sperm antibodies
■ If spermatozoa demonstrate agglutination the presence of sperm
antibodies may be the cause.
■ Sperm antibodies can be present without sperm agglutination
■ Anti-sperm antibodies (ASAs) in semen belong almost exclusively
to two immunoglobulin classes: IgA and IgG.
ANTISPERM ANTIBODIES:
Can occur in the:a)Serum of male or female
b)Seminal plasma
c)Spermatozoa
Effects:a)Lowered progressive
motility.
b) Decreased ability to
penetrate cervical
mucus.
c) Decreased ability to
penetrate egg.
Antisperm antibodies are found in following
conditions:
•Testicular disease
•Autoimmune azoospermatogenesi
•Repeated infections
•Obstruction of ducts
•Trauma
•Genetic predisposition
Techniques of detecting antibodies:
•Mixed antiglobulin reaction test
•Immunobead method
The mixed antiglobulin reaction test
■ Inexpensive, quick and sensitive screening test
■ Principle :
■ A “bridging” antibody (anti- IgG or anti- IgA) is used to bring the
antibody-coated latex
particles into contact with unwashed spermatozoa bearing surface
IgG or IgA.
■ Procedure :
■ The direct IgG and IgA MAR tests are performed by mixing fresh,
untreated semen separately with latex particles or treated red
blood cells coated with human IgG or IgA.
■ To the suspensions is added a monospecific anti-human-IgG or anti-
human-IgA.
■ At least 200 motile spermatozoa are examined
■ If the spermatozoa have antibodies on their surface,
antihuman immunoglobulin will bind IgG or IgA-coated latex
particles to IgA or IgG on the surface of the spermatozoa
■ This will cause attachment of latex particles to spermatozoa, and
motile, swimming
sperms with attached particles will be seen.
■ If the spermatozoa do not have antibodies on their
surface, they will be seen swimming without attached
particles
■ The latex particles will show clumping due to binding of their
IgG to antihuman immunoglobulin.
Immunobead test

■ More time-consuming than the MAR test

■ Provides information about location of antibodies on spermatozoa
■ Sperms are mixed with beads known to be coated with either anti-
IgG ,anti-IgM, or anti-IgA.
■ Microscopic examination of sperm shows the beads attached to
sperm at particular areas.
OTHER CELLS:
Round cells –1) germinal cells (single or double
highly condensed nucleus with
abundant cytoplasm).
2)leucocytes 1- 2/hpf.
Increased no. in infection of
reproductive tract.
RBCs – normally absent.
Present in
1. TB of seminal vesicles
2. rupture of blood vessels
3. Infection of prostate
4.Vit. C deficiency
Epithelial cells from urogenital
tract.
Chemical examination of semen

■ Prostate gland function

– Zinc
– Citric acid
– Acid phosphatase

■ Seminal vesicles
– Fructose
– Prostaglandins
■ Epididymis
– Neutral Alpha glucosidase
Determination of fructose:
Procedure:
Pipette 5 ml of resorcinol reagent in a test tube. [Resorcinol
reagent: resorcinol-50mg, Conc.HCl-33ml, distilled water-67ml]
Add 0.5 ml of semen.
Mix and place in a boiling waterbath for 5min or heat.
Observations: Red coloured ppt. in 30 secs.
In quantitative assays, spectrophotometric method can be
used.
Normal level of fructose: 150-300mg/dl.
Reduced levels: -Seminal vesicle dysfunction
-High sperm count