Semen analysis

Chapter four
 Acknowledgements
• Addisa Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-Ethiopia
 Chapter outline
• Introduction to Cerebrospinal fluid
• Routine laboratory assays
• Collection of sample
• Gross appearance
• Cell counts
• Chemical analysis
• Morphological Examination
• Microbiological Examination
• Serological Examination
 Learning Objectives
Upon completion of this chapter the student will be able to:
1 State the structures involved in sperm production
and their function.
2 Describe the four components of semen with
regard to source and function.
3 Describe the normal appearance of semen and
three abnormalities in appearance.
4 State two possible causes of low semen volume.
5 Discuss the significance of semen liquefaction
and viscosity.
 Learning Objectives
6 Calculate a sperm concentration and count
when provided with the number of sperm
counted, the dilution, the area of the counting
chamber used, and the ejaculate volume.
7 Define round cells, and explain their significance.
8 Describe the appearance of normal sperm,
including structures and their functions.
10 Differentiate between routine and strict criteria
for evaluation of sperm morphology.
11 Given an abnormal result in the routine semen
analysis, determine additional tests that might be
performed.
 Physiology

• Semen is composed of four fractions that are contributed

by
• the testes, 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 specimen
 Semen analysis
 Used in the evaluation of reproductive dysfunction (infertility)
in the male
 Used to select donors for therapeutic insemination
 Is a cost-effective and relatively simple procedure.
 Consists of microscopic and macroscopic components
 Collection and transport of semen
1. Give the person a clean, dry, leak-proof container,
and request him to collect a specimen of semen at
home following 3 days of sexual abstinence
 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.
 Collection and transport, cont’d…
2. Lable the container (name ,date and time of
collection, period of abstinence
 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 . .
 Tests for semen
 Macroscopic
-Physical (volume, viscosity, liquefaction)
-chemical l(eg. ph)
 Microscopic
-stained preparation
- wet-mount
 Semen analysis
 When investigating infertility, the basic analysis of
semen (seminal fluid) usually includes:
 Measurement of volume
 Measurement of pH
 Examination of a wet preparation to estimate the percentage
of motile spermatozoa and viable forms and to look for cells
and bacteria.
 Sperm count
 Examination of a stained preparation to estimate the
percentage of spermatozoa with normal morphology.
 EXAMINATION OF SEMEN

Caution: Handle semen with care because it may

contain infectious pathogens, e.g. HIV, hepatitis
viruses, herpes viruses.
 Macroscopic Examination
Measure the volume
 Normal semen is thick and viscous when ejaculated.
 It becomes liquefied usually within 60 minutes due to a
fibrinolysin in the fluid.
 Failure to liquefy may indicate inadequate prostate secretion.
 When liquefied, measure the volume of fluid in millilitres
using a small graduated cylinder.
 Normal specimens: Usually 2 ml or more
 Macroscopic Examination, cont….

Measure the pH
 Using a narrow range pH paper, e.g. pH 6.4–8.0, spread a drop
of liquefied semen on the paper.
 After 30 seconds, record the pH.
 pH of normal semen: Should be pH 7.2 – 7.8
 When the pH is over 7.8 this may be due to infection.
 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.
 Microscopic Examination
 be performed to obtain estimates of sperm concentration,
motility, and agglutination.
 polygonal cells of the urethral tract and ‘round cells' such as
spermatogenic cells and leukocytes can also be observed
when sperm are counted in a hemocytometer.
 Motility (normal range 50% or above) is expressed as the
percentage of sperm that move.
 Microscopic Examination

Estimate the percentage of motile and viable spermatozoa

Motility
– Place 1 drop of well-mixed liquefied semen on a slide and
cover with cover glass.
– Focus the specimen using the 10_ objective.
– Ensure the spermatozoa are evenly distributed
– if not, re-mix the semen and examine anew preparation.
– Using the 40_ objective, examine several fields
 Microscopic Examination,cont’d…
• to assess motility, i.e. whether excellent (rapid
and progressive) or weak (slow and non progressive).
• Count a Normal motility: Over 50% of spermatozoa are motile
within 60 minutes of ejaculation.
 Reporting of results
• Motility (normal range 50% or above) is expressed as the
percentage of sperm that move.
• Sperm moving rapidly in a straight line with little yaw and
lateral movement are Grade 4
• if they move more slowly, Grade 3.
• Grade 2 sperm move even more slowly and with substantial
yaw.
• Grade 1 sperm have no forward progression.
• Zero progression denotes absence of any motility
 cont’d…
• 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.
• 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.
 Cont…
• 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
 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).
 sperm count
• 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 .
 sperm count
• Wait 3–5 minutes for the spermatozoa to settle.
• Count the number of spermatozoa in an area of 2 sq mm, (i.e.
any 2 large squares within the 9 squares).
• Calculate the number of spermatozoa in 1 ml of fluid by
multiplying the number counted by100, 000.
– Normal count: 20 – 106X106 spermatozoa/ml or more.
– Counts less than 20 - 106 X106/ml are associated with
male sterility. total of 100 spermatozoa, and note out of
the hundred how many are motile.
– Record the percentage that are motile and non motile.
 Staining procedure

• Make a thin smear of the liquefied well-mixed semen on a

slide.
• While still wet,fix the smear with 95% v/v ethanol for 5–10
minutes
• allow to air-dry.
• Wash the smear with sodium bicarbonate formalin solution to
remove any mucus which may be present.
• Rinse the smear with several changes of water.
• Cover the smear with dilute (1 in 20) carbol fuchsin and allow
to stain for 3 minutes.
• Wash off the stain with water.
 Staining cont’d…

• Counter stain, by covering the smear with dilute (1 in 20)

Loeffler’s methylene blue for 2 minutes.
• Wash off the stain with water.
• 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.
 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.
 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.
 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.
Middle piece
Abnormal sperm shapes
 Two heads Cont’d…

● Absent or markedly increased in size.

● Appears divided (bifurcated).
● Angled where it meets tail.
 Morphology cont’d…
Tail
● Absent or markedly reduced in length.
● Double tail.
● Bent or coiled tail.
 Abnormalities of sperm
heads and tails are illustrated
Semen Analysis (spermatogram)
 Exercises
1. The major component of seminal fluid :
2. If the first portion of a semen specimen is not collected,
the semen analysis will have an abnormal:
3. Failure of laboratory personnel to document the time a
semen sample is collected primarily affects the interpretation
of semen:
4. A semen specimen delivered to the laboratory in a
condom has a normal sperm count and markedly
decreased sperm motility. This is indicative of:
5. An increased semen ph may be caused by:
 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. 3 rd 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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