Semen Analysis

Alan T. Koa, MD
Fellow, Philippine Society of Pathologists
Active Consultant, VRP Medical Center
Visiting Consultant, National Kidney and Transplant Institute
Chair, Human Pathology, San Beda College of Medicine
Core Faculty, Ateneo School of Medicine and Public Health
Semen Analysis
Physiology
Specimen Collection
Analysis
Additional Testing

Semen - Physiology
Composed of 4 fractions contributed
individually by testes and epididymis,
seminal vesicles, prostate, bulbourethral
glands
Mixing of all 4 fractions essential for
production of normal semen specimen
Semen - Physiology
Spermatozoa:
Produced in the seminiferous tubules of
the testes
Mature and stored in epididymis
Spermatozoa and fluid from epididymis
contribute 5% of semen volume
Semen - Physiology
Seminal Vesicles:
Contribute 60% of semen volume
Contain high content of fructose that the
spermatozoa readily metabolize
Spermatozoa become mobile when
exposed to seminal fluid
Semen - Physiology
Prostate:
Contributes 20-30% of semen volume
Contains high concentrations of acid
phosphatase, citric acid, zinc and
proteolytic enzymes (responsible for
coagulation and liquefaction of semen
following ejaculation)
Semen - Physiology
Bulbourethral glands:
Contribute 5% of semen volume
Thick, alkaline mucus (neutralize acidity
from prostate secretions and vaginal
acidity)
Specimen Collection
Specimens collected following a period
of sexual abstinence (3-5 days)
Fertility testing: 2-3 samples tested at 2-
week intervals; 2 abnormal samples
considered significant
Specimen Collection
Warm sterile glass
or plastic container
Specimen kept at
RT and delivered to
lab within 1 hour of
collection
Specimen Collection
Record time of collection and specimen
receipt
Fresh semen specimen is clotted
Liquefy within 30-60 mins after collection;
failure to liquefy maybe due to a deficiency in
prostatic enzymes
Analysis begins after liquefaction
Specimen collected by masturbation or
nonlubricant-containing polymeric silicone
(Silastic) condoms
Semen Analysis
Consists of macro and microscopic
examination
Parameters reported include:
appearance, volume, viscosity, pH,
sperm concentration and count, motility,
morphology
Analysis: Appearance
Gray-white color, translucent, musty
odor
Increased white turbidity: presence of
WBCs and infection within reproductive
tract
Varying degrees of red coloration:
presence of RBCs

Analysis: Appearance
Yellow coloration: urine contamination,
prolonged abstinence, medications
Urine is toxic to sperm, thereby affecting
evaluation of motility
Analysis: Volume
2-5 ml
Increased volume: following periods of
extended abstinence
Decreased volume:
Associated with infertility - may indicate improper
functioning of one of the semen-producing organs
Incomplete specimen collection must also be
considered
Analysis: Viscosity
Refers to consistency of fluid and may
be related to specimen liquefaction
Normal specimen should be easily
drawn into a pipette and form droplets
that dont appear clumped or stringy
when discharged from the pipette

Analysis: Viscosity
Ratings: 0 (watery) to 4 (gel-like)
Increased viscosity and incomplete
liquefaction will impede sperm motility
Analysis: pH
Alkaline: 7.2 to 8.0
Increased pH:
infection within
reproductive tract
Decreased pH:
associated with
increased prostatic
fluid
pH pad of urinalysis
reagent strip
Analysis: Sperm
Concentration/Count
Normal sperm concentration: > 20
million sperm/ml
10-20 million/ml is borderline
Total sperm count/ejaculate = sperm
concentration x specimen volume
Normal: > 40 million/ejaculate (20 M/ml
x 2 ml)

Analysis: Sperm
Concentration/Count
Neubauer counting chamber (amount of
dilution and the # of squares counted vary
among labs)
Commonly used dilution is 1:20; dilution is
essential because it immobilizes the sperm
prior to counting
Traditional diluting fluid contains Na
bicarbonate and formalin
Saline and distilled water can also be used


Analysis: Sperm
Concentration/Count
Sperm cells are usually
counted in the 4 corner and
center squares of the large
center square (RBC counting
area)
Both sides of
hemocytometer are loaded
and counted
Counts should agree within
10%
Average of the 2 counts is
used in the calculation
Only fully developed sperm
should be counted
Analysis: Sperm
Concentration/Count
>1 million WBCs/ml is associated with
inflammation or infection of the reproductive
organs (can lead to infertility)
The presence of >1 million spermatids/ml
indicates disruption of spermatogenesis
caused by viral infection, exposure to toxic
chemicals, genetic disorders

Calculation of Sperm
Concentration and Sperm Count
Calculation of sperm concentration is
dependent on dilution used and the size
and number of squares counted
When using 1:20 dilution and counting
5 RBC squares in large center square:
Sperm conc/ml = # of sperm x 1,000,000
Total sperm count = # of sperm/ml x
specimen volume
Analysis: Sperm
Concentration/Count
Makler counting chamber:
Provides a method for counting
undiluted specimens
Sperm are immobilized by heating part
of specimen prior to charging the
chamber
Sperm motility evaluated using the
unheated portion
Analysis: Sperm
Concentration/Count
The method recommended by the WHO
is the Neubauer chamber count
Analysis: Sperm Motility
Sperm capability of forward, progressive
movement is critical for fertility
Undiluted specimen; well mixed,
liquefied semen within 1 hour of
specimen collection
Determine % of motile sperm and
quality of the motility

Analysis: Sperm Motility
% of sperm showing actual forward
movement estimated after evaluating 20
HPFs
Motility is evaluated by both speed and
direction

Sperm Motility Grading
Grade WHO Criteria
4.0 a Rapid, straight-line motility
3.0 b Slower speed, some lateral
movement
2.0 b Slow forward progression,
noticeable lateral movement
1.0 c No forward progression

0 d No movement
Analysis: Sperm Motility
Normal: minimum motility of 50% with a
rating of 2.0 after 1 hour
WHO: Within 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)

Analysis: Sperm Motility
Presence of a high % of immotile sperm
and clumps of sperm requires further
evaluation to determine sperm viability
or presence of sperm agglutinins
Analysis: Sperm Motility
Computer-assisted semen
analysis (CASA)
Provides objective
determination of both
sperm velocity and
trajectory (direction of
motion)
Sperm concentration and
morphology are also
included in the analysis
Found primarily in
laboratories that
specialize in andrology
and perform a high
volume of semen analysis
Analysis: Sperm Morphology
Morphology is evaluated with respect to the structure
of head, neckpiece, midpiece and tail
Normal sperm:
Oval-shaped head 5 um long, 3 um wide
Long, flagellar tail 45 um long
Neckpiece attaches the head to the tail and midpiece
Midpiece is the thickest part of the tail surrounded
by a mitochondrial sheath that produces the energy
required by the tail for motility
Acrosomal cap critical to ovum penetration at the
tip of head(enzyme-containing)
Analysis: Sperm Morphology
Head abnormalities: poor ovum penetration
Neckpiece, midpiece and tail abnormalities:
affect motility
Morphology is evaluated from a thinly
smeared, stained slide under oil immersion
(Wrights, Giemsa, Papanicolaou)
At least 200 sperm should be evaluated and
% of abnormal sperm reported
Analysis: Sperm Morphology
Abnormalities in head structure: double
heads, giant and amorphous heads,
pinheads, tapered heads, constricted heads
Abnormal sperm tails: double, coiled, bent
Abnormally long neckpiece: may cause
sperm head to bend backward and interfere
with motility
Analysis: Sperm Morphology
Krugers strict criteria: requires use of a
stage micrometer or morphometry
Measurement of head, neck and tail size
Size of acrosome
Presence of vacuoles
Not routinely performed in the clinical
laboratory but is recommended by the WHO

Analysis: Sperm Morphology
Normal Values:
>30% normal forms (routine criteria)
>14% normal forms (strict criteria)
Kruger Strict Guidelines
>=15% normal: Normal range - Good prognosis
5-14% normal: Sub optimal range - Prognosis is fair
to good, however, the lower the
percent normal, the lower the
chance of successful fertilization
0-4% normal: Poor prognosis - Will usually need
IVF with intracytoplasmic sperm
injection (ICSI)
Additional Testing
Sperm viability
Seminal fluid fructose level
Sperm agglutinins
Microbial infection
Sperm Viability
Decreased sperm viability is suspected
when a specimen has a normal sperm
concentration with decreased motility
Viability is evaluated by mixing
specimen with an eosin nigrosin stain,
preparing a smear and counting # of
dead cells in 100 sperm
Sperm Viability
Living cells not infiltrated by dye and
remain a bluish-white color
Dead cells stain red against purple
background
Normal viability requires 75% living cells
Seminal Fluid Fructose
Low sperm concentration maybe caused by
lack of support medium produced in seminal
vesicles - indicated by low to absent fructose
level in semen
Resorcinol test to check for presence of
fructose (orange-red color when fructose is
present)
Normal quantitative level: >= 13 umol/
ejaculate
Specimen tested within 2 hours or frozen to
prevent fructolysis
Antisperm Antibodies
Present in both men and women
Detected in semen, cervical mucosa, or
serum
Possible cause of infertility
Male antisperm antibodies are more
frequently encountered
Antisperm Antibodies
Under normal conditions, blood-testes barrier
separates sperm from the male immune
system
When barrier is disrupted (surgery,
vasectomy reversal, trauma, infection),
antigens on sperm produce an immune
response that damages the sperm
Damaged sperm may cause production of
antibodies in the female partner
Antisperm Antibodies
Presence of antibodies in the male:
clumping of sperm
Presence of antisperm antibodies in the
female (demonstrated by mixing semen
with female cervical mucosa or serum
and observing for agglutination): normal
semen analysis accompanied by
continued infertility

Antisperm Antibodies
Tests to detect the presence of antibody-
coated sperm:
1. Mixed agglutination reaction (MAR)
test
2. Immunobead test

Antisperm Antibodies
Tests to detect the presence of antibody-
coated sperm:
Mixed agglutination reaction (MAR) test
screening procedure used primarily to
detect the presence of IgG antibodies

MAR test procedure
Semen sample containing motile sperm is
incubated with IgG AHG and a suspension of
latex particles or treated RBCs coated with
IgG
Bivalent AHG binds simultaneously to both
Ab on sperm and Ab on latex particles or
RBCs, forming microscopically visible clumps
of sperm and particles or cells
<10 % of motile sperm attached to particles is
normal
Antisperm Antibodies
Tests to detect the presence of antibody-
coated sperm:
Immunobead test
More specific procedure
Detects presence of IgG, IgM, and
IgA antibodies
Demonstrates what area of the
sperm the autoantibodies are affecting
Immunobead test procedure
Sperm mixed with polyacrylamide beads
coated with either anti-IgG, anti-IgM, or anti-
IgA
Microscopic exam will show beads attached
to sperm at particular areas
Reported as IgM tail antibodies, IgG head
antibodies, etc.
Presence of beads on < 20% of sperm is
normal
Microbial and Chemical
Testing
Routine aerobic and anerobic cultures
Additional chemical tests: determination of
levels of neutral alpha-glucosidase, Zn, citric
acid, acid phosphatase
Decreased alpha-glucosidase suggests a
disorder of epididymis
Decreased Zn, citrate, acid phosphatase
indicate lack of prostatic fluid
Normal Semen Chemical
Values
Neutral a-
glucosidase
>=20 mU
/ejaculate
Zinc >=2.4 umol/
ejaculate
Citric acid >=52 umol/
ejaculate
Acid phosphatase >=200 units/
ejaculate
Microbial and Chemical
Testing
Seminal fluid contains a high
concentration of prostatic acid
phosphatase, therefore the detection of
this enzyme can aid in determining the
presence of semen in a specimen
Postvasectomy Semen
Analysis
Presence or absence of spermatozoa
Specimens are routinely tested at
monthly intervals, beginning at 2
months postvasectomy and continuing
until 2 consecutive monthly specimens
show no sperm
Sperm Function Tests
Test Description
Hamster egg penetration Sperms are incubated
with species-nonspecific
hamster eggs and
penetration is observed
microscopically
Cervical mucus
penetration
Observation of sperm
penetration ability of
partners midcycle
cervical mucus
Sperm Function Tests
Test Description
Hypo-osmotic
swelling
Sperm exposed to low
sodium concentrations are
evaluated for membrane
integrity and sperm viability
In vitro acrosome
reaction
Evaluation of the acrosome
to produce enzymes
essential for ovum
penetration
Normal Values
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% within 1 hour
Quality >2.0
Morphology >14% normal forms (strict
criteria)
>30% normal forms
(routine criteria)
White blood cells <1.0 million/ml