Seminal fluid analysis

 A semen analysis measures the amount of semen a man produces

and determines the number and quality of sperm in the semen
sample.
 Seminal fluid is not made up of just sperms but also semen plasma
which is part of the ejaculatory fluid and plays a role for
movement and nutrition of the sperm.

 Purpose of the test:

There are basically four indications for the examination of seminal
fluid:
1. The investigation of fertility: male infertility is primarily
responsible in 30%-50% of infertile marriages.
2. To determine the effectiveness of vasectomy.
3. To determine the suitability of semen for artificial insemination.
4. Medico legal: testes to detect semen are frequently requested in
alleged rape or in association with other sexual crimes of violence.

 Fluid Fractions:
1. Bulbourethral & Urethral glands: (produce about 2-5 % of the
fluid volume of semen) are very small mucus secreting glands.

2. Prostate: (produce about 15-45 % of the fluid volume of semen)

Prostate glands secretion is a milky, alkaline fluid that plays a role
in activating sperm, the secretion contains acid phosphatase and
proteolytic enzymes that act on the fluid from the seminal vesicles,
resulting in the coagulation and liquefaction of the semen.

3. Seminal vesicles: (produce about 46-80 % of the fluid volume of

semen) Viscous, yellowish secretion is rich in fructose, vitamin C,

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prostaglandin, and other substances, which nourish and activate the
sperm passing through the tract.

4.Testis & Epididymis: (5%) Spermatocytes are produced in the

testis under the influence of testosterone, and then the epididymis
(is the first part of the duct system) provides a temporary storage
site for the immature sperm that enter it from testis for 30 days.
Stimulus

Hypothalamus

GnRH

FSH-RH LH-RH

Anterior pituitary gland

FSH LH
(Follicular Stimulating hormone) (Luteinizing
hormone) In Testis

Sertoli cells Leydig cells

locally
Spermatogenesis
Testosterone
Spermatocyte

Sperm(in epididymis)

Sperm from epididymis plus seminal plasma from sourses

mentioned above produce seminal fluid that passes through
ejaculation.

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 The sperm within the semen are the cells that actually fertilize
the egg and are therefore the most important to assess. However,
the sperm account for only 1-2 % of the seminal fluid volume.
Problems with the surrounding fluid may also interfere with the
movement and function of the sperm. Therefore, both the sperm
and the fluid must be tested.

 Specimen collection:
 Specimen should be collected into pre warmed, sterile, non-
toxic, wide-mouth container, after a couple has abstained from
sexual activity for 3-5 days.
 Verbal and written instructions should be given to the patient to
ensure appropriate collection & delivery of semen sample to the
laboratory. Ideally the sample should be collected in a room set
aside for this purpose at the clinic laboratory in order to reduce
ejaculation-analysis interval but this is not always possible.
 The patient should be advised to urinate and then wash and dry
his hands and genitals thoroughly prior to ejaculation to avoid
bacterial contamination. It is important to note that
contamination of the semen sample with either soap or water
may adversely affect sperm quality.

 Methods of collection:
1. Masturbation (the method of choice for all seminal fluid tests)
2. The use of condom: it is not recommended for fertility testing
because the condoms may contain spermicidal agents (used to
determine the effectiveness of vasectomy).
3. By coitus interrupts (withdrawal method): the sample may be
mistimed and part of the ejaculate may thus be lost.
 The sample should be clearly labeled with:
1. the patient's name
2. ID or clinic number (if available)
3. Date and time of sample collection.
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 The following should be recorded on the laboratory analysis
form:
1. The period of abstinence (in days), a couple should abstained
from sexual activity for 3-5 days.
2. Date and time of sample collection.
3. Time of liquefaction:
 After ejaculation, the seminal secretions form a coagulum, which
gradually liquefies 10-30 min. In most cases, the semen sample
should become fully liquefied within 30 minutes of production.
 Put sample in incubator 370c during the thirty minutes and see it
every 5 min.
 Once liquefaction is complete then the physical appearance of
the sample should be recorded in the laboratory records. If
liquefaction does not occur then this abnormality should be
noted.

 Coagulation and liquefaction

 Prostatic secretion contains acid phosphatase and proteolytic
enzymes that act on the fluid from the seminal vesicles, also in
the presence of prostaglandin resulting in the coagulation and
liquefaction of the semen.
Coagulation and subsequent liquefaction are believed to be three
stage processes:
1. Coagulation results from the actions of a prostatic clotting
enzyme on a fibrinogen-like precursor formed by the seminal
vesicles.
2. Liquefaction is initiated by enzymes of prostatic origin.
3. The protein fragments are degraded further to free amino acids
and ammonia by the action of several poorly characterized
proteolytic enzymes, including an amino peptidase and pepsin.
Clearly, a semen analysis should not be performed immediately
following sample production. The sample should be mixed well in

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the original container by swirling for several seconds prior to
removing the lid. Do not invert the container.

4. Viscosity:
Estimate the viscosity of the semen by aspirating the semen into the
measuring pipette and allowing the semen to drop by gravity and
will not appear clumped. Observe the length of the thread. With
excessively viscous samples, thorough mixing can be difficult and
accurate estimation of sperm concentration and motility may be
impossible.

5. Volume:
 This is a measurement of the volume of the ejaculate. Normal is
(1.5-6 milliliters).
 Excessively small or large volumes are important in the transport
of semen within the female reproductive tract and should be
noted.
 The volume may be low if a man is anxious when producing a
specimen, if all of the specimen is not caught in the collection
container, or if there are hormonal abnormalities or ductal
blockages.

6. Odor:
Distinctive

7. Color:
Semen is normally a gray- white opalescent fluid. Its opacity is
due to the most part, to its high protein content but is of course
also produced by the many millions of spermatozoa as well as
the cellular debris that is normally suspended within it.

8. PH:
The normal pH of semen is alkaline (7.2- 8.0)
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Microscopic examination
1- Concentration (sometimes referred to as the "count")
 This is a measurement of how many million sperm there are in
each milliliter of fluid.
 There are various techniques for obtaining this number - some
prove to be more accurate than others are.
 Average sperm concentration is more than 60 million per
milliliter (60-150 million/ml).
 Counts of less than 20 million per milliliter (<20 million/ml) are
considered sub-fertile.
 Several terms are used to describe both sperm concentration
and sperm count:
 Azoospermia describe a total absence of spermatozoa in semen.
(After centrifuge sperm count is zero/HPF).
 Oligozoospermia refers to a reduced number of spermatozoa in
semen and is usually used to describe a sperm concentration of
less than 2 million/ml. Sperm count 5-10 sperm/HPF.
 Severe oligospermia, sperm count 1-2 sperm/HPF.
 Polyzoospermia denotes an increased number of spermatozoa in
semen and is usually refers to a sperm concentration in excess of
350 million/ml.
Methods of measuring sperm concentration
A) By using hemacytometer:
 The sperm count is performed in the same manner as blood and
CSF counts; that is by diluting the specimen and counting the
spermatozoa in a neubauer chamber.
 Sperm can be counted by make dilution 1:20 in WBC pipette or
by automatic pipette (which is more accurate) with a solution
containing sodium bicarbonate (5g) and formalin (1ml)
(immobilize & preserve the spermatozoa), tap water (100 ml)
will suffice as a diluent.
 The sperm should then be counted - do not count headless or
"pin-heads" sperm and do not count tailless heads.
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 Traditionally, the sperm concentration is expressed in millions
per milliliter (x106/ml) of semen and the total sperm/ejaculate is
reported in millions (x106) per ejaculate.

 Calculations
1. Using a 1:20 dilution and two large WBC’s squares counted
The sperm concentration/ml = No of sperms counted x 100,000
2. Using a 1:20 dilution and five small RBC’s squares counted
The sperm concentration /ml = No of sperms counted x 1,000,000

B) Direct smear
The application of a drop (50µ) of well-mixed semen to a clean
glass slide under a lightly applied glass coverslip will allow
visualization of the sperm in a specimen of semen.

2- Motility (sometimes referred to as the "mobility")

This describes the percentage of sperm, which are moving. 50% or
more of the sperm should be moving. In order to achieve
fertilization, a sperm must not only be able to move but be capable
of movement that results in forward progression is often also
known as progressive activity.

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There are three classifications of motility
1. Rapid progressive motility (active) - the sperm are moving
swiftly across the field usually in a straight line
2. Sluggish progressive motility - the sperm may be less linear in
their progression and shaking.
3. Immotility (dead) - sperm do not move at all.
 Eosin stain is used to differentiate live (unstained) and dead
(stained) sperm.

 Other cells in semen

 The presence of other cell types in human semen other than
spermatozoa are common and include:
1. leukocytes normally (1-4/HPF), increase number
(leukocytospermia) indicates reproductive tract infection
2. Epithelial cells normally (1-2/HPF)
3. Spermatocytes (Immature germ cells) 1-2/HPF.
4. Erythrocytes (1-2/HPF). Increased number may indicate a
reproductive tract infection or damage to a small capillary during
sample production.
5. Bacteria and protozoan such as Trichomonas vaginalis are
uncommon in human semen but their presence is indicative of
possible male reproductive tract infection and should be reported to
the referring doctor for further evaluation.

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3- Agglutination
 The presence of agglutination should be recorded as this may
indicate immunological infertility. Assess the spermatozoa in 10
random fields - estimate the average percentage of spermatozoa
clumped together to the nearest 5%.
 Only count motile sperm attached to other motile sperm - do not
assess immotile sperm stuck together or motile sperm adhering
to mucus threads, other cells or debris , this is non-specific
aggregation.

4- Morphology
This describes the shape of the sperm. The sperm are examined
under a microscope and must meet specific sets of criteria for
several sperm characteristics in order to be considered normal.
 Generally accepted that a high incidence of morphologically
abnormal spermatozoa in a semen sample is associated with
reduced fertility.
 Human sperm can be visualized using bright field microscopy on
fixed, stained specimens.
 Examples of fixed stained preparations (Wright stain, Leishman
stain, Giemsa stain).
 Normal spermatozoa should
have an oval shaped head (4-
5.5μm long and 2.5-3.5μm
wide).
 The midpiece should be
cylindrical (3-5μm long and
1.0μm wide).
 The tail should also be
cylindrical (45-50μm long and
0.5μm wide) with a narrower
terminal segment (4-6μm
long).
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 There should be no head, midpiece or tail defects, and no
cytoplasmic droplet more than one-third the size of a normal
sperm head.

 Defects to be scored
a) Head shape/size defects - such as large, small, tapering, pinhead
form, amorphous, vacuolated, multiple heads or any combination
of these.
b) Neck and midpiece defects - such as non-inserted or bent tail,
distended, irregular / bent midpiece, thin midpiece (no
mitochondrial sheath), absent tail (free or loose heads) or any
combination of these.
c) Tail defects - such as short, multiple, hairpin, broken, irregular
width, coiled tails, tails with terminal droplets or any combination
of these.
d) Cytoplasmic droplets - greater than one-third the size of a
normal sperm head.

 Each spermatozoa is scored as either normal or abnormal with

each of the defects being tallied separately. If a majority of the
cells have a particular morphological defect this should also be
noted.
 In stained preparations 100-200 sperm should be scored using a
x100 oil immersion bright field objective.

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 Useful biochemical Test
Fructose:
 Fructose is the primary energy source for sperm. It is required
for spermatozoa survival in an anaerobic environment and it
stimulates sperm motility.
 Spermatozoa, which are subjected to centrifugation and thus
separated from the seminal plasma, will not survive
anaerobically unless seminal plasma or carbohydrates source is
added back to separated spermatozoa. Seminal plasma fructose is
produced by the seminal vesicles. Fructose production is
stimulated by testosterone. Since the seminal vesicles do not
have a large storage capacity, collection of several ejaculates
within a few days will yield decreased fructose values. It takes
about two days for fructose levels in the seminal vesicle to be
replenished .
 Fructose measurements are useful diagnostically in men with
low-volume ejaculates. The absence of fructose can indicate the
congenital absence or infections that affect the seminal will also
result in absent or reduced fructose concentration.

 Reagents:
 ZnSO4 (5%)
 Ba(OH)2 (4.76%)
 Resorcinol (1%)
 Conc. HCL
 Water bath (90C), automatic pipette ,tubes.

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 Procedure
 In three tubes B, ST, T we put
B St T
H2O 3ml 0 ml 2.9ml
Sample ------------ ------------ 0.1ml
Working St ------------ 3 ml ------------
ZnSo4 0.5ml 0.5ml 0.5ml
Ba(OH)2 0.5ml 0.5ml 0.5ml

 Then mix, we will show white ppt, leave it 5 min.

 Centrifuge at high speed for 5 min.
 After that we take from the three tubes 2 ml of supernatant of
each, as the table under we do:
B St T
Supernatant 2.0ml 2.0ml 2.0ml
Resorcinol 2.0ml 2.0ml 2.0ml
Conc. HCl 6.0ml 6.0ml 6.0ml
 Mix, put it in water path at least time 10 min. we will show pink
color.
 Cool the tubes under tap water, and then read it on 490 nm wave
length of spectrophotometer.

 Calculation:
Conc. St= 350 mg/dl
Abs. T = 0.502
Abs. St = 0.575
Conc. T =( Abs. T / Abs. St )*Cons. St
= 0.502/0.575*350
= 305.565mg/dl

Normal value: 150- 350 mg/dl

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 Serological Analysis
 Anti-Sperm Antibodies
Antibodies to sperm can be present in the serum of some females as
well as males, the seminal fluid and the cervical mucosa are
statistically associated with an increased risk of infertility. It is
known that antibodies directed toward various sperm antigens can
result in reduced fertility in men.

0.5 ml serum from male 0.5 ml serum from female

0.5 ml seminal fluid 0.5 ml seminal fluid
 Mixing, put tubes in water bath 370c for 4 hours
 Comment:
If sperm activity present, then after mixing seminal fluid with
serum either from male or female sperm activity become zero,
also the formation of coupling either head to head or tail to tail
so we consider the presence of anti sperm antibodies.

 Pyospermia and the microbiology of semen

 Increased number of WBC’s in semen is known as pyospermia
and in occasions, is a cause of infertility in men, and so must be
examined microbiologically as the main source of WBCs mostly
from the prostate this means prostatitis.
 The first step in the microbiological examination is to make a
Gram stain and then make semen culture.

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