Sudan International University

Faculty of Medical Laboratory Sciences

Body Fluid course
Alaaeddin Musaad Mohammed
MSc., BSc.(Hon). MLS

20/5/2015 lectures 9 , 10 & 11

Seminal fluid
analysis
 Fertility vs. Sterility

FERTILE
not probably
pregnant pregnant
PARTNER
fertile pregnant
Male partner

SUBFERTILE

not possibly probably

M ALE Su-
Fertile

pregnant pregnant pregnant

Sterile
STERILE

not not not

pregnant pregnant pregnant

Female
STERILE partner
SUBFERTILE FERTILE
Sterile Su- fertile Fertile
FEM ALE PARTNER
 What? When? Why &
How?

When we know “Where, Why,

When & What, then we can
answer the all important
question How (to solve).
~ Chinese quote...!
 What is the “standard” approach to semen
evaluation?
 International minimum
standards are, by consensus,
the Lab Manual of
World Health Organization.
 Focus is on standardization
with expanded section on
quality control.
 What is semen, exactly?
A mixture of seminal plasma and cells
• Seminal plasma contains:
– Prostatic fluid (~30% of the volume)
– Epididymal plasma (~5% of the volume)
– Seminal vesicle fluid (the remainder of the ejaculate)
• The cells are:
– Spermatozoa
– Germ line cells
– Leukocytes of various types
– Bacteria
– Epithelial cells
– Occasional red cells
 Formation of the sperms
cells
• Formed in the seminiferous
tubules,
• Start as spermatogonia (self-
renewing stem cell of the
male germ cell line) – located
on the basement membrane
• The transformation from the
round germ cell to the sperm
cell occurs during passage to
the centre of the
seminiferous tubule.
 Spermatogenesis
• Is a cascade of cell
divisions:
– Mitosis: spermatogonia to
primary spermatocytes
– First meiotic division:
secondary spermatocytes
– Second meiotic division:
haploid spermatids
• This process takes 70 ± 4
days in the human.
 Spermiogenesis
• Is differentiation of the
round spermatid into a
spermatozoon
• This is the process in
which sperm
morphology is largely
determined.
What the spermatozoon looks like?
• Not the homunculus (left)
• More like the rabbit sperm
(right) – drawn by Antoni
van Leeuwenhoek in 1679
• Human sperm
 spermatozoon
• The human sperm cell is about 70
µm long
• The nucleus is in the head contains
the 23 chromosomes
• It is the head which binds to the egg
at fertilization
• Midpiece: the energy for motility is
generated
• Tail: motility – the beat is initiated
just behind the midpiece, and then
propagated along the tail.
 Sperm transport and seminal plasma
• “Testicular sperm” need to undergo more maturation steps
before they are ready to fertilize.
• Transported from the testes to the epididymis, where they
mature, and acquire the ability to swim.
• Then moved to the vas deferens, for storage
• At ejaculation, the sperm are transported out of the vas and
mix with accessory gland secretions:
– prostatic fluid (pH slightly acidic to neutral; contains citric
acid and zinc)
– seminal vesicle fluid (pH strongly alkaline; contains
fructose).
 Sperm function
 The fertilizing sperm swims through the layers
of cells around the ovum.
 The sperm then loses the front membranes of
its head and binds to the egg membrane.
 its tail stops beating, and the egg incorporates
the whole sperm cell.
 The egg unpacks the sperm, then the male and
female zygote formed.
 SEMEN
ANALYSIS
 What Is A Semen Analysis?

An evaluation of spermatogenesis and spermiogenesis.

• Approach to interpret with regard to:
–diagnosis of specific lesions.
–indicators of dysfunctional and/or functional
potential.
–Requires understanding of the relevance of sperm
patho-physiology.

• In any case, the results must be accurate and reliable.

 SEMEN ANALYSIS
What tests should always be done?
Always:
 Semen volume
 Sperm concentration
 Differential motility
 Morphology

If there is an indication:
• White cells Count and differential
• Vitality testing
 Why Perform Semen Analysis?
Diagnosis of sterility.
Prognosis of fertility.
Identify treatment options:
 Surgical treatment
 Medical treatment
 Assisted conception treatment
Forensic purposes. ( rape cases for example)
Suitability for artificial insemination
Therefore = a screening test to help direct management.
 Semen collection,
preparation, and instruction
 2-7 days abstinence from ejaculation prior to specimen
collection.
 The entire specimen must be collected into a clean
wide mouth container.
 Clearly labeled with the patient name and identifying
information.
 Transport the specimen to the laboratory right after
collection (if collected off-site) Delivered within 1 hour
of collection.
 Avoid temperature extremes.
 How to collect sample?

 Coitus interrupts : withdrawing just before ejaculating

follow by ejaculating into a clean sample cup.
 Coitus by using a condom: A special (silicon) condom
that does not contain any substance that kills sperm
(spermicide). Ordinary condoms should be washed and
cleaned since they usually contain spermicides.
 Assisted ejaculation :induced electro-ejaculation .
 Masturbation: directing the semen into a clean sample
cup. Do not use a lubricant which can kill sperms.
 How to collect sample?
 The patient should give an instruction to be
followed when collecting the sample.
 The ways of collection:
 special or washed condom or sheath, the
specimen is obtained from condom after normal
sexual intercourse.
 Induced method.
 Masturbation.
Sample has reached the lab yet
REMEMBER
SEMEN IS A BODY FLUID
BIOHAZARDOUS
 Semen Analysis Include
• Macroscopic • Microscopic
 Appearance concentration/count
 Viscosity motility
morphology
 Odour
viability
 coagulation + liquefaction
 volume
 pH

• Motility &Viability must be performed within

1½ - 2 hrs of collection
 How to Proceed ?
Sample

macroscopic

Complete
Put in 37ºC incubation till
incubator at Not
Liquified Liquefied Liquefication or
once
for 3 hours

Perform the test as

Fellow
 Macroscopic examination
• By Naked eye  Volume: Using Volumetric
 Semen is viscous, yellow grayish. pipette or in graduated cylinder
Forms gel-like clot immediately. to the nearest 0.1 ml or
 Liquefies completely in 5-60 centrifuge tube free of
contamination. Volume (1.5 - 6
minutes; this must be complete
ml).
before further testing (mix)
 Viscosity: 5ml pipette or plastic
 Appearance: homogenous white-
pipette : Consistency (Viscid –
gray opalescence.
Highly Viscid).
 Brown/red in
 pH: important parameter of
hematospermia
motility and viability 7.2-
 Dense white turbid if
8.0; Using red Litmus paper .
inflammation and high WBC.
 Appearance
Note the following:
Colour (normal = white to grayish-yellow) – if
there is blood present, it may range from pink to
brown
Opacity / translucence (normal = tends to opaque)
Whether mucus streaks or cell clumps are present
 Odour
Different people have different abilities to smell
semen, so this cannot be standardized
However, when the lid is taken off the collection jar, it
should be noted if there is a strong smell of urine or of
putrefaction
Samples collected after a prolonged abstinence period
(i.e. several weeks) are likely to have a stronger odour.
 Liquefaction & Viscosity
Liquefaction is the breakdown of the gel portion of the seminal
plasma – the enzymes for this are in the prostatic fluid
A sample with incomplete liquefaction has a gelatinous
material in a liquid base – this can be seen when the sample is
swirled for the appearance assessment.
Viscosity is related to the fluid nature of the whole sample
This is rated subjectively according to the length of the thread
of semen produced when the sample is allowed to run back
out of the volumetric pipette used to measure the ejaculate
volume.
 Volume
The volume of the sample should be measured to allow an
accurate determination of the sperm number.
This is most easily assessed using a warmed disposable
volumetric pipette (which is sterile and known NOT to be
spermotoxic).
After the sample is measured, allow it to run back into the
collection jar, noting its viscosity (a normal sample will
have some viscosity – i.e. not watery, but it will flow
easily).
 pH
pH is important because sperm die at pH < 6.9
The pH of liquefied semen is normally determined using
test strip or litmus paper.
measure pH after volume and viscosity – by touching the
“emptied” volumetric pipette to the test strip
The normal pH range is 7.2–8.4
Inflammatory disorders of the accessory glands can take
the pH outside of this range.
 Microscopic examination
• Sperm count
• Motility
• Viability
• Morphology
• Other microscopic finding
• Special Cases
 Sperm count
• Prepare the following
dilution:
• 380 µl Count solution* + 20
µl semen sample
• Counting:
• On Heamocytometer (on
WBCs count squares) Shaded are is one cell
Count Here
• = Count in 4 squares X 50000
• Count >20 million/ml
• Total count > 40 million
• While estimating count
• No stain
• Count 200 total sperm and then the motile
• Calculate the percentage of
– Progressive motile
– Sluggishly motile (<5 um/s )
– Non motile
• >50% motile.
 Sperm count solution
• Bufferd 3.5% Formol Saline consists of:
• NaHCO3 5 gm
• Formaline 35% 1 ml
• Crystal Violet 2 ml
• Mix & complete to 100 ml by distelled water
 Motility
The wet preparation:
Place 10µl of thoroughly mixed, liquefied
semen on a microscope slide and cover with a
cover slip and examine with X40 to determine
motility type.
• Rapid progressive:
• Rapid straight :forward movement.
• Sluggish: Slow irrgular movement (Backward
– In circles – forward)
• Non progressive: Movement in situ
• Immotile: No Movement.
 Gradation of sperm motility (WHO)-
2010
• Grade 4 (A): Sperm with progressive motility. These are the
strongest and swim fast in a straight line.
• Grade 3 (B): Sperm with non-linear forward motility. These also
move forward but tend to travel in a curved or crooked motion.
• Grade 2 (C): These have non-progressive motility because they
do not move forward despite they move their tails.
• Grade 1 (D): These are immotile and fail to move at all.

36
Sperm
motility
should be
scheduled
as:
Total motility
after 1 hour.
Total motility
after 2 hours.
Total motility
after 3 hours
from
ejaculation.
 Agglutination
• Reported when motile sperm stick to each
other in a definite pattern.
– Head-head
– Tail-tail
– Head-tail
• Immunological cause of infertility (anti-sperm
Abs)
• Done on several HPF
 Mixed Anti-globulin Reaction Test
• Place 10 ul of semen, Sperm**latex particles and
antiserum on a glass slide.
• Mix well semen and latex particles (5 seconds),
followed by the antiserum (5 seconds).
• Place a coverslip on top of the mixture and incubate the
slide at RT for 2-3 minutes in a damp chamber (e.g. a
Petri dish with a moist cotton or filter paper)
• Count at least 200 sperms.
• Assume clinical significance (infertility due to immune
reaction ) if >50% of sperms have beads attached.

Glass slide

antiserum Latex beads semen

 Viability
• Using Eosin stain • Viability > 75%(50% in
• Put one drop of semen other)
+ one drop of Eosin
stain.
• Examine under
microscope
Viable do not
take up the •Live
stain

Dead take •
up the stain
dead
• Supravital stain:
– Eosin +/- Nigrosin
• Viable do not take up the stain
• This distinguish live non motile from dead; it
is important to compare viability and motility.
• Eosin stain 5 g/l
• NaCl solution 0.9 %
Qualitative method for detection
of fructose in semen

Add few drops of semen

5 ml resorsinol

Boil for 20 – 30 seconds

Red Colour No
Fructose change
Present Fructose
Absent
  Resorsinol 5 gm
 Concentrated HCL 33 ml
 Dissolve Resorsinol crystals in
conc., HCL, Then complete to 100
ml with distilled water.
Normal level of fructose: 150-300mg/dl.
Reduced levels: - Seminal vesicle dysfunction
- High sperm count
 Morphology
• Smear:
– H&E, Papanicolaou, Wright stains
– Feathering like blood smear or 2 slides
– Count and classify 100-200 spermatozoa
– Examine the head, midpiece, tail
• Normal >30%
• Immature
• Abnormal
 Sperm Abnormalities

Head abnormalities: Tail abnormalities:

– absence – coiled
– double head – kinked
– micro/ megalo – Lengthened
 Other microscopic finding

• Pus Cells (WBC) < 1 million/ml

• RBCs normally not found

• Spermatogenic cells
[1] Round cells: 1) germinal cells (single or double
highly condensed nucleus with
abundant cytoplasm).
2)leucocytes < 1 million/ml or 1-2/hpf.
Increased no. in infection of reproductive
tract.
[2] RBCs: - Normally absent.
- Present in 1. TB of seminal vesicles
2. rupture of blood vessels
3. Infection of prostate
4.Vit. C deficiency
[3] Epithelial cells: - From urogenital tract.
 Special Cases
• Normospermia : Normal semen parameters
• Aspermia : absence of semen or No semen
ejaculated .
• Azoospermia : No spermatozoa found in semen .
• Oligospermia : low number of sperm.
• Asthenozoospermia : poor sperm motility and/or forward
progression
• Teratozoospermia : sperm carry more morphological defects
than usual.
• Hematospermia : Blood present in semen
• Leucocytospermia : White blood cells present in semen
• Necrospermia : No live sperm in semen
 Others
Mira1000 Semen Analyzer
(CASA)
Computer Assisted Semen
Analyzer
 Uses video and computer
software technology to
capture the types and speed of
sperm motility.
 Automatic image digitization
and processing
 CASA
 Additional parameters can be measured such as
curvilinear velocity (VCL) , straight line velocity (VSL),
linearity and flagellar beat frequency and amplitude of
lateral head (ALH)
– VCL=mean distance btw 1st sperm- 2nd sperm
position/time
– VSL=distance btw 1st –last sperm position/time
– VAP=average path velocity
– ALH =mean deviation from average path
– LIN =linearity (VSL/VCL)
– STR=straightness (VSL/VAP).
 CASA
 Advantages
– More objective and reproducible measurement.
– Superior documentation of laboratory values.
– Superior in measurement of sperm motility.
 Disadvantages
– Not reliable if sperm density is <2x106/ml. Require dilution if
>40 x 106/ml in isotonic buffer to avoid sperm collision
– Need to record 200 sperms for accurate distribution of
velocity.
– Parameters not standardized between laboratories –difficult
to interpret results
– No improvement on the manual method in distinguishing
fertilizing capacity of semen.
 Semen biochemistry
 Acid phosphatase: marker for prostatic function.
 Citric acid: can indicate prostatic function – low levels
may indicate dysfunction or a prostatic duct
obstruction.
 Zinc: marker for prostatic function .
 Fructose: marker for seminal vesicle function, and is a
substrate for sperm metabolism .
 -Glucosidase: secreted exclusively by the epididymis
and so is a marker for epididymal function .
 Semen for C/S(microbiology)
 Seminal fluid as other body fluids can be cultured for
microbial detection purposes.
 In this situation, microbiological contamination
from non-semen sources (e.g. commensal
organisms from the skin) must be avoided.
 The specimen containers, pipette tips and
pipettes for mixing must be sterile.
 Reporting result
• will include Diagnostic
Semen Analysis reports
– for infertility patients
• Enclosed by reference
ranges written within
result report.
WHO- WHO-1992 WHO-1987 Semen Characteristics
2010
or =1.5 > or = 2> or = 2> Volume (ml)
7.2-7.8 7.2-8.0 7.2-8.0 PH
or = 15 > or = 20> or = 20> Sperm concentration
(million/ml)
or = 39 > or = 40 > or = 40 > Total sperm count
(million/ejaculate)
or = 4 > or = 30 > or = 50 > Morphology (% of
normal)
or = 58 > or = 75 > or = 75 > Vitality (% of living)
1.0< 1.0 < 1.0 < WBC (M/ml)
or = 20 < or = 20 < or = 10 < Immunobead test (%sperm
56
with beads)
 WHO- WHO- WHO-
2010 1992 1987
Semen
Characteristics
or = 32 > or = 25 > or = 25 > Motility Within 1h of
ejaculation
Grade (A)
or = 40 > or = 50 > or = 50 > Motility Within 1h of
ejaculation Grade (A &B)
or = 20 > or = 20 > or = 20 > Neutral a-glucosidase
(u/ejaculate)
or = 2.4 > or = 2.4 > or = 2.4 > Total Zinc (µmol/ejaculate)

or = 52 > or = 52 > or = 52 > Total Citric acid

(µmol/ejaculate)
or = > or = > or = > Total acid phosphatase
200 200 200 (u/ejaculate) 57
 Factors affecting SA results
 Medicines: such as cimetidine (Tagamet), male and female
hormones (testosterone,estrogen), sulfasalazine,
nitrofurantoin, and some chemotherapy medicines.
 Caffeine, alcohol, cocaine, marijuana, and smoking
tobacco.
 Herbal medicines: such as high doses of echinacea.
 Temperature : sperm motility value will be inaccurately
low if the semen sample gets cold.
 Exposure to radiation: some chemicals (such as certain
pesticides or spermicides), and prolonged heat exposure.
 An incomplete semen sample: more common if a sample
is collected by methods other than masturbation.
 Not ejaculating for several days affect the semen volume.
 Quality control
• Quality Assurance Program
– Standard Operating Procedures
– Laboratory Manual
– Documentation
– Sample ID and Tracking
• External QC
– Comparison of tests with an external source
• Internal QC
– Minimized variation by training
– Purchased QC samples with known values
– Video recordings for motility
 CONCLUSION
Semen analysis is an important laboratory test and
should be thought of in the same way as any other
diagnostic assay.

It is used in determining treatment plans for

infertility.

The results can therefore have a huge impact on

the level of intervention, with the associated
emotional and financial costs to the couple.
 Thanks
Any Questions ?