Semen Analysis

SEMEN ANALYSIS:
What? When? How? Why?
Dr Sharon Mortimer, PhD

Director, Oozoa Biomedical Inc
Research Planning & Development Specialist,
Faculty of Medicine, UBC
Editor, “Reproduction, Fertility and Development”
BACKGROUND
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
© Oozoa Biomedical Inc, April 2005

BACKGROUND
Formation of the sperm cell (1)
• Formed in the seminiferous tubules,

develop in close association with the
Sertoli cells
• 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
BACKGROUND
• 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 – so errors will take
about 3 months to show up

BACKGROUND
• Spermiogenesis: differentiation
of the round spermatid into a
spermatozoon
• This is the process in which

sperm morphology is largely
determined

BACKGROUND
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)

BACKGROUND
What the spermatozoon looks like (1)
• Not the homunculus (left)
• More like the rabbit sperm

(right) – drawn by Antoni
van Leeuwenhoek in 1679

BACKGROUND
Head • The human sperm cell is about 70 µm long

Mid-
piece • The nucleus is in the head – contains the 23
chromosomes
Tail • 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

BACKGROUND
• Tail cross-section
outer dense
fibres
dynein
arms
• The dynein arms from one
B subunit
doublet reach forward and
microtubule
touch the next doublet, ratchet
doublet forward, and then let go
A subunit
• Then the next doublet’s arms
reach forward, etc.
central pair • This generates the tail wave,
and hence sperm motility

BACKGROUND
Sperm function
• The ejaculated sperm pass through the cervix, then the

uterus, and enter the oviduct
• The fertilizing sperm swims through the layers of cells
around the egg (cumulus and corona), and reaches the zona
pellucida
• The sperm then loses the front membranes of its head (the
“acrosome reaction”), binds to the zona, then forces its way
through the zona to the egg membrane
• When the sperm head 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
pronuclei form. © Oozoa Biomedical Inc, April 2005
WHAT IS A SEMEN ANALYSIS?
• An evaluation of spermatogenesis and spermiogenesis.
• Traditional descriptive analysis:

• interpretation based on population distributions of characteristics,
• therefore prone to misinterpretation at the individual level.
• Modern approach is to interpret with regard to:

• diagnosis of specific lesions; and
• 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.

WHY PERFORM SEMEN ANALYSIS?
• Diagnosis of sterility
• Diagnosis of infertility
• Prognosis for fertility
• Identify treatment options:
• surgical treatment
• medical treatment
• assisted conception treatment
Therefore = a screening test to help direct management.

What is the “standard” approach to semen
evaluation?
International minimum standards are, by consensus, the
World Health Organization’s Lab Manual.
• Focus is on standardization with

expanded section on quality control.
• Methods amenable for use in any
(“third world”) country.
• Basic infertility work-up.

SEMEN ANALYSIS
Sample Collection
For a meaningful result, semen samples must always be
collected under standardized conditions:
– the container has to be sterile and known NOT to be
spermotoxic (i.e. provided by the lab)
– the man must have had 3 – 5 days of abstinence
– the man must have washed his hands before collection
(particularly if microbiological analysis is requested)
– the man must NOT have used lubricants (except for
Pre~Seed or His~Seed, the only “sperm-friendly” ones)
– the sample must be kept at 37°C until analysis, which begins
ideally within 30 min, but absolutely within 60 min, of
ejaculation
SEMEN ANALYSIS
Sample Handling
• The semen sample should be mixed gently during the

liquefaction period to promote liquefaction
• The sample should NEVER be vortexed (the only exception
is for the fixed prep for concentration assessment)
• The sample should NEVER be “needled” – if it is too viscous
to work with, a known volume of sperm buffer (not PBS)
should be added and the sample mixed gently. The added
volume must be included in the sperm concentration
calculation

SEMEN ANALYSIS
Macroscopic Evaluation
• There are several macroscopic evaluations which give
useful diagnostic information about the sample:
– Appearance
– Odour
– Liquefaction
– Volume
– Viscosity
– pH

SEMEN ANALYSIS
Macroscopic Evaluation - Appearance
• Swirl the sample, still in the collection jar, gently

• Hold the jar up to look through its bottom
• 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

SEMEN ANALYSIS
Macroscopic Evaluation - 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

SEMEN ANALYSIS
Macroscopic Evaluation – 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
SEMEN ANALYSIS
Macroscopic Evaluation - 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)

SEMEN ANALYSIS
Macroscopic Evaluation - pH
• pH is important because sperm die at pH < 6.9
• The pH of liquefied semen is normally determined
using test strips (we use EM Science ColorpHast type,
pH 6.5–10.0)
• We usually 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
SEMEN ANALYSIS
The wet preparation – set-up
• Place 10µl of thoroughly mixed, liquefied semen on a
microscope slide and cover with a 22x22mm No 1½ coverslip
• 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
– Microscope: phase contrast optics and a heated stage

SEMEN ANALYSIS
The wet preparation – assessments
• The characteristics assessed are:
– Motility (to be discussed later)
– Sperm aggregation (random clumping) – “some” is normal,
but large clumps (each with hundreds of sperm) is abnormal
– Spermagglutination (between specific sites) – could suggest
the presence of antisperm antibodies.
– Round cells: should be <1 per 40× field (~ 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
SEMEN ANALYSIS
Motility assessment - Background
• This is the first assessment made on the wet prep
• If >10–15% of the sperm are clumped, just assess the free-
swimming sperm (and note this on the report form)
• A repeat assessment should be made on a second wet prep
• Don’t estimate, count (> 4 fields and 200 sperm per prep)
• Use a 40× objective, and phase contrast optics
• Make a random selection of the fields that are assessed
• Assess fields that are away from the coverslip edge
• Count only those sperm that were in the field at one moment
in time (you have to be quick!)
• There must be agreement to within 10% between duplicates
SEMEN ANALYSIS
Motility assessment - types
• % motile = the proportion of sperm with tail movement

• Progression rating = the grade of progression shown by the
majority of the sperm: this can be from 0 (all immotile) to 4
(all with rapid progression); or from a (rapid progression) to
d (all immotile)
• Differential motility count = proportion of sperm in each of
4 motility classes (rapid progressive; slow progressive; non-
progressive; immotile)

SEMEN ANALYSIS
Motility assessment – differential motility
• Differential motility classification is

based on the distance swum over time:
– Rapid progressive: > 25 µm/s
– Slow progressive: 5 – 25 µm/s
– Non-progressive: < 5 µm/s
– Immotile: no flagellar movement
• The sperm head is about 5 µm long, so

the rapid sperm have a net gain of 5
head lengths / second

SEMEN ANALYSIS
Motility assessment – monitor
• Alternatively, we use a video

monitor with a grid for the
differential motility assessments
– so we do them off the screen.
• Each square is the equivalent of
25 × 25 µm (a stage micro-meter
is used to set this up)
• This approach is very useful for
training and for QC/QA

SEMEN ANALYSIS
Motility assessment – lab sources of error
• Have the definitions been implemented correctly?
• Are staff trained to classify progression?
• Internal quality control?
• How is velocity/progression assessed?
• Effect of temperature :% motile = minimal
% progressive = slight
% rapid = very large
• Representative sample aliquots?
• Adequate number of sperm counted?
• Repeatability of duplicate counts?
• Calculations performed correctly?
• Precision of results?
• External quality assurance / proficiency testing?
• Uncertainty of results? © Oozoa Biomedical Inc, April 2005
SEMEN ANALYSIS
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

SEMEN ANALYSIS
Sperm concentration- background
• Sperm count = total number of sperm in the ejaculate

• Sperm concentration = number of sperm per ml
(not “density”, which is an expression of mass / unit volume)
• Most accurate method is volumetric dilution and
haemocytometry
• Must use a positive displacement pipettor
• We make a 1 + 19 dilution with a fixative solution (allows
the assessments to be batched - usually counted within two
days)

SEMEN ANALYSIS
Sperm concentration - method
• Fix the haemocytometer

coverslip over the chambers –
ensure interference fringes can
be seen
• Vortex the fixed sample, load ~10
µl in both chambers
• Leave in a humid chamber for
10-15 minutes
• Count using a 20× objective and
phase contrast optics

SEMEN ANALYSIS
Sperm concentration - haemocytometer
First large square counted

The number of squares assessed
depends on the number of sperm
counted in the first large square:
– If < 10 counted, the whole grid
is assessed
– If 10-40 counted, 10 squares are
assessed
– If > 40 counted, 5 squares are
assessed

SEMEN ANALYSIS
Sperm concentration - calculations
• If the counts of the two chambers are not within 5% of their

average (i.e. the difference > 1/20 of their sum): discard, remix
the sample, and set it up again
• If the two counts are in agreement, then the sum of the two
counts is divided by the correction factor:
– If 2 × 25 squares counted, divide their sum by 10
– If 2 × 10 squares counter, divide their sum by 4
– If 2 × 5 squares counted, divide their sum by 2
• This gives the sperm concentration in millions per ml
• Sperm count = concentration × total volume

SEMEN ANALYSIS
Sperm concentration – lab sources of error
•Sample aliquot representative of ejaculate?
– semen homogeneous (mixed)?
– accurate sample aliquot (N.B. viscosity)?
– duplicate aliquot?
•Accurate dilution : – volumes of sample aliquot and diluent?
– storage (airtight) / sperm bind to vial?
•Secondary sampling : – mixing of diluted aliquot?
– duplicate aliquots?
•Preparation of counting chambers : – good chamber design/manufacture?
– chamber loaded correctly &/or cover glass placed correctly?
– adequate minimum number of cells?
– repeatability of duplicate counts?
•Calculations correct?
•Precision of results?
•Uncertainty of measurement known?
SEMEN ANALYSIS
Sperm concentration – interpretation
• The WHO Reference values for:
– Sperm concentration is > 20 × 106 sperm/ml
– Sperm count is > 40 × 106 sperm per ejaculate
• A persistently low sperm concentration is associated with
impaired fertility
• If a man has a sperm concentration < 5 × 106 sperm/ml, the
WHO recommends assessment for numerical and structural
abnormalities of sex chromosomes
• Azoospermia can indicate a failure of spermatogenesis or
blockage(s) in the male tract

FALLING SPERM COUNTS
SECULAR TREND IN SPERM COUNTS
150
100
Mspz/ml
50
0
1930 1940 1950 1960 1970 1980 1990
Carlsen et al.: Br. Med. J., 305:609-613, 1992

Falling sperm counts: Apocalypse now?
ANDROLOGY SPECIAL INTEREST

GROUP OF ESHRE (1997)
Pre-Congress “Controversies” Course
“Yes” = Richard Sharpe

“No” = David Mortimer
The motion was lost – not because of any lack of evidence for
adverse effects on male reproductive potential, but because the
data on sperm counts was established as too unreliable for valid
meta-analyses.
Variation in Sperm Counting

SEMEN ANALYSIS
Sperm morphology – background
• Morphology is even more important than motility and
concentration
• Because of the small size of the human sperm head, must
use an air-dried smear which has been stained
• The Papanicolaou method is best
• Prepared samples are assessed using a 100× oil-immersion
objective under bright field optics
• The WHO recommends that 200 spermatozoa are counted
per sample (and says that 2 × 200 is better)
• Fields for counting must be selected at random
• When counting, remember about the normal distribution
SEMEN ANALYSIS
Sperm morphology – morphology types
Variations of normal head shape Asymmetric Non-inserted Distended Thin

insertion tail midpiece midpiece
Small / large head Tapering heads

Cytoplasmic Coiled Duplicate Conjoined
droplet tail tail form
Pyriform heads Vacuolated

head
Short Hairpin Bent

Constricted Reduced Dense
tail tail tail
acrosome staining
Amorphous forms Terminal droplet

SEMEN ANALYSIS
Sperm morphology – assessment
• Even “good” samples will have <20% normal forms

• But we know that these sperm from these samples can
fertilize eggs – so is the morphology any help at all?
• Yes it is – but there are a few things to think about:
– The result will indicate a probability, not an absolute
– You have to count 2000 sperm to discriminate reliably
between, say, 3% and 5% (“Strict Criteria” cut-off 4%)
– The dynamic range of 4 – 15% is not very large, and means
that it is difficult to make a meaningful assessment
• So we use an extra “index” for morphology assessment
SEMEN ANALYSIS
Sperm morphology – assessment schemes
• Tygerberg scheme defines normal sperm and
counts the number that meet these criteria.
vs • WHO’92 looks for, and counts, defects that might
affect sperm function in each region of a sperm –
normal sperm are those that have no recognizable
defects.
• Expert direct comparison study between the
schemes* revealed little difference.
• Issues with counting error and uncertainty of
measurement, especially if only 100 sperm counted.
• Need additional information, e.g. Acrosome Index
or Teratozoospermia Index (TZI) when dynamic
range of % normal forms is very limited.
* Mortimer & Menkveld (2001) J. Androl. 22:192-205.

SEMEN ANALYSIS
Sperm morphology – TZI
• The Teratozoospermic Index is an expression of the average
number of abnormalities per abnormal sperm
• Each sperm cell is assessed for an abnormality in the head,
neck/midpiece, or tail, and for a cytoplasmic droplet
• If it does not have any of these abnormalities, it is “normal”
• If it does have an abnormality, it is “abnormal”, and we
score each abnormality. So, if a cell has an abnormal head
and tail, it is counted as 1 cell, and 2 abnormalities
• Then, (total # abnormalities) / (total # sperm) = TZI
• A TZI > 1.80 has been associated with poor sperm fertilizing
ability in vivo and in vitro
SEMEN ANALYSIS
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 – colorimetric assay
(WHO)
• Fructose: marker for seminal vesicle function, and is a
substrate for sperm metabolism – spectrophotometric assay
(WHO)
• -Glucosidase: secreted exclusively by the epididymis and so
is a marker for epididymal function – spectrophotometric
assay (WHO) © Oozoa Biomedical Inc, April 2005
SEMEN ANALYSIS
Sperm Vitality
• Used in samples with low total motility (<50%)
• Distinguishes between samples with live, but immotile sperm
(e.g Kartagener syndrome), and those with lots of dead
sperm (could result from: sperm senescence; exposure to
detergents or lubricants; or spermotoxic antibodies)
• Also provides a check on the accuracy of motility
assessments: % live should be slightly higher than % motile
• Usually performed using a vital stain, such as eosin Y, with a
counterstain (nigrosin) – although could use a fluorescent
DNA stain, such as H33258.

SEMEN ANALYSIS
Leukocyte tests
• Cytochemical stain for peroxidase activity – benzidine-
cyanosine is the easiest to use (and it is available in a kit).
Only stains those cells with peroxidase activity so does not
detect activated polymorphs or lymphocytes
• Immunocytochemistry: double antibody assay (primary
antibody is a mouse MAb to CD45), reaction is with alkaline
phosphatase:anti-alkaline phosphatase complex (available in
a kit)
• The tests are run on whole semen
• The normal range is < 1 × 106 / ml
• Values above this are considered to be clinically significant
SEMEN ANALYSIS
Retrograde ejaculation
• In some men, the semen passes back into the bladder at
ejaculation - this is confirmed by examination of a sample of post-
ejaculatory urine
• The man must take sodium bicarbonate the day before, and the
day of, his appointment – to alkalinize his urine
• Before collection, he should pass urine and then wait until he feels
there is some urine in his bladder before masturbating
• He then collects a urine sample for analysis
• Assess volume and pH of the urine
• Centrifuge (600g for 10 min), resuspend pellets to a total of 20ml
with sperm buffer + protein
• Re-spin, resuspend pellets and combine – add buffer to a final
volume of 1ml
• Perform a standard semen analysis with this suspension
SEMEN ANALYSIS
Post-vasectomy analysis
To determine the sperm concentration when few/none expected:
– centrifuge as much of the sample as possible (1000 g for 15 min)
in a disposable conical centrifuge tube – note the exact volume
used
– Remove most of the supernatant - note volume removed
– Make at least 5 wet preps (10µl of resuspended pellet under a 22
× 22mm coverslip)
– Search through at least 5 wet preps
– If anything that looks like a sperm is seen – the report is “not
clear”
– Final decision is the responsibility of the Pathologist

SEMEN ANALYSIS
What tests should always be done?
Always:
• Semen volume
• Sperm concentration
• Differential motility
• Morphology
If there is an indication:
• White cells
• Vitality
ABNORMAL SEMEN ANALYSIS
Treatments to correct abnormalities
• There is some evidence accumulating that there are some

treatments which can improve semen analysis results,
including:
– antioxidants
– carnitine
– reducing scrotal temperature
• However, there is still no good evidence that these changes
in semen analysis results are related to improved fertility
• Therefore, two abnormal semen analyses will often lead to
a couple being recommended fertility treatments
FERTILITY WORK-UP
• Fertility is a “couple” problem, and so both partners are
assessed
• For the woman, the investigations may include hormone
assessments (such as a day 3 FSH), a hysterosalpingogram,
and/or a laparoscopy
• Both partners also undergo physical examinations
• For these functional assessments, the investigation is based
on finding where the problem(s) lie – i.e. predicting failure
• The treatment chosen is the one most likely to circumvent
the problem(s) – i.e. increasing the likelihood of success

FERTILITY vs STERILITY
FERTILE
not probably
pregnant pregnant
pregnant
MALE PARTNER
SUBFERTILE
not possibly probably

pregnant pregnant pregnant
STERILE
not not not

pregnant pregnant pregnant
STERILE SUBFERTILE FERTILE
FEMALE PARTNER

FERTILITY TREATMENT OPTIONS
• If a partner is sterile (i.e. no gametes), then the couple

would need donor gametes to achieve a pregnancy
• If one or both partners are sub-fertile, then the treatment
options are:
– no treatment, or ovulation induction
– intra-uterine insemination (+ ovulation induction)
– in vitro fertilization (includes ICSI)

INTRA-UTERINE INSEMINATION (IUI)
• IUI is the least invasive of the ARTs - involves the selective

washing of semen to isolate the motile spermatozoa (can’t
put whole semen into the uterus)
• Up to 15 million motile spermatozoa are inseminated
• Advantages:
– relatively inexpensive – simple procedures
– minimal use of FSH
– can be used in consecutive cycles
– can usually start treatment virtually immediately
• Disadvantages:
– lower success rate per cycle than other ARTs
IN VITRO FERTILIZATION (IVF)
Overview
• There are many types of IVF
• For virtually all types, the woman is treated with “fertility
drugs” to stimulate the development of a group of eggs (the
average is around 10 – but the range can be enormous)
• Just prior to ovulation, the oocytes are retrieved
• That afternoon, they are inseminated with prepared sperm
• Inseminated eggs checked the next day for fertilization
• The fertilized eggs are kept in culture for up to 5-6 days
• Embryo transfer / possibly cryopreservation
Fertilization and embryo development

Intracytoplasmic sperm injection (ICSI)
• ICSI and its variants - the

highest-level ART
• One sperm is injected
directly into an egg
• Only mature eggs injected
• After the insemination, the
rest of the lab procedures are
the same as for “standard”
IVF

Risks and rewards
• IVF gives the highest chance of pregnancy per cycle –
although this is still controlled by the woman’s age
• Traditionally there was a high risk of multiple pregnancy
with IVF – as several embryos were transferred at once
• The new culture systems give embryos with a much higher
chance of implanting – so fewer are transferred
• The advent of ICSI has allowed men with very poor
fertility prognoses (including azoospermia) to become
biological fathers
• This can bring the requirement for genetic counselling
prior to treatment (Y-chromosome deletions, etc.)
ART SUCCESS AND FEES
Pregnancy rates (women < 37 years old):
• No treatment / fertile couple: around 15-20% per cycle
• IUI + COH: 20% per cycle
• IVF (includes ICSI): 50-60% per cycle (was about 10%)
Cost in BC (excluding drugs, which can be $2000–$4000):

• IUI + COH: $300 – $400
• IVF: $4500 – $4700
• ICSI: $5750 – $6250
• ICSI with aspirated sperm: $6250 – $6500 (+ urologist fees)
TREATMENT OPTIONS
Normal semen analysis
The recommended treatment will depend on the woman’s

test results, and could be:
• No treatment: if her results were normal, and she is young,
and they have been trying for only a short time (< 1 year)
• IUI: if the couple has already been trying for some years and
the woman has normal results
• IVF: if the woman is in her mid to late thirties, or if she has
blocked tubes

TREATMENT OPTIONS
Low concentration (oligozoospermia)
• IUI: if enough motile sperm can be recovered from the
ejaculate (and if the woman’s results are normal)
• IVF: if enough sperm can be recovered from the ejaculate,
and if the woman is older, or has blocked tubes.
• ICSI: only need to be able to isolate as many motile sperm as
there are eggs, so used in cases of extreme oligozoospermia
• ICSI with aspirated sperm: in the case of ejaculatory
azoospermia (i.e. there are sperm in the testes but not in the
ejaculate)

TREATMENT OPTIONS
Low motility (asthenozoospermia)
• IUI: less likely, and depends on whether enough sperm with
good progressive motility can be recovered, and if the
woman’s results are normal
• IVF: if enough motile sperm can be recovered from the
ejaculate, with a final total motility of >90% with good
progression (and if hyperactivation is seen)
• ICSI: quite likely, as the sperm may not be able to generate
enough power to break through the outer layer of the egg
(the zona pellucida)

TREATMENT OPTIONS
Poor morphology (teratozoospermia)
• If the sperm morphology is very poor, then ICSI is the
most likely option
• This is due to the high likelihood of failure of fertilization
by IUI or IVF, related to functional failure in sperm-egg
interactions, including:
– Geometric interference in sperm-egg binding (head too
round, or too long)
– Interference in penetration of the zona (low energy, poor
transmission of force due to midpiece or tail defects)

RESEARCH & NEW TECHNIQUES
Sperm kinematics
• The way sperm swim
affects their fertility
• To get through the cervix,
they have to swim in a
straight path
• To get through the outer
layer of the egg, they
have to generate a lot of
power – this is seen as
hyperactivated motility

Sperm kinematics
• The sperm tracks are analyzed and a number of kinematic
parameters are derived, including:
– Velocity (VCL, VSL and VAP)
– Velocity ratios (expression of the path shape and regularity)
– Amplitude of lateral head displacement
– Beat/cross frequency
• The proportion of sperm in a sample which meet particular
kinematic criteria is used to predict (failure) of:
– Mucus-penetrating ability
– Hyperactivation (a marker of sperm function)
CASA
• Computer-aided sperm
analysis
• Able to assess the
kinematics of hundreds
of sperm in a couple of
minutes
• Means that these tests
can be part of the
infertility work-up

ROS and sperm

Sperm DNA Damage
Sperm Chromatin Structure Assay: SCSA™
© SCSA Diagnostics Inc, Brookings, SD, USA

Evenson et al. (1980) Science 240:1131-1133 © Oozoa Biomedical Inc, April 2005
Structured management
• Use the test results to predict where blocks to fertilization

are occurring
• A treatment plan to go around these blocks is created for
each couple
• It is designed to maximize the chance of achieving a
successful pregnancy in the most practicable and
economical way
• The treatment plan is implemented after consultation with
the couple

The WHO and Structured Management
(2000)

STANDARDIZATION & QUALITY
CONTROL
Fundamental requirements of all laboratory tests :

• appropriate and robust methodology
• careful training
• operator experience
} accuracy & precision
• suitable assay control limits
• regular internal comparisons = IQC
• regular external comparisons = EQA
• cost effectiveness
College of Reproductive Biology Inaugural Meeting, Houston, May 1997

ISO 15189:2003
Medical laboratories – Particular requirements for quality and competence.
5.5.1 Appropriate procedures that meet the needs of the users of the
laboratory’s services shall be used.
5.5.2 Only validated procedures shall be used.
5.5.3 All procedures must be documented and available:
a) purpose j) sources of interference
b) principle k) calculation of results (includes
c) performance specifications uncertainty of measurement)
d) primary sample(s) l) reference intervals
e) sample container m) reportable interval for patients
f) equipment & reagents n) alert &/or critical values
g) calibration o) interpretation by laboratory
h) procedural steps p) safety precautions
i) QC procedures q) potential sources of variability
5.5.4 Performance specifications must related to intended use.
5.5.5 Periodic review of biological reference intervals

QC IN SEMEN ANALYSIS
Mean ± SD% differences between 4 trained andrology scientists
using WHO manual/visual semen analysis methods on 60
determinations. (Calgary Diagnostic Semen Lab, ca. 1990)
Concentration Total motility Prog motility
A -2.3 ± 7.4 +0.3 ± 3.0 +0.4 ± 2.6

B -1.7 ± 4.9 -0.8 ± 3.1 -0.8 ± 2.9
C +4.5 ± 7.3 -1.0 ± 3.3 -0.6 ± 3.3
D -0.5 ± 7.0 +1.6 ± 2.9 +1.0 ± 2.8

TRAINING IN SEMEN ANALYSIS

GOAL-ORIENTED TRAINING
• Originally elaborated in Calgary during the mid/late 1980s

as a means to facilitate the training of new staff.
• Subsequently applied in numerous locations where we were

involved in training andrology lab staff:
e.g. Sydney (Australia), London (UK), Stockholm (Sweden),
Boston (USA), Bangkok (Thailand).
• Adopted by the ESHRE Andrology SIG Education Sub-

committee as the basis for their Basic Semen Analysis
Courses (25 run by the end of 2003).

ESHRE Basic Semen Analysis Courses

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

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SEMEN ANALYSIS:

What? When? How? Why?

Dr Sharon Mortimer, PhD

A mixture of seminal plasma and cells

© Oozoa Biomedical Inc, April 2005

• Formed in the seminiferous tubules,

• Start as spermatogonia (self-renewing

• The transformation from the round

© Oozoa Biomedical Inc, April 2005

• This is the process in which

© Oozoa Biomedical Inc, April 2005

• “Testicular sperm” need to undergo more maturation steps

© Oozoa Biomedical Inc, April 2005

• Not the homunculus (left)

• More like the rabbit sperm

© Oozoa Biomedical Inc, April 2005

Head • The human sperm cell is about 70 µm long

Tail • It is the head which binds to the egg at

© Oozoa Biomedical Inc, April 2005

© Oozoa Biomedical Inc, April 2005

• The ejaculated sperm pass through the cervix, then the

• An evaluation of spermatogenesis and spermiogenesis.

• Traditional descriptive analysis:

• Modern approach is to interpret with regard to:

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

© Oozoa Biomedical Inc, April 2005

© Oozoa Biomedical Inc, April 2005

• Focus is on standardization with

© Oozoa Biomedical Inc, April 2005

• The semen sample should be mixed gently during the

© Oozoa Biomedical Inc, April 2005

© Oozoa Biomedical Inc, April 2005

• Swirl the sample, still in the collection jar, gently

© Oozoa Biomedical Inc, April 2005

• Different people have different abilities to smell semen,

© Oozoa Biomedical Inc, April 2005

• Liquefaction is the breakdown of the gel portion of the

• Viscosity is related to the fluid nature of the whole sample

• The volume of the sample should be measured to allow

© Oozoa Biomedical Inc, April 2005

© Oozoa Biomedical Inc, April 2005

• % motile = the proportion of sperm with tail movement

© Oozoa Biomedical Inc, April 2005

• Differential motility classification is

• The sperm head is about 5 µm long, so

© Oozoa Biomedical Inc, April 2005

• Alternatively, we use a video

© Oozoa Biomedical Inc, April 2005

• The WHO’s Reference values for motility are:

• Assuming that all of the collection and laboratory factors

• However, this should be confirmed by a repeat semen

© Oozoa Biomedical Inc, April 2005

• Sperm count = total number of sperm in the ejaculate

© Oozoa Biomedical Inc, April 2005

• Fix the haemocytometer

© Oozoa Biomedical Inc, April 2005

First large square counted

© Oozoa Biomedical Inc, April 2005

• If the counts of the two chambers are not within 5% of their