MLS 419: AUBF LAB Composition of Semen

FINALS WEEK 1 – LESSON 1

Semen Analysis 5% Spermatozoa
INTRODUCTION 60-70% Seminal Fluid Fructose and Flavin
Spermatogenesis 20-30% Prostate Fluid Acid phosphatase, citric
- Formation of Spermatozoa (Sperm) in acid, zinc, and proteolytic
seminiferous tubules enzymes
- Spermatogenesis begins with spermatogonia →
undergo mitosis → some spermatogonia remain 5% Bulbourethral fluid Thick alkaline mucus
near the basement membrane of seminiferous Chemical Components
tubules in an undifferentiated stage to serve as a • Spermine and choline
reservoir of cells for future cell division and • Acid phosphatase
subsequent sperm production • Fructose – energy for the flagella to propel them
- The rest of spermatogonia lose contact with the thru the female reproductive tract
basement membrane and squeeze through the tight o w/o fructose the sperm cannot swim and
junction of the blood testis barrier → undergo will remain immotile
developmental changes and differentiate into • Flavin – responsible for the gray appearance of the
primary spermatocytes until they become sperm semen
cells. • Potassium, Citric Acid and Ascorbic acid
• Ergothionine
Physiology • Phosphorylcholine
• Proteolytic enzyme
o Coagulation and liquefaction of the semen
following the ejaculation

Purpose
- Evaluation of reproductive dysfunction in males –
INFERTILITY TESTING
- Select donors for therapeutic insemination
- Monitor success of surgical procedures:
o Varicocelectomy
o Vasectomy

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- Testes (seminiferous tubules) – secretion of
sperm; Paired glands
- Epididymis – sperm maturation and development
of flagella; store sperms
- Ductus deferens (vas deferens) – transports
sperm to the ejaculatory duct
- Seminal vesicle – transport medium for the sperm;
rich in fructose and flavin.
o Produces the major fraction of semen
- Prostate gland – propels the sperm through the
urethra; contains acid phosphatase, citric acid,
zinc, and proteolytic enzymes responsible for
both the coagulation and liquefaction of the semen;
muscular gland.
- Bulbourethral glands – secretes alkaline mucus
o Located below the prostate gland
- Note: These glands contain the fraction of semen
and mix together in ejaculation
Sample Collection
• PREPARATION
1. Abstinence: 2 – 7 days (Note: No prolonged
abstinence)
2. Sterile glass or plastic container (warm)
• Collected in a private room near the laboratory
- In order to limit the exposure of the semen to
fluctuation in temperature and the time
between the collection and analysis.
• Give a clear written and verbal instructions
concerning the collection of semen
• Deliver to the laboratory within 1 hour after
collection
• Record the patient’s name, and birth date, the
period of sexual abstinence, the completeness of
the sample, difficulties with collection, and the time
specimen collection and specimen receipt
• METHODS OF COLLECTION
- Masturbation
- Coitus interrupts
- Common condom collection
- Silastic condom collection
- Aspiration from the vaginal vault after coitus
NOTE:
- If married, collection from sex is better than
masturbation because of the degree of
arousal.
- The first part of the ejaculate is mostly
collected because it is more concentrated

• PRESERVATION
 ➢ Awaiting analysis LIQUEFACTION
▪ semen stored at body temperature • After ejaculation, semen is typically a semi-solid
(incubator) coagulated mass.
➢ For artificial insemination • Liquefy within 30 to 60 minutes (RT) after
▪ Frozen and stored for 1 year at -85degC at collection (Normal)
sperm bank o Rarely, it may take 60 minutes or more
• In liquefaction, semen becomes more homogenous
SEMENALYSIS PARAMETERS and quite watery; small areas of coagulation remain
• Liquefaction (final stage).
• Appearance • Failure of liquefaction within 60 minutes
• Volume o Deficiency in prostatic enzymes and
• Viscosity should be reported.
• pH o If complete liquefaction is not observed
• Sperm Concentration and Count after 60 minutes, this should be reported.
• Motility • Note:
• Morphology 1. Normal Liquefied semen samples may
contain jelly like granules (gelatinous
Parameters Reference Values
bodies) - no clinical significance
Volume 2 to 5 mL 2. Presence of mucus strands may interfere
with semen analysis
Viscosity Pours in droplets **Semen taken from home are normally liquefied by the
time they arrived in the lab**
pH 7.2 to 8.0

Sperm > 20 million/mL If after 2 hours the specimen has not liquefied:
Concentration o Dulbecco’s phosphate-buffered saline
o Proteolytic enzymes such as alpha-
Sperm Count > 40 million/ejaculate chymotrypsin or bromelain
Motility > 550% within 1 hour
o “these treatments may affect biochemical
t e s t s , s p e r m m o t i l i t y, a n d s p e r m
Quality > 2.0 or a, b, c morphology, so their use must be
documented.”
Morphology > 14% Normal forms (strict criteria)

> 30% normal forms (routine criteria)

Round Cells < 1.0 million/mL

APPEARANCE
COLOR
Gray-white, Pearly-
white, Light Yellow, NORMAL
Opaque

Shades of Yellow Correlate with flavin concentration

Associated with certain drugs, over

Deep Yellow abstinence, jaundice, urine
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contamination

Brown or Red Presence of red blood cells

Increased white Presence of white blood cells and

turbidity infection within the reproductive tract
➢ Less opaque, if the sperm concentration is
very low
➢ If required, the specimen culturing is
performed prior to continuing with the semen
analysis
 pH
• Reflects the balance between the pH values of
different accessory gland secretions.
• Measured after liquefaction (preferably after 30
minutes) – (WHO) or within 1 hour of ejaculation
due to loss of CO2 occurs after the production –
(Strasinger)
• pH paper range 6.0 to 10.0 should be used
⎯ mix the semen well
⎯ spread a drop onto pH paper
⎯ read within 30 seconds
▪ wait for the color of the
impregnated zone to be uniform
⎯ compare the color with the calibration
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strip to read pH
VOLUME
• Normal volume: 2-5 mL • Normal pH: 7.2- 8.0
o the volume of the ejaculate is contributed
• Increased pH: Infection
• Decreased pH: increased prostatic fluid,
mainly by the seminal vesicle and prostate
ejaculatory duct obstruction, poorly develop seminal
glands, and small amount from the
vesicle
bulbourethral gland and epididymis.
• It can be measured by pouring the specimen into a
INITIAL MICROSCOPIC EXAMINATION
clean graduated cylinder calibrated in 0.1 – mL • Phase-contrast microscope
increments • Scanning at a total magnification of 100x (10x
o Precise specimen volume is essential in
objective lens with 10x ocular lens)
any evaluation of semen because it allows • Provides overview
the total number of spermatozoa and non
o Mucus strand formation
sperm cells in the ejaculate to be
o Sperm aggregation or agglutination
calculated.
• Note: Measuring volume by aspirating the sample o Presence of cells other than spermatozoa
(Epithelial cells, round cells, and isolated
from the specimen container into a pipette or
sperm head or tails)
syringe is not recommended • Then be observed at 200x or 400x magnification
o Assessment of a sperm motility
Low Volume High Volume o Determination of the dilution required for
accurate assessment of sperm number
- Obstruction of the - active exudation in
ejaculatory duct cases of active Making a wet preparation
- congenital bilateral inflammation of the • Mix the semen sample well
absence of vas accessory organs • Remove an aliquot of semen immediately after
deferens - extended abstinence mixing, allowing no time for the spermatozoa to
- collection problems settle out of suspension.
(loss of fraction of the • Remix the semen sample before removing replicate
ejaculate) aliquots.
- partial retrograde
ejaculation or androgen
deficiency
VISCOSITY
• Consistency of the fluid
• Aspirating into a wide-bore plastic disposable pipette "
• Normal viscosity: form small discrete droplets that • Place a standard volume of semen, e.g. 10uL, onto
do not appear clump or stringy when falling by gravity a clean glass slide
by a pipette and observing the length of any thread. • Cover it with a coverslip, e.g. 22 mm × 22 mm for
• Abnormal viscosity: drop from a thread more than 10uL, to provide a chamber approximately 20um
2 cm long (viscous) deep. The weight of the coverslip spreads the
• Ratings: 0 (watery) – 4 (gel like) sample.
o Viscosity can also be reported as low, • Take care to avoid the formation and trapping of air
normal, or high. bubbles between the coverslip and the slide.
• Note: High viscosity can interfere with determination • Assess the freshly made wet preparation as soon
of sperm motility, sperm concentration, detection of as the contents are no longer drifting.
antibody-coated spermatozoa and measurement of
biochemical markers.
Aggregation of spermatozoa
• Adherence either of immotile spermatozoa to each
other or of motile spermatozoa to mucus strands,
non-sperm cells or debris is non-specific
aggregation and should be recorded.
Cellular elements other than spermatozoa
• Epithelial cells in genitourinary tract
• Leukocyte and immature germ cells (collectively
referred as round cells)
• Examined the stained smear at 1000x magnification
• Can be more precisely identified and quantified by
detecting peroxidase activity or the antigen CD45

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• Motile spermatozoa sticking to each other, head-to-
head, tail-to-tail or in a mixed way.
• Motility is vigorous with frantic shaking motion but
sometimes are so agglutinated with limited motion
• Any motile spermatozoa stick to each other by their
heads, tails or midpieces should be noted.
• Major type of agglutination (grades 1-4); site of
attachment (grades A-E)

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GRADE DEGREE DESCRIPTION
< 10 spermatozoa per agglutinate,
1 Isolated many free spermatozoa

10 – 50 spermatozoa per
2 Moderate agglutinate, free spermatozoa

Agglutinates of > 50 spermatozoa,

3 Large some spermatozoa still free

All spermatozoa agglutinated and

4 Gross agglutinates interconnected

Sperm Motility
- Assessed ASAP after liquefaction (preferably at 30
minutes, WHO) or within 1 hour following ejaculation
- Normal: 60% or higher progressively motile
sperm

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 Sperm Morphology
1. Mix the semen sample well
2. Remove an aliquot of semen immediately after
• Head. Neckpiece, midpiece,
tail
mixing, allowing no time for the spermatozoa to • Abnormalities:
settle out of suspension.
o Head – poor ovum
3. Remix the semen sample before removing a
penetration
replicate aliquot.
o N e c k p i e c e ,
4. For each replicate, prepare a wet preparation
Midpiece, and Tail –
approximately 20 um deep
5. Wait for the sample to stop drifting (within 60 affects motility
• Head – oval-shaped; approx.
seconds).
6. Examine the slide with phase-contrast optics at 5um long and 3 um wide
• Tail – approx. 45 um long
×200 or ×400 magnification.
7. Assess approximately 200 spermatozoa per
• Critical to ovum penetration:
Acrosomal cap – encompass
replicate for the percentage of different motile
half of the head; cover 2/3 of
categories.
8. Compare the replicate values to check if they are sperm nucleus
• Neckpiece – attaches the head to the tail and the
acceptably close. If so, proceed with calculations;
midpiece.
if not, prepare new samples. • Midpiece – 7 um long; thickest part because it is
Categories of Sperm Movement surrounded with mitochondrial sheath
o Examination of sperm morphology is also
Spermatozoa moving actively, essential, because sperm that are
Progressive motility (PR) either linearly or in a large circle, morphologically incapable for fertilization
regardless of speed
also results to infertility
All other patterns of motility with an • NORMAL VALUES:
Nonprogressive Motility (NP) absence of progression o Routine Criteria: > 30% normal
o Kruger’s Strict Criteria: > 14% normal;
Immotility (IM) No movement
measures head, neck and tail
Note: **When discussing sperm motility, it is important to ▪ Requires the use of stage
specify total motility (PR + NP) or progressive motility (PR). micrometer or morphometry
o Normally at least 70% of sperm
Must watch! 26:50 sperm motility under microscope demonstrate normal morphology

Semen smearing methods for sperm morphology

(a) “Feathering” method for undiluted semen. The semen
drop (S) spreads along the back edge of the angled slide
and is pulled forwards over the slide to form the smear. (b)
Pipette method for washed samples. A drop of the sperm
suspension (SS) is spread over the surface of the slide by
pushing the horizontal pipette (P).

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Common abnormalities of Sperm Heads and Tails

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Note: **Images are available in Stras page 211, chapter 10

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" center boundary line
Sperm Concentration
• “total sperm number” vs. “sperm concentration”
• Sperm concentration
⎯ Number of spermatozoa per unit volume of
semen and is a function of the number of
spermatozoa emitted and the volume of
fluid diluting them.
• Total sperm number
⎯ Total number of spermatozoa in the entire
ejaculate and is obtained by multiplying the
sperm concentration by the semen volume
⎯ NORMAL VALUES: > 40 million/ejaculate
• Normal values: 20-250 million sperm per milliliter
⎯ Concentration of 10-20 million/milliliter is
considered as borderline
• Makler counting chamber – undiluted semen, heat
instead (inaccurate results)
• Improved Neubauer Counting Chamber
⎯ 1:20 dilution (common)
⎯ Diluting fluids: cold water (most
common), formalin, Na bicarbonate, 0.5%
chlorazene, 1% formalin in 3% trisodium
citrate
⎯ Sperm are counted in the same manner of
counting in the CSF.
✓ 5 RBC squares: 4 corner and
center squares of the large center
square (same to manual RBC count)
✓ Computation: # sperm cells
counted x 1,000,000 (mL)
✓ 2 WBC squares: only 2 corner
large squares (same to manual
WBC count)
✓ Computation: #sperm cells
counted x 100,000 (mL)
Considerations:
• Additions of stain (crystal violet) to diluting fluid
helps visualization using bright-field microscope.
• Note: Only FULLY DEVELOPED sperm should be
counted
o Immature and WBCs – “round cells” must
not be included)
• > 1 million WBCs/mL – inflammation or infection
• > 1 million spermatids/mL – disruption of
spermatogenesis caused by viral infections,
exposure o toxic chemicals, and genetic disorders.
Inclusion Criteria:
• Count only whole spermatozoa (w/ the head and
tail)
• Whether or not sperm cell is counted is determined
by the location of its head (*the location of its tail is
unimportant)
o Sperm cell is counted if its head lies on the
boundary line
• To avoid counting the same sperm cells in adjacent
square
o A sperm cell with its head on the line
dividing two squares should be only, if that
line is one of the two perpendicular
boundary line
o for example: cells may be counted if most
of the sperm head lies on the lower or left
center boundaries = which form an
“L-shape”
o DO NOT COUNT cells on the upper right
Sample:
1. Using a 1:20 dilution, an average of 60 sperm are
counted in the five RBC counting squares on the
both sides of the hemocytometer. Calculate the
sperm concentration per milliliter and the total
sperm count in a specimen with a volume of 4mL.
Ans:
60 sperm counted x 1,000,000 = 60,000,000 sperm/mL
60,000,000 sperm/mL x 4 mL = 240,000,000 sperm/
ejaculate (NORMAL)
Counting Sperm Cells in WBC Squares
• The middle of the three lines define the square’s
boundary (black line-left panel). All spermatozoa
within the central square are counted, as well as
those with their heads between the twi inner lines
(white circles). Do not count those whose heads lie
between the outer two lines (black circles)

• Sperm cells (spermatozoon) with most of its head
lying on the central line is COUNTED ONLY IF that
line is the lower of or left-hand of the square (white
circles, middle panel) BUT NOT IF it is the upper or
right hand line of the square (black circles-right
panel)
• ❗ Always use the L-shaped pattern in counting
your cells
Sample:
1. Using a 1:20 dilution, an average of 600 sperm
are counted in the 2 WBC counting. Calculate the
sperm concentration per milliliter and the total
sperm count in a specimen with a volume of 2mL.
Solution:
Staining Principles : Traditional Fixation and Sequential
Staining
to fix the cells, it also hydrates them
Ethanol
to rehydrate the fixed smears to permit
Graded ethanol water-soluble haematoxylin staining
to rehydrate dried smears to permit
purified water water-soluble haematoxylin staining
haematoxylin to stain the nucleus blue
to remove unbound nuclear haematoxylin
tap water
to remove non-specifically bound dye
acidic ethanol from the cytoplasm (destaining)
to reduce the acidity and return blue
tap water colour to the nucleus
to return blue color to the nucleus (if tap
Scott’s solution water is insufficient)
to dehydrate smears to permit ethanol-
Ethanol soluble Orange G/EA-50 staining
to stain the cytoplasm pink
Orange G
to stain the cytoplasm pink
EA-50
to dehydrate the stained smears
Graded ethanol gradually to permit the use of ethanol-
soluble mountants
to permit the use of ethanol-insoluble
Xylene mountants
After staining the slides can be viewed MOUNTED or
UNMOUNTED (with/without coverslip)

STEPS IN PAPANICOLAOU STAINING:

Answer: 120,000,000 sperm/ejaculate

Related Terms
• ASPERMIA = no ejaculate at all
• OLIGOSPERMIA = less than 20 million per mL
• NECROSPERMIA = immotile/dead sperms
• AZOSPERMIA = complete absence of sperm

Staining Methods
once the semen stains has been air-dried they must
be fixed and stained to highlight the details of the
spermatozoa

Different Stains:
• Papanicolaou Stain (recommended)j
• Shorr or Diff Quick Stain (recommended)
• Wright’s Stain
• Giemsa Stain

for ALL THESE STAINS:

• the head is stained pale blue (acrosomal
region) and is stained dark blue (post
acrosomal region)
• Midpiece = red stain
• Tail = blue or reddish
the excess residual cytoplasm usually located
behind the head and around the midpiece is
stained pink or red in papanicolaou stain and
reddish-orange in Shorr or Diff Quick Stain
STEPS IN SHORR STAINING: SEMINAL FLUID FRUCTOSE—————————————
• Low fructose levels are caused by abnormalities of
the seminal vesicles, bilateral congenital absence
of the vas deferens, obstruction of the ejaculatory
duct, partial retrograde ejaculation, and androgen
deficiency .
• Screened for the presence of fructose using the
resorcinol test that produces an orange color
when fructose is present
• A normal quantitative level of fructose is equal to
or greater than 13 µmol per ejaculate.
• determined using Spectrophotometric methods.
Specimens for fructose levels should be tested
within 2 hours of collection or frozen to prevent
fructolysis.
• Procedure:
1. prepare reagent (50 mg resorcinol in 33 mL
concentrated HCl diluted to 100mL with
water)
2. mix 1mL of semen with 9 mL of reagent
3. boil
4. observe for orange red color

ANTISPERM ANTIBODIES——————————————-
• may be present in both men and women
• Detected in semen, cervical mucosa,
or serum : possible cause of infertility
• Blood-testes barrier separates sperm from the male
immune system. (When disrupted in surgery,
PAS STAINED SEMEN vasectomy, trauma, infection— antigens on sperm
produce an immune response)
• Damage sperm may cause the production of
antibodies in female.
• In male, clumps of sperm are observed in routine
semenalysis.
o Sperm-agglutinating antibodies cause to
SHORR STAINED SEMEN stick in a head-to-head, head-to-tail, or tail-
to-tail pattern.
o Agglutination grading: “few”, “moderate”,
or “many”
• In female, normal semen analysis accompanied by
continued infertility.
o Mixing the semen with female cervical
H-E STAINED SEMEN mucosa or serum and observing for
agglutination

Tests for Antisperm Antibodies

MIXED AGGLUTINATION REACTION (MAR TEST)
• screening procedure to detect IgG antibodies
OTHER TESTS & TECHNIQUES • Semen + IgG (AHG) + latex particles or treated
RBCc coated with IgG
SPERM VITALITY——————————————————- • Bivalent AHG binds to both antibody on the sperm
• Assessed within 1 hour of ejaculation. and antibody on latex particles or RBCs, forming
• Evaluated by eosin-nigrosin stain, preparing a microscopically visible clumps of sperm and
smear, and counting the number of dead cells in particles or cells.
100 sperm using a brightfield or phase-contrast • Normal: < 10% of motile sperm attach to
microscope. particles
• Living cells remain bluish white whereas dead
cells stain red against the purple background
• Normal vitality requires 50% or more living cells
• Presence of a large proportion of vital but immobile
cells may indicate a defective flagellum
• High number of immotile and nonviable cells may
indicate epididymal pathology
IMMUNOBEAD TEST
• more specific to detect IgG, IgM, and IgA
antibodies and demonstrates what area of sperm
(head, neckpiece, midpiece, or tail) the
autoantibodies are affecting.
o Head- directed Ab: interfere penetration into
cervical mucosa or ovum
o Tail-directed Ab: affect movement through
the cervical mucosa

• Sperm + polyacrylamide beads (coated with anti-

IgG, anti-IgM, anti-IgA)
• Microscopic examination -beads attach to sperm at
particular areas
• Reported as “IgM tail antibodies, “IgG head
antibodies,” and so forth
• Normal: presence of beads on less than <50%
of the sperm

MICROBIAL TESTING————————————————- *TCA = Trichloroacetic acid

• >1 million WBCs/ mL indicates infection within
reproductive system, prostate.
• Routine Aerobic/anaerobic cultures and test for
Chlamydia trachomatis, Mycoplasma hominis,
Ureaplasma urealyticum = most frequently
performed
CHEMICAL TESTING————————————,,,,,,,,——-
• Decreased neutral alpha-glucosidase,
glycerophosphocholine and L-carnitine suggest a
disorder of epididymis.
• Decreased zinc, citric acid, glutamyl
transpeptidase and acid phosphatase indicate lack
of prostatic fluid.
• Spectrophotometric methods- quantitate cirtric acid
and zinc
POST VASECTOMY ANALYSIS——————————/,.—-
• much less involved procedure when compared with
infertility analysis
• concern only in presence or absence of
spermatozoa
• the length of time required for complete sterilization
can vary greatly among patients and depends on
both time and number of ejaculations
• finding viable sperm in a post-vasectomy patient is
not uncommon and care should be taken not to
overlook even a single sperm
• Specimens are routinely tested at monthly
intervals, beginning at 2 months postvasectomy
and continuing until two consecutive monthly
specimens show no spermatozoa
• Wet preparation using phase microscopy
• Negative wet preparation is followed by specimen
centrifugation for 10 minutes and examination of
the sediment.

SPERM FUNCTION TEST————…………………———,,,

advances in assisted reproduction and in vitro fertilization
have resulted in a need for more sophisticated semen
analyses to assess not only the characteristics of sperm but
also the functional ability
SPECIAL CASE
• In cases of alleged rape -microscopically examining
most commonly performed in specialized andrology
the specimen for the presence of sperm with xylene
laboratories:
and examine under phase microscopy.
• Hamster egg penetration assay
• Motile sperm- detected for up to 24 hours after
intercourse • Cervical mucus penetration test
• Hypo-osmotic swelling test
• Nonmotile sperm- 3 days
• In-vitro acrosome reaction
• As sperm die off, heads remain and present for 7
days after intercourse.
• Seminal fluid contains high concentration of
prostatic acid phosphatase.
• Seminal glycoprotein p30 (prostatic specific antigen
[PSA]) - present even in the absence of sperm;
more specific detection
• Further information, perform ABO blood grouping
and DNA analysis)