SEMEN ANALYSIS (WHO

PROTOCOL)
PRESENTER: DR POOJA
MODERATOR: DR SAURABH
 Introduction
• Infertility affects around one in eight couples of reproductive age, with a
male factor being solely responsible in 20%.

• Semen analysis (SA) represents the most basic evaluation of male

infertility.

• It is important for assessment of male reproductive function and genital

tract patency to enable appropriate treatment for male subfertility and to
monitor treatment response.
Importance of Semen Analysis as a tool :
(1) Investigation of infertility
(2) Assess male reproductive health and function to guide management
(3) Guide the choice of assisted reproductive technology(ART)
procedure
(4) For selection of donors for artificial insemination and
(5) Measure the efficacy of male contraception (vasectomy)
Fraction of semen contributed by various glands:
• Testes: male gametes or spermatozoa are produced, & constitute 5% of semen
volume.
• Seminal vesicles (50%)of the semen volume, alkaline viscous, yellowish
secretion rich in fructose, prostaglandin, protein kinase which nourish and
activate the sperm.
• Prostate (40%) of the semen volume, consist of citric acid, zinc, acid phosphatase
and proteolytic enzymes that is responsible for liquefaction of semen.
• Bulbourethral glands (2-5%)- small mucus secreting glands.
The results of laboratory measurements of ejaculate characteristics will
depend on : whether a complete ejaculate is collected.
• During ejaculation, the first fractions expelled are mainly sperm-rich
prostatic fluids, whereas later fractions are dominated by seminal vesicular
fluid.
• Therefore, losing the first (sperm-rich) portion of the ejaculate has more
influence on the results of analysis than losing the last portion would have.
Basic semen examination involves the following steps

• Pre-examination procedures –
The pre-examination procedures comprise:
1) patient information
2) sample collection
3) sample reception
4) initial sample handling
 Patient information :

• The man should be given clear written and spoken instructions

concerning the collection of the semen sample.

• Period of abstinence is important as it affects both the quantity and

motility of spermatozoa.
• Therefore the ejaculate should be collected after a minimum of 2 days
and a maximum of 7 days of ejaculatory abstinence.
• A clean dry, wide mouth container should be used.

• Sample transportation : Collection should be done in the proximity of

lab. If not so then, transport must not allow the sample temperature
to go below 20 °C or above 37 °C

• Lubricants should be avoided: Interfere with motility

 Examination of semen sample
• Initial macroscopic examination
• Microscopic examination
• Functional test
• Immunologic analysis
Macroscopic evaluations:

• Volume
• Appearance
• Liquefaction
• Viscosity
• pH
Volume
• Normal: >1.5 ml per ejaculation

• Low semen volume is characteristic of obstruction of the ejaculatory duct or

congenital bilateral absence of the vas deferens (CBAVD).
• Low semen volume can also be the result of collection problems (loss of a
fraction of the ejaculate), partial retrograde ejaculation or androgen
deficiency.

• High semen volume may reflect active exudation in cases of active

inflammation of the accessory organs.
Appearance:
 A normal liquefied ejaculate has a macroscopically homogeneous,
cream/grey opalescent appearance.

 It may appear less opaque if the sperm concentration is very low.

The colour may also be different –

 slightly yellowish after longer abstinence times,
 red-brown when red blood cells are present (haemospermia), or
 clearer yellow in a patient with jaundice or taking certain vitamins or drugs
 Liquefaction
• A temperature of 37 °C will facilitate liquefaction. Also, a slow, swirling
movement of the sample container will help liquefaction to complete.

• Complete ejaculate liquefaction is normally achieved within 15–30

minutes at room temperature.
• If liquefaction is not complete within 30 minutes, this should be
recorded, and noted in the final report.

• The ejaculate could then be left in 37 °C for another 30 minutes.

• If liquefaction is not complete after 60 minutes, this should also be

included in the final report.
 Viscosity
• After liquefaction, viscosity of ejaculate
can be estimated by gently aspirating it
into a wide-bore (approximately 1.5 mm
diameter) plastic disposable pipette,
allowing the semen to drop by gravity
and observing the length of any thread.

• A normal liquefied ejaculate falls as

small discrete drops.
• If viscosity is abnormal, the drop will
form a thread more than 2 cm long
Between 30 and 60 minutes after ejaculate collection
• Assess liquefaction and macroscopic appearance of the semen.
• Prepare a wet preparation for assessing microscopic appearance,
sperm motility.
• Make dilutions for assessing sperm concentration
• Measure semen pH (if indicated).
• Assess sperm vitality (if the percentage of motile cells is low).
• Perform the mixed antiglobulin reaction (MAR) test for anti-sperm
antibodies (if required).

• Assess the presence of leukocytes cells (if required).

• Centrifuge semen aliquots (if biochemical markers are to be assayed)

 WITHIN 3 HOURS OF EJACULATE COLLECTION

• Determine sperm concentration (can be done later, preferably the

same day)

• Send samples to the microbiology laboratory (if required).

• Ideally, investigations should commence within 30 minutes after
collection, but at least within 60 minutes.

• Ejaculates may contain dangerous infectious agents [e.g. human

immunodeficiency virus (HIV), hepatitis viruses or herpes simplex
virus] and should therefore be handled as a biohazard.
Microscopic Examination

• Sperm Motility

• Sperm Viability or Vitality

• Sperm Count

• Sperm Morphology
 Representative sampling
• Although liquefied ejaculates macroscopically may appear
completely homogenous, there may still be small but significant
compartments with different composition of sperm and secretions.

 It is therefore essential to:

• use replicate aliquots of at least 50 µl for dilution: for sperm
concentration assessment.
• use replicate aliquots of at least 10 µl : for sperm motility assessment.
Making a wet preparation

1. Place a 10 µl well-mixed aliquot onto a clean microscope slide that

preferably is prewarmed to 37 °C (e.g. in the sample incubator).

2. Place a coverslip by dropping it carefully horizontally over the drop.

3. Assess the freshly made wet preparation as soon as the contents

are no longer drifting
Sperm motility
• The extent of progressive sperm motility is related to pregnancy rates.
• The velocity of motile spermatozoa is temperature dependent. It is
therefore essential to standardize the temperature during motility
assessment.
• Avoid assessing areas close (< 5 mm) to the edge of the coverslip to
prevent drying artefacts affecting the motility assessment.
• Field choice should be random.
• Avoid choosing fields based on the number of spermatozoa seen.
Start the scoring of a given field at a
random instant (do not wait for
spermatozoa to swim into the field
to begin scoring).

Systematic selection of fields for

assessment of sperm motility, at
least 5 mm from the edges of the
coverslip
At least 5 different fields should be assessed.

• The sum of the four grades should be 100.

• Report the average percentage for each motility grade to the nearest
whole number.

The recommended categories are:

1) rapidly progressive spermatozoa : moving fast forward in a straight line.
2) slowly progressive spermatozoa : slow linear or non linear movement
3) non-progressive: active tail movement present , progression absent
4) immotile – no active tail movements.
• Sperms of grades (c) and (d) are considered to be poorly motile
(asthenospermia).

• Normally, ≥ 25% of sperms show rapid progressive motility, or ≥

50% of sperms show rapid progressive and slow progressive motility.

• If the proportion of motile spermatozoa is < 50%, then proportion of

viable sperms should be determined by examining an eosin
preparation
Assessment of sperm clumping
• There are two different types of sperm clumping:

• Sperm aggregates : Adherence either of immotile spermatozoa to each other or

of motile spermatozoa to mucus strands, non-sperm cells or debris.
 Sperm agglutinates
• Agglutination specifically refers to motile spermatozoa sticking to
each other, head to-head, tail-to-tail or in a mixed way.
• Any motile spermatozoa that stick to each other by their heads, tails
or mid-pieces should be noted.
Sperm Count
• Dilution for the assessment of sperm number with a fixative is
necessary to immobilize spermatozoa.

• Dilution is also important to create a specimen that is more aqueous

for more reliable loading into the haemocytometer.
• At least 50 µl semen should be mixed with the diluent solution to
ensure reliable representativity and sufficient suspension volume for
thorough sample mixing
Principles for counting in a haemocytometer grid
• Count only whole spermatozoa (with a head and a tail).
• If there are many headless sperm tails (so-called “pinhead”
spermatozoa) or heads without tails, their presence should be recorded
in the report.
• The concentration can be estimated in relation to whole spermatozoa
(for instance, “45 headless tails per 100 spermatozoa”).
• Only spermatozoa in contact with the lower or left boundaries are
counted; not those in contact with the upper or right boundaries
• Semen is diluted 1:20 with sodium bicarbonate formalin diluting fluid
(Take 1 ml liquefied semen in a graduated tube and fill with diluting
fluid to 20 ml mark. Mix well).

• A coverslip is placed over the improved Neubauer counting chamber

and the counting chamber is filled with the well-mixed diluted semen
sample using a Pasteur pipette.
• The chamber is then placed in a humid box for 10-15 minutes for
spermatozoa to settle.
• Using the 40× objective and iris diaphragm lowered sufficiently to
give sufficient contrast, number of spermatozoa is counted in 4 large
corner squares
 Sperm vitality
• In samples with poor motility, the vitality test is important to
discriminate between immotile dead sperm and immotile live sperm.

• Percentage of live spermatozoa is assessed by identifying those with

an intact cell membrane, by dye exclusion or by hypotonic swelling.

• Vitality test using eosin–nigrosine: This one-step staining technique

uses nigrosin to increase the contrast between the background and
the sperm heads, which makes them easier to discern.
A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will
not be stained, while a non-viable or dead cell will have damaged cell membrane, will
take up the dye, and will be stained pink-red.
• Calculate the percentage of live cells.
• Report the percentage of vital spermatozoa, rounded off to the
nearest whole number
 Vitality test using hypo-osmotic swelling :
• As an alternative to dye exclusion, the hypo-osmotic swelling test may
be used to assess vitality.
• This is useful when staining of spermatozoa must be avoided, e.g.
when choosing spermatozoa for intracytoplasmic sperm injection
(ICSI).
• The hypo-osmotic swelling test presumes that only cells with intact
membranes (live cells) can swell in hypotonic solutions.
Spermatozoa with intact membranes swell within 5 minutes in hypo-
osmotic medium, and all flagellar shapes are stabilized by 30 minutes
Sperm Morphology

• Smear is prepared by spreading a drop of seminal fluid on glass slide, stained,

and percentages of normal and abnormal forms of spermatozoa are counted.
• The staining techniques used are Papanicolaou, eosin-nigrosin, hematoxylin-
eosin, and Rose Bengal-toluidine blue stain.
• At least 200 spermatozoa should be counted under oil immersion.
• Percentages of normal and abnormal spermatozoa should be recorded.
• Morphological defects have been associated with increased DNA
fragmentation, an increased incidence of structural chromosomal
aberrations, immature chromatin and aneuploidy.

• Coiled tails (more than 360°) may indicate epididymal dysfunction

IMMUNOLOGIC ANALYSIS
• Antisperm Antibodies
• The immunological tests done on seminal fluid include

1) Sperm MAR test (mixed antiglobulin reaction): Detect IgG and IgA
antibodies against sperm surface in semen sample.
2) Immunobead test: Antibodies bound to the surface of the
spermatozoa can be detected by antibodies attached to immunobeads
(plastic particles with attached anti-human immunoglobulin that may
be either IgG, IgA,or IgM).
Genetic and genomic tests
Sperm aneuploidy test
Normally sperm have a haploid complement of chromosomes (22
autosomes and 1 sex chromosome (X, Y)).
In an aneuploid sperm there is loss or gains of one or more autosomes
and/or sex chromosomes.

The only aneuploidies consistent with a viable but affected birth result
from changes in chromosome number of chromosomes 13, 18, 21, X and Y.
Robertsonian translocations are one well-known cause of increased levels
of aneuploid sperm
• Fluorescent in situ hybridization is a cytogenetic clinical diagnostic
assay that assesses the frequencies of chromosomal abnormalities
SEMEN ANALYSIS RECORDING FORM

• Patient name, Date of collection

• Sample ID
• Age
• Referring clinic/clinician/date of referral
• Information from patient: Abstinence days
• Collection At lab? Y/N
• Collection time hour:minute
• Severe infection/inflammatory disease in last 6 months?
• Medication for severe or chronic disease?
• Ejaculate volume (ml) Net weight of ejaculate
• Examination begun- hour:minute
• Ejaculate – Macroscopic examination
• Visual appearance (1, normal; 2, abnormal)
• Viscosity (1, normal; 2, abnormal)
• Treatment (e.g. to induce liquefaction)
• Ejaculate – Microscopic examination
• Aggregations (0, none; 1, some; 2, plenty)
• Agglutination
• Liquefaction (1, normal; 2, abnormal)
• pH
• Sperm Concentration (106 per ml)
• Motility
• Rapid progressive (a) (%)
• Slow progressive (b) (%)
• Non-progressive (c) (%)
• Immotile (d) (%)
• Vitality
• Vitality (when < 40% motile) (% live)
• Morphology
• Normal forms (%) (or typical or ideal)
• Any additional comments
Thank you