TOPIC OUTLINE Prostate gland

I. Semen Physiology ● Acidic fluid (20%-30%)

II. Specimen Collection ● Contains acid phosphatase, citric acid,
a. Methods of Collection zinc, and proteolytic enzymes
III. Physical and Microscopic Examination of
Sperm. ○ This enzyme is used to test if the
a. Appearance fluid submitted is really semen.
b. Volume and Viscocity ○ Enzymes coagulate semen prior to
c. Sperm Count and Concetration ejaculation and cause liquefaction
d. Sperm Motility after ejaculation
e. Sperm Morphology
f. Sperm Viability
○ In Clinical chemistry it is used as
IV. Chemical tests a marker for prostate cancer
a. Seminal Fructose ■ high conc. of acid
b. Antisperm antibodies phosphatase in blood
c. Immunobead Test
d. Semen Detection
Bulbourethral glands
e. Post Vasectomy Analysis
f. Semen Function Test ● Thick, alkaline fluid (5%) to neutralize acid
V. Semen Analysis and Quality Control from prostate and acid pH of vagina;
otherwise, no sperm motility
Semenalysis
SEMEN PHYSIOLOGY

Semen consists of four components

● Contributed separately by the:
● Testes and epididymis
● Seminal vessels
● Prostate glands
● Bulbourethral glands
● Pag konti lang ang prostatic fluid, there
will be a problem in liquefaction and may
cause problems in the motility of sperm
● Not enough energy from the seminal
vessels will lead to infertility

Spermatozoa
● Produced in seminiferous tubules of testes
● Mature and stored in the epididymis
● Contribute 5% of volume

Seminal vessels
● Majority of fluid (60%)
● Provide fructose for sperm metabolism
● Sperm are not motile without this fluid

Composition of Semen
● Spermatozoa 5%
● Seminal Fluid 60%–70%
● Prostate Fluid 20%–30% SPECIMEN COLLECTION
● Bulbourethral Glands Fluid 5%
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 ● Must be a complete specimen ○ add enzyme if did not liquefy within
○ Incomplete spx may have an effect 30 to 60 mins
in sperm collection count ○ may have decreased amount of
■ must give patient proper prostate liquid
and complete instructions ○ sperm cells may have a problem
● Majority of sperm is in first part of with their motility
ejaculate ● No lubricated, anti spermicidal condoms
● Detailed instructions to patients ● Semen specimens are reservoirs for
● 2–3 days abstinence; no longer than 5 hepatitis and HIV
days ○ use gloves and observe necessary
○ > 5 days = sperm not that motile precautions
● Laboratory provides containers and ideally ● Discard as biohazardous waste
a (private) room for collection
● Home: deliver to laboratory within 1 hour;
keep specimen warm Appearance
○ 37 C
○ instruct px to not contaminate the
Color Interpretation
spx with water or urine
Methods of Collection Normal Gray-white,
Methods of Collection translucent
● Self-production or masturbation - BEST
because it prevents contamination White turbidity Infection ; perform
● Coitus interruptus – withdrawal method culture
○ px must have coitus w/ partner,
then pull out Red Blood cells, Abnormal
○ Disadvantage: spx may not be
complete Yellow Urine, Prolonged
● Vaginal vault aspiration - Aspiration of abstinence,
seminal fluid from the vaginal vault after Medications
coitus
○ let px and his partner have sex, -Urine is toxic to sperm = no motility.
then finish in vagina -Clots remaining after 1 hour: wait for liquefaction
○ fluid is aspirated from the vagina before analyzing.
○ Disadvantage: prone to -chymotrypsin may be used to increase
contamination motility.
● Condom method – only non
lubricant-containing rubber or Volume and Viscocity
polyurethane condoms should be used Volume and Viscosity
○ use non-lubricant ● Normal: 2 – 5 mL
■ may immobilize sperm ● Measure in a graduated cylinder
20cells if contaminated with ○ after 30-60 mins of liquefaction
lubricant/water/oil ● Decreased = infertility, incomplete
○ use polyurethane condoms that do collection
not contain spermicides ● Viscosity: droplets with thin threads from a
pipette are normal
● Record time of collection and time of ● Rate 0 (watery) to 4 (gel-like) or low,
receipt normal, high
● Clotted specimen should liquefy in 30–60 ○ clotted semen - affects motility of
minutes sperm cells
○
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 pH ○ 60,000,000 sperm concentration x
3 mL volume = 180,000,000 sperm
● Normal : 7.2–8.0 count
● Over 8.0 = infection
● Acid = increased prostate fluid ● Azoospermia - complete or total absence
● Check with pH paper (or reagent strip) of spermatozoa seen in
● Necrospermia – presence of sperm cells
Sperm Concentration and Count whether completely dead or immobile
● Oligospermia - deficiency in the number
of sperm cells or presence of few motile
-Sperm Concentration = number sperm/mL cells seen.
● using Neubauer counting chamber or
hemocytometer Sperm Motility
-Sperm Count = number sperm per ejaculate
● computed ● Need to have sperm with forward,
● Normal concentration = 20-160 progressive movement
million/mL, 10-20 million borderline ● Examine within 1 hour; evaluate undiluted
● Normal count = concentration x volume on glass slide with cover slip
○ >40 million/ejaculate ○ undiluted to observe the motility
○ Example: 40 million (conc), 3 mL ● Estimate percentage with progressive,
(vol) = 120 million/ejaculate forward motion in (atleast) 20 fields.
● Concentration performed in Neubauer Grading scale:
chamber, same as red blood cell count—5
0 no movement
small center squares
● Diluting fluid = sodium bicarbonate and 1 no forward progression
formalin, must immobilize sperm
○ also cold water 2 slow forward movement,
● Dilution: 1 to 20 more lateral
● Count both sides of chamber, sides must
agree within 10%; use the average 3 slower speed, some
● With this method, if an average of 60 lateral movement
sperm are counted multiply by 1,000,000
4 rapid straight line motility

CASA - Computer Assisted Semen Analysis

● Provides objective determination of both

sperm velocity and trajectory (direction
of motion)
● Sperm concentration is also included in
the analysis

Sperm Morphology

● Must multiply sperm per μL by 1000 to Critical to fertilization

reach sperm/mL when using a different Evaluate head, neck, midpiece, tail
number of squares Parts:
Sperm count 1. Head: oval with acrosomal cap at end
and covering half of the head

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 a. Contains enzymes for ovum ● Round bodies
penetration ○ White blood cells (WBCs) and
2. Neckpiece attaches head to midpiece and spermatids (immature sperm)
tail; ○ Differentiate on morphology smear
3. The midpiece is surrounded by sheathe of ○ Count number of each separately
mitochondria (energy) for tail in 100 cells
movement ■ C = Number x
4. Tail is a long flagellum for movement/ concentration / 100
○ Normal: < 1,000,000
■ More than that may indicate
infertility
-Observe on thin smear under oil
-Wright stain or Pap stain are the stains used
-Count 200 and report number of abnormal
-Strict criteria measures, acrosome, tail, neck,
and midpiece recommended by World Health
Organization (WHO)

Kruger’s criteria
● Normal (Nonstrict): >30% normal
● Strict: > 14% normal
Sperm Viability

Eosin-nigrosin stain are the stains used.

● Dead cells stain red
● Normal cells stain blue-white.
● Count number/100 cells

● Normal: 75% living (blue-white)

● Corresponds to motility
● Seminal fructose tests

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 ● AHG combines with particles and antibody
coated sperm forming clumps.
● Normal <10% motile sperm attach to
particles

Immunobead Test

More specific
● nasasabi kung saan mismo sa
sperm yung may problem
Demonstrates antibodies to head, neck (problem
Seminal Fluid Fructose in ovum penetration), midpiece, and tail
(problems in motility)
● Low sperm concentration may be caused ○ Beads are coated with antihuman
by lack of the support medium produced in globulin
the seminal vesicles. This can be ○ Microscopic examination shows
indicated by a low to absent fructose level where on sperm antibodies are
in the semen. attacking
● Resorcinol test - for screening for the ○ Tail = movement; head =
presence of fructose penetration
● Positive: orange-red color ○ Normal: beads on <20% of sperm
● Specimens: should be tested within 2 ■ >20% = infertile
hours or frozen
Microbial and Chemistry Testing
Antisperm Antibodies
● Present in both men and women ● >1 million WBCs usually cause prostate
● Male more common: surgery, vasectomy infection: culture and test for: Mycoplasma
reversal, trauma hominis,
● Sperm normally do not encounter the ● Chlamydia trachomatis,
immune system, so body considers them ● Ureaplasma urealyticum
foreign ● Gonorrhea
The damaged sperm may cause the production of
antibodies in the female partner. Chemistry tests: neutral α-glucosidase, zinc,
citric acid, acid phosphatase
Suspect male antibodies when clumps of sperm ● important for sperm cells to thrive, and has
are seen; female no clumping an impact in the viability of these cells

Female: mix female serum with sperm and check

DECREASE RESULT:
for agglutination
● simplest test to confirm antisperm ↓ Neutral α Epididymis
antibody -glucosidase
● agglutination = positive for antisperm
antibody against the sperm of the man ↓ zinc, citric acid, acid ↓ Prostatic fluid
phosphatase
Immunoassays: males
Mixed agglutination reaction (MAR) test
● Incubate sperm with antihuman globulin
(AHG) and IgG-coated latex particles.
● Purpose of coating is to make the reaction
more visible.

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 Normal Semen Chemical Values ● Hypo-osmotic swelling – test for
membrane integrity and viability of sperm
in low-sodium concentration
Neutral a-glucosidase ≥20 mU/ejaculate
● In-vitro acrosome reaction - evaluation
Zinc ≥2.4 µmol/ejaculate of the acrosome to produce enzymes
essential for ovum penetration
Citric acid ≥52 µmol/ejaculate
Other Tests
Acid phosphatase ≥200 Units/ejaculate ● Spinbarkeit test
○ Test for the tenacity of mucus (of
Semen Detection the female partner)
● Sim Huhner test
● Is semen present in specimen? ○ Post-coital test
● Measure specimen acid phosphatase ○ Test for the ability of sperm cells to
content penetrate the cervical mucosa
● Observe microscopically for sperm Semen Analysis Quality Control
○ Enhance with xylene; use phase
microscopy ● Clinical Laboratory Improvement
● Detection of seminal plasma glycoprotein, Amendments (CLIA) rates semen
p30 analysis as high complexity
● DNA analysis ● Commercial controls and training
materials are available
Post Vasectomy Analysis ● College of American Pathologists
(CAP) offers proficiency testing
● Vasectomy is for couple who do not want
anymore children:
○ vas deferens is tied.
○ unsuccessful operation can still
lead to release of sperm.
● Takes several months for all sperm to be
gone, based on time and ejaculations
● Begin in 2 months; continue until 2
months are negative
○ only then can the operation be
called successful
● Wet preparation under phase; if negative,
centrifuge for 10 minutes, examine again
● Only one sperm is required for
fertilization

Semen Function Test

● Hamster egg penetration - sperm

incubated with species non-specific
hamster eggs and penetration observed
microscopically
● Cervical mucus penetration - Uses the
mid-cycle cervical mucus and observe
sperm penetration ability

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