Semen analysis

Introduction

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

and determines the number and quality of sperm in the semen
sample.
 A semen analysis is usually one of the first tests done to help
determine whether a man has a problem fathering a child
(infertility).
 A problem with the semen or sperm affects more than one-third
of the couples who are unable to have children (infertile).
Purpose of seminal fluid analysis
 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. Medicolegal: 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 (2-5%) are very small
mucus secreting glands, add alkaline mucus to neutralize
prostatic acid and vaginal acidity
2. Prostate: (produce about 13-33 % 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,
prostaglandin, and other substances, which nourish and activate the
sperm passing through the tract. This component has high flavin
content, which is largely responsible for the fluorescence of semen.
4. Testis & Epididymis: (5%) Spermatozoa 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. This fraction still in
the inactive form until ejaculation due to the high content of
carnitine, glyceryle-phosphorylcholine and diminished oxygen
supply.
 Coagulation and liquefaction
 Coagulation and subsequent liquefaction are believed to be three
stage processes:
 Coagulation results from the actions of a prostatic clotting enzyme on a
fibrinogen-like precursor formed by the seminal vesicles.
 Liquefaction is initiated by enzymes of prostatic origin.
 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 the original container by
swirling for several seconds prior to removing the lid. Do not invert the
container.
 Specimen collection
 Specimen should be collected into prewarmed (21oC), sterile, non-
toxic, wide-mouth container, after a couple has abstained from sexual
activity for 2-3 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.
4. TESA: Testicular sperm extraction (TESE)
 Labeling
 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.
• The following should be recorded on the laboratory analysis
form:
1. The period of abstinence (in days).
2. If sample collection was complete or incomplete.
3. The time interval from collection to analysis.

• The sample should be transported upright, at body

temperature if possible, and should be delivered to laboratory
as soon as possible after collection and certainly within one
hour of ejaculation. If the sample is cold on receipt, this should
be noted in laboratory records. Patients should be advised not to
expose the sample to extremes of temperature.
 Examination of seminal fluid
 When evaluating semen specimens in cases of infertility,
the following parameters are routinely measured:
 volume
 viscosity
 pH
 sperm count (concentration)
 motility
 morphology.
 Macroscopic examination
 After ejaculation, the seminal secretions form a
coagulum, which gradually liquefies 10-20 min.
In most cases, the semen sample should become
fully liquefied within 60 minutes of production.
 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.
 Viscosity of the ejaculate
 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 Normal droplets form
a thin thread when released from the pipette.
 Droplets with threads longer that 2 centimeters are considered highly
viscous.
 Ratings of 0 (watery) to 4 (gel-like) can be assigned to the viscosity
report.
 Viscosity can also be reported as low, normal, and high.
 Increased viscosity and incomplete liquefaction impede sperm
motility
 Volume
 Normal is (2-5 milliliters). Using either a graduated cylinder with a conical
base or a disposable wide- mouthed pipette (accurate to 0.1ml) measure the
ejaculate volume to the nearest 0.1ml.
 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.
 Color of seminal fluid
 Semen is normally a gray-yellow 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.
 PH
 The normal pH of semen is slightly alkaline (7.2- 8.0)
but increases with time.

 Increased pH is indicative of infection within the

reproductive tract.
 A decreased pH is associated with increased prostatic
fluid.
Microscopic examination
 Microscopic examination
1. Concentration (sometimes referred to as the
"count")
2. Motility (sometimes referred to as the "mobility")
3. Agglutination
4. Morphology
 Concentration "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 subfertile.
 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 20 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
1. 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.
2. 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.
3. The sperm should then be counted - do not count headless or "pin-heads"
sperm and do not count tailless heads .
4. 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
 Using a 1:20 dilution, an average of 60 sperm are counted in the five RBC
counting squares on both sides of the hemocytometer. Calculate the sperm
concentration per milliliter and the total sperm count in a specimen with a
volume of 4 mL.
 60 sperm counted x 1,000,000 = 60,000,000 sperm/mL
 60,000,000 sperm/mL x 4 mL = 240,000,000 sperm/ejaculate
B. Direct smear
 The application of a drop 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.
 Motility "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.
 There are four classifications of motility
1. Rapid progressive motility - the sperm are
moving swiftly across the field usually in a
straight line
2. Slow or sluggish progressive motility - the
sperm may be less linear in their progression
3. Non-progressive motility - sperm are also
described as twitching or shaking
4. Immotility - sperm do not move at all.
 Vitality Assessment
1. Eosin-nigrosin (dead sperm stain pink/red)
2. Eosin (1%) (dead sperm stain pink/red)
3. Trypan (0.4%) blue (dead sperm stain blue)
4. Hypo-osmotic swelling test (HOS) (live sperm shows tail curling

 Test 1, 2 and 3 for diagnostic uses. Usually 1:1 ratio of semen to dye
mixture, mix well and smear onto a slide. Read immediately at x40
objective, count 200 sperms
 Test 4 is use to choose live (immotile) sperm for ICSI. Dead sperms
will not react in HOS while live sperm will take up fluids causing their
tails to curl within 5 min and stabilize at 30 min. Therefore viable
sperms may be selected for ICSI
 Eosin stain is used to differentiate live (unstained) and
dead (stained) spermatozoa
 Other cells in semen
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.
 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.
 Morphology
 This describes the shape of the sperm.
 70% of the sperm should be normal by these criteria.
 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 (Papanicolaou stain,
Vital staining with eosin/nigrosin, 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).
 There should be no head, midpiece or tail defects, and no
cytoplasmic droplet more than one-third the size of a normal
sperm head.
 Normal spermatozoa structure
 Defects to be scored
 Head shape/size defects - such as large, small, tapering,
pinhead form, amorphous, vacuolated, multiple heads or any
combination of these.
 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.
 Tail defects - such as short, multiple, hairpin, broken, irregular
width, coiled tails, tails with terminal droplets or any
combination of these.
 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
Abnormalities of sperm
heads and tails are illustrated
 Hematoxylin-Eosin Staining
o Hematoxylin-Eosin
 Fairly good
differentiation
o Fix slide in EtOH/MeOH 95% 20 min  The acrosomal area and
o Wash in running tap water 5 min cytoplasmic fragments is
o Dry on absorbent paper stained pink and the
post-acrosomal area is
o Hematoxylin (Sigma, HHS-128) 20 min stained dark purple.
 Abnormally stained
o Wash in running tap water 5 min sperms (nuclear/
o Acid alcohol (99 ml 70% EtOH + 1 Dip (2) chromatin material) may
ml H2SO4) be differentiated.
o Eosin (Sigma, HT1102128) 5 min  Takes longer and need
o EtOH 70% 2 min experience to produce
o EtOH 90% 2 min (2) good staining
o Absolute EtOH (99.9%) 2 min (2)
o Xylene 2 min (2)
Abnormal Sperms
Abnormal Sperms
Triple head .1
sperm
Acrosome .2
reacted sperm
Sperm with no .3
acrosome
Sperm with a .4
tapering head
and swollen
mid-piece