SEMEN

Urinalysis and Body Fluids

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Table of Contents:
1. Physiology

2. Specimen Collection

3. Semen Analysis

4. Additional Testing

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PHYSIOLOGY

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PHYSIOLOGY
SEMEN IS COMPOSED OF FOUR FRACTIONS CONTRIBUTED BY:
TESTES

EPIDIDYMIS

SEMINAL VESICLES

PROSTATE GLAND

BULBOURETHRAL GLANDS

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 The secretion of sperm primarily takes place in the testes,
particularly the

SEMINIFEROUS TUBULES

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 Germ cells for the production of spermatozoa
are located in the epithelial cells of the
seminiferous tubules.
In the epididymis, the sperm mature and
develop flagella.
The entire process takes approximately 90
days.

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 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.

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 Seminal vesicles - produce most of the fluid
present in semen (60% to 70%)
This fluid serves as the transport medium for
the sperm
Prostate gland - produces acidic fluid which is
20% to 30% of the semen volume
Bulbourethral glands - contribute 5% of the fluid
volume

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 The milky acidic fluid contains high
concentrations of acid phospatase, citric acid,
zinc, and proteolytic enzymes responsible for
both the coagulation and liquefaction of the
semen following ejaculation

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 The fluid contains a high concentration of
fructose and flavin.
Fructose - for energy of the sperm.
In the absence of fructose, sperm do not
display motility in semen analysis.
Flavin - responsible for gray appearance
of semen.

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SPECIMEN
COLLECTION
AND HANDLING

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 The variety in the composition o the semen
fractions makes proper collection of a
complete specimen essential for accurate
evaluation of male fertility.

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 Most of the sperm are contained in the first
portion of the ejaculate.
When a part of the portion of the ejaculate
is missing:
Sperm count - decreased
pH - increased
Specimen - will not liquefy

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 Specimens are collected following a period of
sexual abstinence of at least 2 days to not
more than 7 days.

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 Specimen should be kept at room
temperature.
Delivered to the laboratory within 1 hour
of collection.

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 Must be recorded:
Patient’s name
Date of birth
Period of sexual abstinence
Completeness of the smple
Difficulties with collection
Time of collection
Specimen receipt

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 Specimen should be collected by
masturbation.
If not possible, only nonlubricant-
containing rubber or polyurethane
condoms should be used.

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 All semen specimens are potential reservoirs
for HIV and hepatitis viruses, thus, standard
precautions must be observed.
Intra-uterine insemination (IUI), in vitro
fertilization (IVF) - sterile materials and
techniques must be used

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 SEMEN ANALYSIS

Semen analysis for fertility evaluation consists

of both macroscopic and microscopic
examination. Parameters reported include
appearance, volume, viscosity, pH, sperm
concentration and count, motility, and
morphology.

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 SEMEN ANALYSIS
Appearance
· Gray- white color – normal semen
· Almost clear – very low sperm concentration
· White turbidity – presence of white blood cells and
reproductive tract infection
o During microscopic examination, WBCs must be
differentiated from immature sperm (spermatids)
· Varying amounts of red coloration- presence of red blood
cells and are abnormal.
· Yellow coloration- urine contamination, specimen
collection following prolonged abstinence, and medications. 07
 Liquefaction
A fresh semen specimen is clotted and should liquefy within 30 to 60

minutes after collection; therefore, recording the time of collection is

essential for evaluating semen liquefaction. Failure of liquefaction to occur

within 60 minutes may be caused by a deficiency in prostatic enzymes and

should be reported.

Analysis of the specimen cannot begin until liquefaction has occurred.

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. Liquefaction
After 2 hours of no liquified specimen, equal volume of Dulbecco’s

phosphate-buffered saline or proteolytic enzymes such as alph-

chymotrypsin or bromelain may be added to induce liquefaction.

May affect:

· biochemical tests

· Sperm motility

· Sperm morphology
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 Liquefaction
Dilution of semen with bromelain must be counted for when

calculating sperm concentration:

· Jelly-like granules (gelatinous bodies)- may be present in

liquified semen and have no clinical significance.

· Mucus strands- may interfere with semen analysis.

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 VOLUME
2-5mL – normal semen volume
· Can be measured by pouring the specimen into a clean graduated
cylinder calibrated 0.1 mL increments.
Increased volume- seen in periods of extended abstinence.
Decreased volume- associated with infertility and improper functioning
seminal vesicles
 VISCOSITY
Specimen viscosity- refers to the consistency of the fluid and may be related

to specimen liquefaction.

Incomplete liquified- clumped and highly viscous

Normal semen specimen- easily drawn into a pipette and form discrete

droplets

· Do not appear clumped or stringy when falling by gravity from the pipette

· Thread droplets longer than 2cm – highly viscous and abnormal

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· The pH of semen indicates the balance between the pH values from the acidic prostatic

secretion and the alkaline seminal vesicles secretion.

· should be measured within 1 hour of ejaculation due to the loss of CO, that occurs.!

· The normal pH of semen is alkaline with a range of 7.2 to 8.0.

· Increased pH - infection within the reproductive tract.

· A decreased pH- may be associated with increased prostatic fluid, ejacula-tory duct

obstruction, or poorly developed seminal vesicles.

· I Semen for pH testing can be applied to the pH pad of a urinalysis reagent strip and the color

compared with the manufacturer's chart. Dedicated pH testing paper also can be used

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SPERM
CONCENTRATION
 Sperm Concentration

Normal Value = 20-250 million /ml

Borderline = Between 10-20 million /ml

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 1. Improved Nuebauer counting chamber

Dilution: 1:20 using a mechanical

pipette

Diluents: Formalin, Sodium Carbonate

Saline, Distilled water

Purpose of Diluents: To immobilize

the sperm

METHODS:
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 2. Maker Counting Chamber

For undiluted specimen

Uses heat to immobilize

METHODS:
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Formula for sperm
concentration

Total cells counted x Dilutiom

x 1000

Total area counted (mm2) x Depth (0.1)

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SPERM COUNT
SPERM Normal Value = >40 million
COUNT sperm/ejaculate

Formula :Sperm concentration x

Specimen volume
Specimen should be liquefied first
Normal value = >50% motile within 1
hour
Quality = > 2.0

Sperm Motility
GRADE WHO CRITERIA GRADE ALTERNATIVE GRADING
4.0 A Rapid, straight line motility Progressive motility (PM) Sperm moving linealrly
or in a large circle
Slower speed, Non progressive motility
3.0 B Sperm moving with an
some lateral movement (NP)
absence of progression
Slow forward progression, No movement
2.0 C Immolity (IM)
noticeable
lateral movement
1.0 C No foraward progression

No movement
0D
CASA ( Computer Assisted Semen Analysis)

Provides objective determination of sperm

velocity, trajectory, concentration and
morphology
SPERM MORPHOLOGY

Sperm morphology is evaluated from a thinly smeared,

stained slide under oil immersion. Smears are made by
placing approximately 10 micro liter f semen near the
frosted end of a clean microscope slide
Air-dried slides are stable for 24 hours
At least 200 sperm should be evaluated and the
percentage od abnomal sperm reported.
Normal Values
A. Routine criteria = > 30% normal
B. Kruger’s Strict criteria = >14% normal

Stains for sperm morphology:

a. Wright’s stain
b. Giemsa stain
c. Shorr stain
d. Papanicolau’s stain = best stain
 Acrosomal cap
part of the sperm that contains enzyme for
ovum penetration
Size : 1/2 of head / 2/3 of nucleus
Head = Length 5 micrometer, width 5 micrometer
Normal head appearance = oval
Tail= 45 micrometer long
Midpiece: 7 micrometer
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ADDITIONAL TESTING
The most common are tests for sperm vitality, seminal fluid fructose
level, sperm agglutinins, and microbial infection.
 SPERM VITALITY
link between sperm concentration, motility, and vitality Slide
Assessment within 1 hour of ejaculation
Use of eosin-nigrosin stain
Preparation of smear for evaluation

Vitality Evaluation
Counting dead cells in 100 sperm
Utilizing bright-field or phase-contrast microscope

Staining Process
Living cells remain bluish white
Dead cells stain red against a purple background

Normal Vitality
Requirement: 50% or more living cells
Correlation with motility

Differentiation between living and dead cells based on staining

Living cells remain bluish white
Dead cells stain red against a purple background
SEMINAL FLUID FRUCTOSE
LEVEL
Causes of Low Sperm Concentration
Lack of support medium from seminal vesicles as a potential cause
Indicators: Low to absent fructose levels in semen

Underlying Causes of Low Fructose Levels

Abnormalities of seminal vesicles
Bilateral congenital absence of vas deferens
Obstruction of ejaculatory duct
Partial retrograde ejaculation
Androgen deficiency

Fructose Screening
Utilization of the resorcinol test
Visual indication: Orange color when fructose is present (Procedure 10-3)

Normal Quantitative Fructose Level

Definition: Equal to or greater than 13 µmol per ejaculate
Assessment using spectrophotometric methods
 Antisperm Antibodies
Causes of Antisperm Antibodies
Disruption of the blood–testes barrier
Events leading to immune response: surgery, vasectomy reversal, trauma, and infection

Male Antisperm Antibodies

More frequently encountered than in females
Clumps of sperm observed during semen analysis as a potential indicator

Sperm-Agglutinating Antibodies
Cause sperm clumping in head-to-head, head-to-tail, or tail-to-tail pattern
Grading of agglutination as "few," "moderate," or "many"

Female Antisperm Antibodies

Normal semen analysis with continued infertility
Detection by mixing semen with female cervical mucosa or serum

Testing for Antisperm Antibodies

Overview of immunoassay kits for semen and serum testing
Focus on two frequently used tests: Mixed Agglutination Reaction (MAR) and Immunobead
Test
MICROBIAL INFECTION
Microbial Testing
Leukocyte count as an indicator of infection, especially in the prostate
Routine cultures for aerobic and anaerobic bacteria
Specific tests for Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum

Chemical Testing in Semen

Analysis of various chemical components:
Neutral α-glucosidase
Free L-carnitine
Glycerophosphocholine
Zinc
Citric acid
Glutamyl transpeptidase
Prostatic acid phosphatase

Spectrophotometric Methods
Utilization for quantitating citric acid and zinc levels

Seminal Fluid Markers

Prostatic acid phosphatase as an indicator of semen presence
More specific marker: Seminal glycoprotein p30 (prostatic specific antigen [PSA])
 Postvasetomy Semen Analysis
Primary concern: presence or absence of spermatozoa

Testing Frequency
Monthly intervals starting at 2 months postvasectomy
Continuation until two consecutive monthly specimens show no spermatozoa

Testing Methods
Wet preparation using phase microscopy
Examination for motile and nonmotile sperm
Caution against false negatives
Specimen centrifugation for 10 minutes
Examination of the sediment for further confirmation

Patient Education
Informing patients about the variability in sterilization timelines
Stressing the importance of follow-up testing
Additional testing for
Abnormal Semen
Analysis

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THANK YOU!