Chapter Eight

Semen analysis

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 Chapter Course outline
• Introduction about semen

• Composition of semen

• Collection and transportation of semen sample

• Microscopic and macroscopic examination of semen sample

• Chemical and microbiological examination of semen sample

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 Learning Objectives
Upon completion of this chapter the student will be able to:
1. Describe the components of semen with regard to source and
function
2. List reasons for semen analysis
3. Outline instructions to give to a patient for the correct method
for collecting a semen specimen for laboratory analysis
4. Differentiate between routine and strict criteria for evaluation of
sperm morphology
5. List the normal values for: semen volume, viscosity, pH, sperm
count, motility and morphology
6. Determine additional tests that might be performed
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What is semen? Brainstorming
A mixture of seminal plasma and cells
 Seminal plasma contains:
 Prostatic fluid (20-30%)
 Epididymal plasma (5%)
 Seminal vesicle fluid (60-70%)
 Boulbourethral fluid (5%)
 The cells are:
 Spermatozoa (2-5%)
 Germ line cells
 Leukocytes of various types
 Epithelial cells
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Physiology
Seminal plasma
• Is composed of four fractions
• Contributed by:
 Epididymis, seminal vessels, prostate, and
bulbourethral glands
• Each fraction differs in its composition, and the mixing of
all four fractions during ejaculation is essential for the
production of a normal semen
Sperm cell: Formed in the seminiferous tubules

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Diagram of the male genitalia

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Spermatogenesis
• Begins at puberty and continues
throughout adult life of a male.
• Sperm are produced within the
seminiferous tubules.
• Sertoli cells (nurse cells) provide
support and nutrients for the germ cells
as they undergo mitosis and meiosis
• Primordial germ cells differentiate
into spermatogonia (diploid cells)
• Spermatogonia during mitosis
division become two primary
spermatocyts
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Iyasu.S 8
Cont’..…
• Each primary spermatocytes
(diploid cells ) through meiosis I
produce two secondary
spermatocytes (haploid cells).
• Each secondary spermatocytes
during meiosis II divides to
produced two spermatid (haploid
cells with 23 chromosomes).
• As a result of the two meiotic
divisions, each primary
spermatocyte produces four
spermatids.
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 Cont’..…
• Changes that transform spermatids
into sperm :
1. Chromatin condensation
2. Flagellum formation
3. Acrosome cap development
4. Discarding excess cytoplasm
• Transported from the testes to the
epididymis, where they mature
• Spermatogenesis takes 70 ± 4 days
in the human

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Iyasu.S 11
Cont’….
• Then moved to the vas deferens, for storage
• At ejaculation, the sperm are transported out of the vas and
mix with secretions of the accessory gland
• Accessory gland secretions:
 Seminal vesicles – provides fructose & nutrients (N.B:- Spermatozoa
become mobile when exposed to seminal fluid)

 Prostate gland – Provides enzyme, acid phosphatase, citric acid, zinc,

and proteolytic enzymes (for liquefaction)
 Bulbourethral glands- thick alkaline mucous-like fluid that
neutralizes acids from the prostatic secretions and vaginal acidity

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Iyasu.S 13
Significance of semen analysis
 Used in the evaluation of reproductive dysfunction
(infertility) in the male
 Prognosis for fertility
 To asses Post vasectomy
 Forensic analysis of fluid as being semen
 In unproven rape

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Specimen Collection
 Sterile container
 Label the container
 (Name, date and time of collection, period of abstinence)
 The man must have had 3 – 5 days of abstinence
 Complete specimen: Majority of sperm are in first part of
ejaculate
 Best if collected at laboratory site
 Specimens should be collected by masturbation

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Cont’…..
 Condom is used to collect the fluid
 This must be well-washed to remove the powder which
coats the rubber
 It must be dried completely before being used
 known NOT to be spermotoxic
 No lubricants
 Deliver the specimen to the laboratory within 1 hour
 Fluid should be kept as near as possible to body temperature
 This is best achieved by placing the container inside a
plastic bag and transporting it in the person's armpit

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Sample Handling
 The semen sample should be mixed gently during the
liquefaction period to promote liquefaction
 The sample should NEVER be vortexed
 If it is too viscous to work with, a known volume of
sperm buffer should be added and the sample mixed
gently
 The added volume must be included in the sperm count
calculation

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Sample Handling

Caution: Handle semen with care because it may

contain infectious pathogens.
E.g. HIV, hepatitis viruses, herpes viruses.

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Tests for semen
 There are several macroscopic evaluations which give
useful diagnostic information about the sample:
 Appearance
 Liquefaction
 Volume
 Viscosity
 pH

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 Macroscopic Evaluation - Appearance

1. Normal semen
 Has a gray-white color
 Appears translucent, and
 Has a characteristic musty odor
2. Yellow coloration may be caused by:
 Urine contamination
 Specimen collection following prolonged abstinence, and
 Medications
3. Increased white turbidity indicates the presence of WBCs
and infection within the reproductive tract
4. If blood is present it may appear red, pink to orange, are
abnormal

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 Liquefaction
• A fresh semen specimen is clotted and should liquefy within 30 to
60 minutes after collection
• Recording the time of collection is essential for evaluation of
semen liquefaction
• Analysis of the specimen cannot begin until after liquefaction has
occurred
• If after 2 hours the specimen has not liquified, proteolytic
enzymes such as alpha-chymotrypsin may be added to allow the
rest of the analysis to be performed
• Failure of liquefaction to occur may be caused by a deficiency in
prostatic enzymes and should be reported
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Viscosity
• Refers to consistency of the fluid and may be related to
specimen liquefaction
• Normal specimen should be easily drawn into a pipette
and form droplets that don’t appear clumped or stringy
when discharged from the pipette
• Ratings: 0 (watery) to 4 gel-like)
• Increased viscosity and incomplete liquefaction will
impeded sperm motility
• Pours in droplets normally

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Volume…………….

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Volume
 When liquefied, measure the volume of fluid in millilitres
using a small graduated cylinder
 Normal specimens: Usually 2 ml – 5ml
 Increased volume: following periods of extended
abstinence
 Decreased volume: associated with infertility; may
indicate improper functioning of one of the semen
producing organ , primarily the seminal vesicles
 Incomplete specimen collection must be also be
considered

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pH
• The normal pH of semen is alkaline with a range of 7.2 to 8.0.
Alkaline to off-set acid vaginal environment
• Increased pH is indicative of infection within the reproductive
tract
• When the pH is below 7.0 and the semen is found to contain
no sperm, this may indicate dysgenesis (failure to develop) of
the vas deferens, seminal vesicles or epididymis.
• A decreased pH is associated with increased prostatic fluid
 Semen for pH testing can be applied to the pH pad of a
urinalysis reagent strip

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Microscopic Analysis
• Motility
• Concentration / cell count
• Morphology
• Agglutination
• Viability
Generally performed 30 - 60 min after collection
Must be after liquefaction has occurred

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The wet preparation – set-up
• Place 10µl of thoroughly mixed, liquefied semen on a microscope slide and cover with a
22x22mm
• The characteristics assessed are:
 Motility
 Sperm aggregation (random clumping) – “some” is normal, but large clumps
(each with hundreds of sperm) is abnormal
 Sperm-agglutination (between specific sites) – could suggest the presence of
anti-sperm antibodies
 Round cells: (~ 1 million/ml)
 If more abundant, a leukocyte test should be run
 Epithelial cells: usually present in small numbers
 Erythrocytes: should not be present
 Debris: particles smaller than sperm head, may be plentiful
 Bacteria and protozoa: presence indicates infection
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The wet preparation – assessments
• There are several important points to keep in mind:
The quality of sperm motility is affected by temperature
The lower the temperature, the poorer the motility, and
then cold shock starts to occur at around 15°C
So great care must be taken to ensure that the slides and
coverslips, as well as the pipette tips are kept at 37°C
The assessment must start as soon as the flow stops
 If this is >1 minute, then a new wet prep must be made

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Motility
• Motility is a very necessary quality of sperm
 Must propel through uterus & fallopian tubes which is quite a long
distance
• Must be evaluated within the 1st hour following collection
• Will decrease over time
• Evaluation:
 Manual Subjective evaluation
 Objective evaluation:
 High-resolution video photography / CASA (computer
assisted semen analysis)

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Motility…
Estimate the percentage of motile and viable spermatozoa
• Place 1 drop of well-mixed liquefied semen on a slide and
cover with cover glass.
• Focus the specimen using the 10X objective.
• Ensure the spermatozoa are evenly distributed
• If not, re-mix the semen and examine a new preparation.
• Using the 40X objective, examine several fields

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Motility assessment - types
• Motility is evaluated by both speed and direction
• Grading can be done using a scale of 0 to 4, with 4 indicating
rapid, straight-line movement and 0 indicating no movement

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 Motility assessment – interpretation
• The WHO’s Reference values for motility are:
– 50% or more with progressive motility; or
– 25% or more with rapid progressive motility

• Assuming that all of the collection and laboratory factors have

been controlled, a poor motility result may have negative
implications for fertility
• However, this should be confirmed by a repeat semen analysis,
and the result should be interpreted with the rest of the semen
analysis results

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Cont’….
Gram stain smear
 The spermatozoa remain motile for several hours
• Perform gram stain smear:
 When more than 60% of spermatozoa are non motile,
 When more than a few leucocytes and
 > 6 red blood cell/ HPF
 Look for the type of bacteria that exist in the semen

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Cont’..…
• If motility is less than 50%, a viability stain of eosin Y with
nigrosin as a counterstain is done
• Dead sperm will stain red, whereas live sperm will exclude the
dye and appear unstained
 Post-vasectomy
• In samples with no visible sperm, such as post-vasectomy
semen, the entire sample should be centrifuged, and the
pellet examined for intact or damaged sperm fragments

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Viability
Procedure
• Mix one drop of semen with 1 drop of 0.5% eosin solution on
a slide
• After 2 minutes examine microscopically
• Use the 40X objective to count the percentage of viable and
non-viable spermatozoa
• Viable spermatozoa remain unstained, non-viable
spermatozoa stain red
• Normal viability: 75% or more of spermatozoa should be
viable (unstained)

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Cont’….

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 Sperm count
– Normal value = 20 – 160 million/mL
– Make 1 to 20 dilution with sodium bicarbonate and formalin,
then count it using hemacytometer

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Cont’……
 Sperm count = total number of sperm in the ejaculate
 Total sperm count/ ejaculate= sperm concentration X
specimen volume
 Sperm concentration = number of sperm per ml

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Cont’…..
Sperm dilution fluid
• The traditional diluting fluid contains sodium
bicarbonate and formalin, which immobilize and
preserve the cells; however, good results can also be
achieved using saline and distilled water
• If they are mobile, you wouldn’t be able to count
them properly

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Cont’….
• Using a graduated tube or small cylinder, dilute the semen 1
in 20 as follows:
– Fill the tube or Thoma pipette to the 1 ml mark with well-
mixed liquefied semen
– Add sodium bicarbonate-formalin diluting fluid to the 20
ml mark, and mix well
– Using a Pasteur pipette or Thoma pipette, fill an Improved
Neubauer ruled chamber

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Cont’….
• Fix the haemocytometer
coverslip over the
chambers
• load ~10 µl in both
chambers
• Leave in a humid
chamber for 10-15
minutes

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 Sperm count…
 Standard method to begin calculation of # cells
(mature sperm) per microliter:

ave. # cells counted x dilution

# squares counted x volume of each square
 Normal count 20 -160X106 spermatozoa/ml or more
 Counts less than 20 X106/ml are associated with male
sterility
 Azoospermia is complete absence of spermatozoa in semen

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Iyasu.S 44
Example
• Using a 1:20 dilution, an average of 60 sperm are
counted in the five RBC counting squares on both sides
of the hemocytometer
1. Calculate the sperm concentration per milliliter
2. Calculate the total sperm count in a specimen with a
volume of 4 mL

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Cont’…
• In a 1:20 dilution, average 600 sperm are counted in
two WBC counting squares
1. Calculate the sperm concentration per milliliter
2. Calculate the total sperm count in a specimen with
a volume of 2 mL

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Sperm morphology
• Sperm morphology is evaluated with respect to the
structure of the head, neckpiece, midpiece, and tail
• The normal sperm has:
 An oval-shaped head approximately 5 µm long and 3 µm
wide and
 A long, flagellar tail approximately 45 µm long
• Abnormalities in head morphology are associated with
poor ovum penetration, whereas neckpiece, midpiece,
and tail abnormalities affect motility

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Cont’….
• Sperm morphology is evaluated from a thinly
smeared , stained slide under oil immersion
• Staining can be performed using
Wright’s,
Giemsa,
Papanicolaou stain or
carbol fuchsin

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Cont’……..
Staining procedure of carbol fuchsin
1. Make a thin smear of the liquefied well-mixed semen on a slide
2. While still wet , fix the smear with 95% v/v ethanol for 5–10
minutes
3. Allow to air-dry
4. Wash the smear with sodium bicarbonate formalin solution to
remove any mucus which may be present
5. Rinse the smear with several changes of water
6. Cover the smear with dilute (1 in 20) carbol fuchsin and allow
to stain for 3 minutes.
7. Wash off the stain with water
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Cont’….
8. Counter stain, by covering the smear with dilute (1 in 20)
Loeffler’s methylene blue for 2 minutes
9. Wash off the stain with water
10. Allow the smear to air-dry
Staining results
 Nucleus of head . . . . . . . . . . . . . . . . . . . . Dark blue
 Cytoplasm of head . . . . . . . . . . . . . . . . . . . Pale blue
 Middle piece and tail . . . . . . . . . . . . . . . . . Pink-red
 Alternative stains: Other staining techniques used to stain
spermatozoa include Giemsa and Papanicolaou.

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 Reporting morphology of spermatozoa
 Examine the preparation for normal and abnormal
spermatozoa using the 40X objective
 Use the100X objective to confirm abnormalities
 Count 100 spermatozoa and estimate the percentage showing
normal morphology and the percentage that appear
abnormal

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Normal spermatozoa
• Measure 50–70 µm in length
• Each consists of an oval-shaped
head (with acrosomal cap)
which measures
3–5 X 2–3 µm
• A short middle piece
• A long thin tail (at least 45 µm in
length)
• In normal semen, at least 50%
of spermatozoa should show
normal morphology
• Most specimens contain no
more than 20% abnormal forms
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Abnormal spermatozoa
The following abnormalities may be seen:
Head
 Greatly increased or decreased in size
 Abnormal shape and tapering head (pyriform)
 Acrosomal cap absent or abnormally large
 Nucleus contains vacuoles or chromatin is unevenly distributed
Two heads
 Additional residual body, i.e. cytoplasmic droplet
 Appears divided (bifurcated)
 Angled where it meets tail

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Cont’…..
Tail
 Absent or markedly reduced in length
 Double tail
 Bent or coiled tail

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Abnormal sperm shapes

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Abnormalities of sperm heads and tails

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 Cont’…..
• WBC, RBC, bacteria presence should be noted & may
indicate infection
• Round cells (neutrophils and immature sperm) should be
noted as well.

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Normal spermatozoa
• Normal values
 > 30% normal forms using routine criteria
 > 15% normal forms (strict criteria or the Kruger’s
criteria)
• Kruger’s strict criteria
Measurement of head, neck and tail size
Size of acrosome
Presence of vacuoles
Not routinely performed but recommended by WHO

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Additional tests: semen biochemistry
• Acid phosphatase: marker for prostatic function
 Useful in rape cases
• Citric acid: can indicate prostatic function – low levels may indicate
dysfunction or a prostatic duct obstruction
• Zinc: marker for prostatic function – colorimetric assay (WHO)
• Fructose: marker for seminal vesicle function, and is a substrate for
sperm metabolism – spectrophotometric assay (WHO)
 Reorcinol test—check for presence of fructose (orange red color)
Normal: >= 13 umol/ ejaculate
• -Glucosidase: secreted exclusively by the epididymis and so is a
marker for epididymal function – spectrophotometric assay (WHO)
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Cont’…..

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Reading assignment

Read about
Antisperm Antibodies
and
microbiological analysis of semen

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Iyasu.S 62
 Lab sources of error in semen analysis
 Failure to collect the 1st portion (since 70% of sperms is in the first
part of the ejaculate)
 Failure to document the time
 Use of lubricant
 Use of spermicidal
 Contamination
 Transportation error
 Temperature
 Not abstaining 3-5 days
 Wrong dilution
 Inappropriate pipetting
 Duplicate aliquots?
 Incorrect charging
 Incorrect calculation and reporting
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Study questions
1. Describe the major component of seminal fluid
2. What if the first portion of a semen specimen is not
collected?
3. Failure of laboratory personnel to document the time during
4. semen sample collection primarily affects the interpretation
of……
5. A semen specimen delivered to the laboratory in a condom
has a normal sperm count and markedly decreased sperm
motility. This is indicative of:
6. An increased semen ph may be caused by:

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 References:

• Urinalysis and body fluids / Susan King Strasinger, 5th ed. 2008
• District laboratory practice in tropical countries. 2nd ed. Part I. Monica
Cheesbrough, 2005
• Text book of urinalysis and body fluids. Doris LR, Ann EN, 1983
• Urinalysis and body fluids: A color text and atlas. Karen MR, Jean JL. 1995
• Clinical chemistry: Principles, procedures, correlation. 3rd ed. Michael L. Bishop
et al. 1996
• Tietz Text book of clinical chemistry. 3rd ed. Carl AB, Edward RA, 1999
• Clinical chemistry: Theory, analysis, correlation 4th ed. Lawrence AK. 2003
• ASCP Document
• Urinalysis lecture note . Mistire W. , Dawite Y.

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