Semen

Analysis
What is
Semen Analysis?
Semen analysis is an integral part of
infertility investigations taken as a
surrogate measure for male
fecundity in clinical andrology, male
fertility, and pregnancy risk
assessments.
 Advances in the field of andrology &
assisted reproductive technology (ART),
and increased concern over fertility,
particularly by couples choosing to have
children later in life, have resulted in
increased emphasis on the analysis of
semen
Primary reasons for analysis
• Fertility testing
• Post-vasectomy semen analysis
• Forensic analysis
Semen Composition
 Testis
• Produce the sperms (seminiferous tubules-Germ cells for
spermatozoa production)
• Specialized Sertoli cells (nurse cells for developing sperm)
provide support & nutrients for spermatogenesis
• When spermatogenesis is complete, the immature sperm
(nonmotile) enter the epididymis.

Physiology
Epididymis
• where sperms mature and develop flagella; 90 days
• The sperm remain stored in the epididymis until
ejaculation, at which time they are propelled through the
ductus deferens (vas deferens) to the ejaculatory ducts.
• ejaculatory ducts = receive both the sperm from the
ductus deferens & fluid from the seminal vesicles
 Seminal vesicles
• supply the majority of the vol. of the fluid; transport
medium for sperm
• secretes a viscous liquid that furnishes fructose, flavin &
Physiology other nutrients to maintain the sperms
⚬ fructose = energy source needed for the flagella to
propel; absence, sperm do not display motility
⚬ Flavin = gives gray appearance
⚬ Proteins = coagulation of ejaculate
 Prostate Gland
• surrounds the upper urethra & aids in propelling the sperm
through the urethra by contractions during ejaculation
• secretes a milky fluid that contains acid phosphatase, citric
Physiology acid, zinc & proteolytic enzymes that act on the fluid from
the seminal vesicles, resulting in the coagulation &
liquefaction of the semen
• 20-30% of the semen vol is acidic
 Bulbourethral glands
• about 5% of the fluid vol. ; thick, alkaline mucus that helps to
neutralize acidity from the prostate secretions & the vagina
Physiology • it is important for semen to be alkaline to neutralize the
vaginal acidity present as a result of normal bacterial
vaginal flora
• Without neutralization, sperm motility would be diminished
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Specimen Collection
Majority of sperm are contained in the first portion of the ejaculate
Detailed instruction in spx collection:

• Should be collected in sterile containers or warm sterile glass

⚬ Missing 1st portion = decreased sperm count; falsely increased pH, &
specimen will not liquefy
⚬ Missing last portion = decreased semen vol., falsely increased sperm
count, falsely decreased pH & the specimen will not clot
Specimen Collection
Majority of sperm are contained in the first portion of the ejaculate
Detailed instruction in spx collection:
• 2-3 day period of sexual abstinence (7 days = max)
• When performing fertility testing, 2-3 samples are usually tested at 7days-
3-week intervals, w/ two abnormal samples considered significant
• The laboratory should provide the patient with warm sterile glass or plastic
containers.
• Specimens awaiting motility analysis should be kept at 37oC.
Specimen Collection
• Specimens should be collected by masturbation. If not possible, nonlubricant-
containing rubber or polyurethane condoms
⚬ Condoms – not recommended for fertility testing
￭ may contain spermicidal agent
￭ ·would not affect results if only to assess the presence of sperm (viable
or nonviable) in postvasectomy cases
• If possible, the specimen is collected in a room provided by the laboratory
⚬ if this is not appropriate, the spx should be kept at RT & delivered w/in 1 hour
Specimen Collection
• Record time of collection (not time of receipt)
⚬ A fresh semen specimen is clotted & should liquefy w/in 30 mins. after
collection
￭ time of collection is essential for evaluation of semen liquefaction
⚬ Analysis of the specimen cannot begin until after liquefaction has occurred.
 Volume

Viscosity

pH

Parameters Sperm Count

Motility

Morphology
 Physical
Examination
A. Appearance
• Normal = gray-white color, Pearly-white, appears
translucent, & musty odor
• Clear = sperm conc. is very low
• Increased white turbidity = presence of WBCs &
infection w/in the reproductive; must be differentiated
from spermatids ; LE reagent strip test
• Red coloration = associated w/ the presence RBCs) &
are abnormal
• Yellow = urine; prolonged abstinence & medications
A. Appearance
Liquefaction
• Seminal fluid is clotted & liquefy w/in 30 to 60 mins after collection (recording
the time of collection is essential)
• No liquefaction w/in 60 mins = deficiency in prostatic enzyme
• Analysis of the specimen cannot begin until liquefaction has occurred
A. Appearance
Liquefaction
• No liquefaction after 2 hrs = add equal vol. of physiologic Dulbecco’s
phosphate-buffered saline or proteolytic enzymes (alphachymotrypsin or
bromelain) to induce liquefaction. However, it affect biochemical tests, sperm
motility, & sperm morphology (must be documented); dilution w/ bromelain
must be accounted for when calculating sperm conc.
• Jelly-like granules (gelatinous bodies) may be present but no clinical
significance
• Mucus strands = may interfere with analysis
B. Volume
• 2-5 mL
• Measured by pouring the specimen into a clean
graduated cylinder calibrated in 0.1-mL increments
• Increased volume = extended abstinence.
• Decreased volume = infertility & improper
functioning of one of the semen producing organs,
primarily the seminal vesicles
C. Viscosity
• refers to the consistency of the fluid & related to
liquefaction
• Normal = easily drawn into a pipette & form small
discrete droplets
• >2 cm droplets forming threads è highly viscous
• Ratings of 0 (watery) to 4 (gel-like)
• can also be reported as low, normal, or high
D. pH
• indicates the balance between the pH values from the
acidic prostatic secretion & the alkaline seminal
vesicles secretion
• measured w/in 1 hour of ejaculation due to the loss of
CO2 that occurs
• normal = alkaline with a range of 7.2 to 8.0
• Increased pH = infection w/in the reproductive tract
• Decreased pH = increased prostatic fluid,
ejaculatory duct obstruction, or poorly developed
seminal vesicles
Microscopic
Examination
A. Sperm Concentration & Count
• the actual # of sperm present in a semen spx is a
valid measurement of fertility.
• Normal Values
• sperm conc.: >20-250 million sperms per mL
• BORDERLINE: 10-20 million/mL
• sperm count : >40 million per ejaculate
• Multiplying sperm conc. by the specimen vol.
• Performed in the same manner as CSF counts:
• by diluting the specimen & counting the cells in a
Neubauer chamber
A. Sperm Concentration & Count

Factors that affect sperm concentration

• Days of abstinence
• Infection &
• Stress
*fertility study = more than one semen spx. be evaluated
A. Sperm Concentration & Count
• Dilute the specimen 1: 20
• 2 frequently used methods:
1. counting the sperm in the 5 RBC squares
2. counting the sperm in 2 large WBC squares
When 5 RBC squares are used:
# of sperms counted X 1 million = sperm per mL
When 2 large WBC squares are used:
# of sperms counted X 100, 000 = sperm per mL
A. Sperm Concentration & Count
• Dilution:
- immobilizes the sperm prior to counting
- diluting fluids:
1. sodium bicarbonate
2. formalin
3. saline & distilled water
• Both dilutions and counts should be performed in duplicate
on completely liquefied specimens to ensure accuracy
A. Sperm Concentration & Count
Neubauer hemocytometer
• method recommended by the (WHO)
• Both sides are loaded, settle for 3-5 mins & counted
(agree w/in 10%);
• An average of the two counts; if counts do not agree,
repeat dilution & the counts
• phase or bright-field microscopy
• Add stain (crystal violet) to the diluting fluid for
visualization when using bright-field microscopy
A. Sperm Concentration & Count
Neubauer hemocytometer
A. Sperm Concentration & Count
Neubauer hemocytometer
• Count only fully developed sperm
• Immature sperm & WBCs, “round” cells, not be included
(identify & count separately)
• Stain (spermatids vs leukocytes)
• >1 M leukocytes /mL = inflammation or infection of the
reproductive organs that can lead to infertility
• >1 M spermatids /mL = indicates disruption of
spermatogenesis, caused by viral infections, exposure
to toxic chemicals, & genetic disorders
A. Sperm Concentration & Count
Makler Counting Chamber
• provides a method for counting undiluted specimens
• a chamber w/ a 1-mm2 grid divided into 100 squares
(0.1 X 0.1 mm2) engraved in the cover plate
• sperm are immobilized by heating part of the
specimen prior to charging the chamber
• sperm motility using the unheated portion of the
specimen can also be evaluated in the chamber
A. Sperm Concentration & Count

Makler Counting Chamber

B. Motility
• Once presented to the cervix, the sperm must propel
themselves through the fallopian tubes to the ovum
• examine the undiluted spx microscopically &
determine the percentage of sperm showing active
motility & the quality
• Sperm should be evaluated on their progressive
forward mov’t
• Motility due to brownian mov’t should be disregarded
B. Motility
• Report: % of motile sperm
grade the motility:
a. on a scale of 0-4
b. By word descriptions (immotile- progressive)
c. a-d (WHO)
B. Motility
B. Motility
• examined~ 20 high power fields or 200 sperms/slide
• Evaluated by both speed & direction
• Normal: minimum motility of 50% w/ a rating of 2.0
after 1 hour
• WHO uses a rating scale of a, b, c, d
• Interpretation states that w/n 1 hour, 50% or more
sperm should be motile in categories a, b, and c, or
25% or more should show progressive motility (a and
b)
B. Motility
• high % of immobile sperm & clumps of sperm requires
further evaluation to determine sperm vitality or the
presence of sperm agglutinins
• computer-assisted semen analysis (CASA) = provides
objective determination of both sperm velocity &
trajectory
• Sperm conc. & morphology are also included in the
analysis
C. Morphology
• Evaluated w/ respect to head, neckpiece,
midpiece, & tail
• Abnormalities in head = associated w/ poor
ovum penetration
• neckpiece, midpiece, & tail abnormalities =
affect motility
• Normal sperm: oval head measuring ~ 3
wide X 5 um long, flagellar tail (45 um)
• At least 200 sperm should be evaluated &
the percentage of abnormal sperm reported
 Head
• critical to ovum penetration is the enzyme-containing
acrosomal cap located at the tip of the head
• encompass approx. half of the head & cover approx
two thirds of the sperm nucleus
3 Distinct Parts Neckpiece
of Sperm Cell • attaches the head to the tail & the midpiece

Midpiece
• approx 7 um long; thickest part of the tail is
surrounded by a mitochondrial sheath that
produces the energy required by the tail for motility

Tail
• Approx 45 um long including the neck
C. Morphology
Abnormalities in head structure are associated with
poor ovum penetration and include:
• double heads
• giant heads
• amorphous heads
• pin heads
• tapering heads
• constricted heads
C. Morphology
Abnormalities in tail structure:

• double tails
• coiled tails

An abnormally long neckpiece may cause the sperm

head to bend backward & interfere w/ motility
C. Morphology
C. Morphology
C. Morphology
• Addit’l parameters in the evaluation of sperm
morphology include measurement of head, neck, and
tail size, size of the acrosome, & the presence of
vacuoles (Kruger’s strict criteria) (>14% normal forms)
• requires the use of a stage micrometer or morphometry
• not routinely performed but is recommended by the
WHO
• an integral part of assisted reproduction evaluations
• Normal values: depend on the method of evaluation
used
• routine criteria = > 30% normal forms
C. Morphology
• Should be reported from a stained specimen examined
under OIO
• Recommended stain: Papanicolaou
• Acceptable results can be obtained by using:
1. Papanicolau stain
2. Wright’s stain
3. Giemsa stains
C. Morphology
Spermatids
• immature sperm
• must be differentiated from WBCs
• spherical
• may or may not have tails
• the presence of a high number of these forms is
considered ABNORMAL
• sperm usually mature within the epididymis prior
to their release.
C. Morphology
Calculation of Round Cells
• Differentiation & enumeration of round cells (immature
sperm & leukocytes) can also be made during the
morphology examination
• count the # of spermatids or leukocytes seen in
conjunction w/ 100 mature sperm
• the amount per mL can be calculated using the
formula: N=Number of spermatids or
neutrophils per 100 mature sperms
!×#
•𝐶= S=Sperm concentration M/mL
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C. Morphology
ROUND CELLS
• - >1 million WBCs per milliliter per ejaculate indicates
an inflammatory condition associated with infection
and poor sperm quality and may impair sperm motility
and DNA integrity.
 Volume 2 – 5 mL
Viscosity Pours in droplets
pH 7.2-8.0
Sperm Concentration >20 million/ mL
Sperm Count > 40 million/ejaculate
Motility >50 w/in 1 hours
Quality > 2.0 or fair (2.0 or A, B, C)

>14% normal forms è strict criteria

Morphology
>30% normal formsèroutine criteria

Round cells <1 million/ mL

Terminologies in Semen Analysis
• Oligospermia – sperm concentration <15 million/ml
• Asthenozoospermia – <40% grade (PR+NP) or < 32 PR%
• Teratozoospermia – <4% spermatozoa normal forms
• OAT – Oligo-astheno-teratozoospermia
• Azoospermia – no spermatozoa in semen
• Polyzoospermia – ++ high sperm concentration,
>200M/ml
Terminologies in Semen Analysis
• Hypospermia – semen volume < 1.5 ml
• Hyperspermia – semen volume > 6.0 ml
• Aspermia – no semen volume
• Pyospermia/Leukospermia – leukocytes present in
semen, >1M/ml
• Hematospermia – red blood cell present in semen
• Necrozoospermia – “dead” sperm
 Additional Testing
Should abnormalities be discovered in any of the routine parameters,
additional tests may be requested:

Sperm Seminal fluid Sperm Microbial

viability fructose level agglutinins infection
A. Sperm Vitality
abnormal: if there is NORMAL sperm conc + DECREASED motility
spx. + Eosin-Nigrosin è count dead cells (100 sperms)
• Dead cellS è red against purple background
• living cells è bluish white
- Living cells are not infiltrated by the dye & remain a bluish
white color
- Normal vitality = 50% living cells & should correspond to the
previously evaluated motility
A. Sperm Vitality
B. Seminal Fluid Fructose Level
Decreased Sperm count
è caused by a lack of support medium provided in
the seminal vesicle
è indicated by a low to absent fructose level
Fructose screening: resorcinol test (orange color when fructose
is present)
Normal level: >13 µmol per ejaculate
Fructose analysis: spectrophotometric methods
- spx – should be tested w/in 1-2 hours or frozen(fructolysis will
occur)
C. Antisperm Antibodies
• can be present in both men & women
• detected in semen, cervical mucosa, or serum & are
considered a possible cause of infertility
• not unusual for both partners to demonstrate Abs,
male antisperm Abs are more frequently encountered
C. Antisperm Antibodies
• Under normal conditions, the blood-testes barrier
separates sperm from the male immune system.
When this barrier is disrupted, (following surgery,
vasectomy reversal (vasovasostomy), trauma, &
infection, the Ags on the sperm produce an immune
response that damages the sperm
• The damaged sperm may cause the production of
antibodies in the female partner
C. Antisperm Antibodies
♦ Abs in MALE plasma è clumps of sperm are observed
during a routine semen analysis
♦ Anti-sperm Abs in the FEMALE è NORMAL semen
analysis + continued infertility
• semen + cervical mucosa or Female serum è
Agglutination
C. Antisperm Antibodies
2 frequently used tests for Ab-coated sperm:

1. Mixed agglutination reaction (MAR) test &

2. Immunobead test
C. Antisperm Antibodies
Mixed agglutination reaction (MAR) test
• screening procedure for the detection of (IgG) Abs
• semen w/ motile sperm is incubated w/ IgG antihuman
globulin (AHG) & a suspension of latex particles or treated
RBCs coated w/ IgG
• The bivalent AHG binds simultaneously to both the Ab on
the sperm & the Ab on the latex particles or RBCs, forming
microscopically visible clumps of sperm and particles or
cells
• normal result: <10% motile sperm attached to the particles
C. Antisperm Antibodies
Immunobead test
• more specific procedure (detect the presence of IgG, IgM,
& IgA Abs & demonstrates what area of the sperm (head,
neckpiece, midpiece, or tail) the autoantibodies are
affecting
• Head-directed Abs = interfere w/ penetration into the
cervical mucosa orovum
• tail-directed Abs = affect movement through the cervical
mucosa
• sperm are mixed wi/ polyacrylamide beads known to be
coated w/ either anti- IgG, anti-IgM, or anti-IgA
C. Antisperm Antibodies
Immunobead test
• Microscopic examination of the sperm shows the beads
attached to sperm at particular areas
• Depending on the type of beads used, the test could be
reported as “IgM tail antibodies,” “IgG head antibodies,” &
so forth
• Normal: presence of beads on <20% of the sperm
 Gelatin Agglutination Test -
classic method

Sperm Immobilization Test

Tests for Double-Fluorochrome

evaluation of Sperm-Cytotoxic Ab Assay
Male Anti- ELISA
sperm Abs
Mixed Antiglobulin Reaction

Immunobead Test
D. Microbial & Chemical Testing
• >1 million leukocytes/mL indicates infection w/in the
reproductive system, frequently the prostate
• Routine aerobic & anaerobic cultures & tests for
Chlamydia trachomatis, Mycoplasma hominis, &
Ureaplasma urealyticum
E. Forensic Medicine
• in cases of alleged rape
• Microscopically examine the specimen for the presence of
sperm enhanced w/ xylene &examining under phase
microscopy.
• test the material chemically for acid phosphatase content
è can confirm the presence of semen in a specimen
• glycoprotein p30 = more specific method
• ABO blood grouping
• DNA analysis
E. Forensic Medicine
Other tests:
1. Florence test = test for Choline
• derived from prostate, inhibits bacteria
• Reagent s: Iodine, KI = (+) dark rhombic crystals
2. Barbiero’s test = test for Spermine (more specific)
• Reagent : Picric acid, TCA = (+) yellow- leaf
shaped needles/crystals
3. Flourescence under UV light
Living - Green fluorescence
Dead - Red fluorescence
F. Postvasectomy Semen Analysis
• concern only for the presence or absence of spermatozoa
• common to find viable sperm in a postvasectomy patient
(care not to overlook even a single sperm)
• specimens are routinely tested at monthly intervals
beginning at 2 months postvasectomy & continuing until 2
consecutive monthly specimens show no spermatozoa
F. Postvasectomy Semen Analysis
Recommended testing

a. Microscopic examination of wet preparation using

phase microscopy for the presence of motile and nonmotile
sperm
b. If negative wet preparation, microscopic examination
of sediment from the centrifuged specimen is done (10 mins
centrifugation)
G. Sperm Function Tests
• Advances in assisted reproduction & IVF have resulted in a
need for more sophisticated semen analysis to assess not
only the characteristics of sperm but also the functional
ability
Computer Assisted SA (CASA)
• Uses video and computer software technology to capture
the types and speed of sperm motility.
• Automatic image digitization and processing
• Additional parameters can be measured such as
curvilinear velocity (VCL) , straightline velocity (VSL), linearity
and flagellar beat frequency and amplitude of lateral head
(ALH)
Computer Assisted SA (CASA)
• 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)
Computer Assisted SA (CASA)
Advantages

• More objective and reproducible measurement

• Superior documentation of laboratory values
• Superior in measurement of sperm motility
Computer Assisted SA (CASA)
Disadvantages
• Not reliable if sperm density is <2x106/ml or >50x106/ml, lots of
debris/immotile sperm. 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
END