AUBF BS MLS

URINALYSIS AND BODY FLUIDS / STRASINGER 3rd Year

TRANS UNIT X: SEMEN

 Sperm remain stored in epididymis until ejaculation
o At which time they are propelled through the ductus
OUTLINE deferens (vas deferens) to ejaculatory ducts
Physiology  Ejaculatory ducts receive both sperm from ductus
Specimen Collection
Specimen Handling
deferense and fluid from seminal vesicles
Semen Analysis  Seminal vesicles produce most of fluid present in semen
Appearance (60% to 70%)
Liquefaction o Such fluid is the transport medium for sperm
Volume o It also contain high concentration of fructose and flavin
Viscosity  Spermatozoa metabolize fructose
pH o Serves for energy needed for flagella to propel the
Sperm Concentration and Sperm Count
Sperm Motility
through female reproductive tract
Sperm Morphology  In case of fructose absence, sperm do not display motility
Additional Testing in semen analysis
Sperm Vitality  Flavin is responsible for gray appearance of semen
Seminal Fluid Fructose  Various proteins secreted by seminal vesicles are involved
Antisperm Antibodies in coagulation of the ejaculate
Microbial and Chemical Testing
Postvasectomy Semen Analysis
 Muscular prostate gland, located below bladder, surrounds
Sperm Function Tests upper urethra and aids in propelling sperm through urethra
Semen Analysis Quality Control by contractions during ejaculation
 Approx. 20% to 30% of semen volume is acidic fluid
produced by prostate gland
INTRODUCTION  Milky acidic fluid contains high concentrations of acid
 Advances in andrology, assisted reproductive technology phosphatase, citric acid, zinc and proteolytic enzymes
(ART), and increased concern over fertility especially by responsible for coagulation and liquefaction of semen
couples choosing to have children resulted in increased following ejaculation
emphasis on semen analysis  Bulbourethral gland located below prostate contributes
 Specialized Andrology Laboratories – clinical about 5% of fluid volume in form of thick, alkaline mucus
laboratories where routine semen analysis is performed o Helps neutralize acidity from prostate secretions and
especially for patients with abnormal results to further test vagina
to determine need for in vitro fertilization (IVF)  Important for semen to be alkaline
 Clinical Laboratories personnel may also be employed in o Neutralize vaginal acidity present as a result of normal
andrology labs to perform routine and specialized testing bacterial vaginal flora
 Clinical Laboratories also performs postvasectomy  Without such, sperm motility would be diminished
semen and forensic analysis to determine semen
presence aside from fertility testing

PHYSIOLOGY
 Semen
o Composed of four fractions
 Contributed by the testes, epididymis, seminal
vesicles, prostate gland, and bulbourethral glands
 Each fraction differs in composition
 Mixing of all four fractions during ejaculation is
essential for production of normal semen
specimen
 Testes: paired glands in scrotum
o Contains seminiferous tubules for secretion of sperm
 External location of scrotum contributes to lower scrotum
temperature
o Optimal for sperm development
 Germ cells for spermatozoa production are located in the
epithelial cells of seminiferous tubules
 Specialized Sertoli cells: provide support and nutrients for
germ cells as they undergo mitosis and meiosis
(spermatogenesis)
 Once spermatogenesis was complete, immature sperm
(nonmotile) enters epididymis Figure 10–1. The male genitalia. Top, sagittal view; bottom,
o In epididymis, sperm mature and becomes flagella anterior view.
o Process takes approx. 90 days
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 TRANS: SEMEN

Table No. 10-1 Semen Composition

Spermatozoa 5% SPECIMEN HANDLING
Seminal fluid 60% to 70%  All semen specimens are potential reservoirs for HIV and
Prostate fluid 20% to 30% hepatitis viruses
Bulbourethral glands 5% o Standard precautions must be observed at all times
during analysis
SPECIMEN COLLECTION  Specimens are discarded as biohazardous waste
 Variety in composition of semen fractions makes proper  Sterile materials and techniques must be used when semen
collection of complete specimen culture is to be performed or when the specimen is to be
o Essential for accurate evaluation of male fertility processed for bioassay, intra-uterine insemination (IUI), or
 Most sperm are contained in the first portion of the ejaculate in vitro fertilization (IVF)
o Making complete collection essential for accurate
testing of fertility and postvasectomy specimens SEMEN ANALYSIS
 When part of first portion of ejaculate is missing  Semen analysis for fertility evaluation consists of
o Sperm count: decreases macroscopic and microscopic examination
o pH: falsely increased  Parameters reported:
o Specimen will not liquefy o Appearance, volume, viscosity, pH, sperm
 When part of last portion of ejaculate is missing concentration and count, motility, morphology
o Semen volume: decreased
o Sperm count: falsely increased APPEARANCE
o pH: falsely decreased  Normal semen has gray-white color, translucent, and has a
o Specimen will not clot musty odor
 Patients should receive detailed instruction for specimen  If sperm concentration is very low, specimen may appear
collection almost clear
 Specimens are collected following period of sexual  Increased white turbidity: indicates presence of white
abstinence of at least 2 days to not more than a week blood cells (WBCs) and infection within reproductive tract
o Specimens collected following prolonged abstinence  If required, specimen culturing is performed before
tend to have higher volumes and decreased motility continuing with semen analysis
 In performing fertility test, World Health Organizations  During microscopic examination, WBCs must be
recommended two or three samples be collected not less differentiated from immature sperm (spermatids)
than 7 days or more than 3 weeks apart, with two abnormal o Leukocyte esterase reagent strip test may be useful
samples considered significant to screen for presence of WBCs
 Laboratory should provide patient with warm sterile glass or  Varying amounts of red coloration are associated with red
plastic containers blood cells (RBCs) presence and are abnormal
 If possible, specimen is collected in a room provided by  Yellow coloration may be caused by urine contamination,
laboratory specimen collection following prolonged abstinence, and
o If it is inappropriate, specimen must be kept at room medications
temperature and delivered to laboratory within 1 hour  Urine is toxic to sperm
of collection o It affects evaluation of motility
 Laboratory personnel must record patient’s name and
birthdate, period of sexual abstinence, completeness of Table No. 10-2. Reference Values for Semen Analysis
sample, difficulties with collection and times of specimen Volume 2 to 5 mL
collection and specimen receipt Viscosity Pours in droplets
 Specimens awaiting analysis should be kept at 37°C pH 7.2 to 8.0
 Specimens should be collected by masturbation Sperm >20 million/mL
o If it is impossible, only nonlubricant-containing concentration
rubber or polyurethane condoms should be used Sperm count >40 million/ejaculate
 Ordinary condoms are not acceptable since they Motility >50% within 1 h
contain spermicides Quality >2.0 or a, b, c in Table 10–3
Morphology >14% normal forms (strict criteria)
TECHNICAL TIP. Coitus interruptus is not a reliable means of
>30% normal forms (routine criteria)
semen collection because the first portion of the ejaculate, which
Round cells <1.0 million/mL
contains the highest number of spermatozoa, may be lost and
the low pH of the vaginal fluid may affect sperm motility
LIQUEFACTION
Summary 10-1. Semen Production  Fresh semen specimen is clotted and should liquefy within
STRUCTURE FUNCTION 30 to 60 minutes after collection
Seminiferous tubules Spermatogenesis o Recording the time of collection is essential for
of testes evaluating semen liquefaction
Epididymis Sperm maturation  Failure of liquefaction to occur within 60 minutes: may
Ductus deferens cause deficiency in prostatic enzyme and should be
Propel sperm to ejaculatory ducts
reported
Seminal vesicles Provide nutrients for sperm and fluid
 Specimen analysis cannot begin until liquefaction has
Prostate gland Provide enzymes and proteins for
occurred
coagulation and liquefaction
o If after 2 hours specimen has not liquefied, equal
Bulbourethral Add alkaline mucus to neutralize
volume of physiologic Dulbecco’s phosphate-
glands prostatic acid and vaginal acidity
buffered saline/proteolytic enzyme (e.g. alpha-
chrymotrypsin or bromelain) may be added to induce
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 TRANS: SEMEN
liquefaction then allow rest of analysis to be
performed
 Such treatments can affect biochemical tests,
sperm motility and morphology (such use must be
documented)
 Dilution of semen with bromelain must be accounted when
calculating sperm concentration
 Jelly – like granules (gelatinous bodies) may be present in
liquefied semen and have no clinical significance
 If present, mucus strands interfere with semen analysis
VOLUME
 Normal semen volume ranges between 2 and 5 mL
o Measured by pouring specimen into clean graduated
cylinder calibrated in 0.1-mL increments
 Increased volume may be seen following periods of
extended abstinence
 Decreased volume is frequently associated with infertility
o May indicate improper functioning of one of semen-
producing organs, primarily the seminal vesicles
 Incomplete specimen collection must be considered
VISCOSITY
 Specimen viscosity refers to consistency of fluid and may
be related to specimen liquefaction
 Incompletely liquefied specimens are clumped and highly
viscous
 Normal semen specimen should be easily drawn to pipette
and form small discrete droplets that do not appear
clumped or stringy when falling by gravity from pipette
 Droplets forming threads longer than 2 cm are considered
highly viscous and are recorded as abnormal
 Ratings of 0 (watery) to 4 (gel-like) can be assigned to the
viscosity report
 Viscosity can as well be reported as low, normal or high
 Increased viscosity and incomplete liquefaction impede
testing for
o Sperm motility, sperm concentration, antisperm
antibody detection, and measurement of biochemical
markers
PROCEDURE 10-1
SEMEN DILUTION WITH PHYSIOLOGIC SALINE
Prepare an equal volume of diluent and semen (1 part diluent
and 1 part semen) using Dulbecco’s phosphate-buffered
saline. Repeated pipetting of the prepared dilution will induce
liquefaction. Preparation of Dulbecco’s Phosphate-Buffered
Saline
1. Using a 1-L volumetric flask, add the following:
a. 750 ml of purified water
b. 0.20 g of potassium chloride (KCL)
c. 0.20 g of potassium dihydrogen phosphate (KH2PO4)
d. 0.10 g of magnesium chloride hexahydrate (MgCl2.6H2O)
e. 8.0 g of sodium chloride (NaCl)
f. 2.16 g of disodium hydrogen phosphate heptahydrate
(Na2HPO4.7H2O)
g. 1.00 g of D-glucose
2. In a 10-mL volumetric flask, dissolve 0.132 g of calcium
chloride dehydrate (CaCl2.2H2O) in 10 mL of purified water.
3. To prevent precipitation, add calcium chloride dehydrated
solution to the 1-L flask slowly, stirring continuously.
4. Adjust the pH to 7.4 with 1 mol/L sodium hydroxide
(NaOH).
5. Make up to 1000 mL with purified water
PROCEDURE 10-2
DIGESTION WITH BROMELAIN
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1. Prepare 10 IU/mL bromelain in Dulbecco’s phosphate-
buffered saline.
a. Into a 100-mL volumetric flask, add 1000 IU of bromelain.
b. Add 60 mL Dulbecco’s phosphate-buffered saline.
c. Mix to dissolve. It takes about 15 to 20 minutes. Bring volume
to calibration mark using buffered saline.
2. Dilute one part semen 1:2 with the 10 IU/mL bromelain (1 part
semen + 1 part bromelain solution).
3. Stir with a pipette tip.
4. Incubate at 37°C for 10 minutes.
5. Mix the sample well before analysis.
TECHNICAL TIP. Gently mixing the sample container during
liquefaction can help produce a homogeneous sample
PH
 pH of semen indicates balance between pH values from
acidic prostatic secretion and alkaline seminal vesicles
secretion
 It should be measured within 1 hour of ejaculation due to
loss of CO2 that occurs
 Normal pH of semen is alkaline with range of 7.2 to 8.0
o Increased pH: infection within reproductive tract
o Decreased pH: associated with increased prostatic
fluid, ejaculatory duct obstruction, or poorly developed
seminal vesicles
SPERM CONCENTRATION AND SPERM COUNT
 Even though fertilization is accomplished by one
spermatozoon, actual number of sperm present in semen
specimen is valid measurement of fertility
 Factors affecting sperm concentration
o Days of sexual abstinence before collection,
infection, or stress
 More than one semen specimen should be
evaluated for infertility studies
 Reference values for sperm concentration: greater than 20
to 250 million sperm/milliliter
o Concentrations between 10 and 20 million/milliliter are
considered borderline
 Total sperm count for ejaculate can be calculated by
multiplying sperm concentration by specimen volume
o Total sperm counts greater than 40 million/ejaculate
are considered normal (20 million per milliliter × 2 mL)
 Neubauer counting chamber: it is where sperm
concentration is performed
 Sperm are counted similarly as cells in cerebrospinal fluid
count (CSF)
o Diluting specimen and counting cells in Neubauer
chamber
 Dilution amount and number of squares counted vary
among laboratories
 1:20 is the most common dilution prepared using
mechanical (positive-displacement) pipette
 Semen dilution is essential, it mobilizes sperm before
counting
 Traditional diluting fluid contains sodium bicarbonate and
formalin, immobilizes and preserves cells
o Good results can also be achieved using saline and
distilled water
 Neubauer hematocytometer
o Using this, sperms are counted in four corner and
center squares of the large center square, similar to
manual RBC count
o Both sides of hematocytometer are loaded and allowed
to settle for 3 to 5 minutes, then, they are counted and
counts should agree within 10%
o Average of the two counts is used in calculation
 22:36:55
GMT not agree, dilution and count are repeated
CSU BS MLS 3RD YEAR 3
 TRANS: SEMEN
 Counts are performed with phase or bright-field microscopy
 Stain addition (e.g. crystal violet) to diluting fluid aids in
visualization when using bright-field microscopy
o Only fully developed sperm should be counted
 Immature one and WBCs are referred as “round”
cells which should not be included
 Stain included in diluting fluid aids in differentiating between
immature sperm cells and leukocytes
 Count greater than 1 million leukocytes/millimeter:
associated with inflammation or infection in reproductive
organs, leads to infertility
 Presence of more than 1 million spermatids/milliliter
indicates disruption of spermatogenesis
o Caused by viral infections, exposure to toxic
chemicals and genetic disorders
Figure 10–2. Areas of the Neubauer counting chamber used for
red and white blood cell counts. W, typical WBC counting area;
R, typical RBC counting area.
CALCULATING SPERM CONCENTRATION AND
SPERM COUNT
 Calculation of sperm concentration depends on dilution
used and size and numbers of squares counted
 When using 1:20 dilution and counting five squares (RBCs)
in large center squares as described, number of sperm can
be multiplied by 1 million to equal the sperm
concentration/milliliter
 Unlike blood cell counts, sperm concentration is reported in
millions/milliliter rather microliters
EXAMPLES
 Using a 1:20 dilution, an average of 60 sperm are counted
in the five RBC counting squares on both sides of the
hemocytometer. Calculate the sperm concentration per
milliliter and the total sperm count in a specimen with a
volume of 4 mL
60 sperm counted × 1,000,000 = 60,000,000 sperm/mL
60,000,000 sperm/mL × 4 mL = 240,000,000 sperm/
ejaculate
 In a 1:20 dilution, 600 sperm are counted in two WBC
counting squares. Calculate the sperm concentration per
milliliter and the total sperm count in a specimen with a
volume of 2 µ L.
600 sperm counted × 20 (dilution) = 60,000 sperm/µL
(2 sq mm × squares counted) × (volume counted)
0.1 mm (depth)
60,000 sperm/µL × 1000 = 60,000,000 sperm/mL
60,000,000/mL × 2 mL = 120,000,000 sperm/ejaculate
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and disposable counting chambers that do not require
specimen dilution
 Comparison of methods and standard Neubauer counting
chamber method showed poor correlation with Neubauer
method and among themselves
 WHO states
o Validity of these alternative counting chambers
must be established by checking chamber
dimensions, comparing results with the
improved Neubauer hemocytometer method,
and obtaining satisfactory performance as
shown by an external quality control program
SPERM MOTILITY
 Presence of sperm capable of forward, progressive
movement is critical for fertility
o Once presented to cervix, sperm must propel
themselves through cervical mucosa to uterus,
fallopian tubes, and ovum
 Sperm motility should be assessed by using well mixed,
liquefied semen specimen w/in 1 hour of collection
 Practice of examining sperm motility at timed intervals over
extended period showed to serve no useful purpose
 Percentage of sperm showing actual forward movement
can be estimated after evaluating approx. 20 hpf
 Alternate procedure: examine 200 sperm/slide, then
count percentage of different motile categories using
manual cell counter
 Motility: evaluated by speed and direction
 Grading can be done using a scale of 0 to 4 (Table 10–3)
 Minimum motility of 50Z% with rating of 2.0 after 1 hr is
normal
 WHO’s rating scale of a, b, c, d (Table 10–3)
 Interpretation states that w/in 1 hr
o 50% or more of sperm must be motile in a,b and c
categories
o 25% or more should show progressive motility in a and
b categories
 WHO Laboratory Manual for the Examination and
Processing of Human Semen (2010): recommends
simpler system for grading motility that doen’t include speed
o Due to difficulty in standardized reporting
 Motility
o Graded as progressive motility (PM), nonprogressive
motility (NP), and immotility (IM)
o Must be specified as total motility (PM and NP) or
progressive motility (PM)
 Presence of high percentage of immobile sperm and
clumps of sperm needs further evaluation, determine sperm
vitality or presence of sperm agglutinins
 Instrumentation capable of performing computer-assisted
semen analysis (CASA) was developed
 CASA provides objective determination of sperm velocity
and trajectory
o Sperm concentration and morphology are included in
the analysis
 CASA instrumentation is found in andrology laboratories
and perform high volume of semen analysis
Table No. 10-3. Sperm Motility Grading
GRADE WHO CRITERIA SPERM MOTILITY ACTION
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
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 TRANS: SEMEN

Table No. 10-4. Alternative Sperm Motility Grading Criteria sample should be incubated at this temperature and the
Progressive Sperm moving linearly or in a preparation made with prewarmed slides and cover slips
motility (PM) large circle
Nonprogressive Sperm moving with an absence
motility (NP) of progression
Immotility (IM) No movement

SPERM MORPHOLOGY
 Presence of sperm that are morphologically incapable of
fertilization results to infertility
 Sperm morphology is evaluated with respect to structure of
head, neckpiece, midpiece and tail
 Abnormalities in head morphology are associated with poor
ovum penetration, whereas neckpiece, midpiece, and tail
abnormalities affect motility
 Normal sperm has oval-shaped head approx. 5 µm long
and 3 µm wide and long, flagellar tail approx. 45 µm long
 Enzyme – containing acrosomal cap: critical to ovum
penetration located at the tip of the head
o Acrosomal cap must encompass approx. half of the
head and cover approx. two thirds of the sperm nucleus
o Neckpiece attaches head to tail and midpiece
 Midpiece is approx. 7.0 µm long and is the thickest
Figure 10–3. Normal spermatozoon structure
part of the tail since it is surrounded by
mitochondrial sheath which produces energy
required by tail for motility
 Morphology of sperm is also evaluated from thinly smeared,
stained slide under oil immersion
o Smears made by placing approx. 10 µL of semen near
the frosted end of a clean microscope slide, placing
second slide with clean, smooth edge in front of semen
drop at 45° angle and draw the slide back to the edge
of the drop of semen, allowing the semen to spread
across the end, when semen is evenly distributed
across spreader slide, lightly pull the spreader slide Figure 10–4. Spermatozoon with double head, hematoxylin-
forward with continuous movement across first slide to
eosin (×1000)
produce smear
o Staining can be performed using Wright’s, Giemsa,
Shorr, or Papanicolaou stain
 Matter of laboratory preference
 Air-dried slides are stable for 24 hours
 At least 200 sperm should be evaluated and the percentage
of abnormal sperm reported
 Routinely identified abnormalities in head structure
o Double heads, giant and amorphous heads,
pinheads, tapered heads, and constricted heads
 Abnormal sperm tails are frequently doubled, coiled, or bent
 Abnormally long neckpiece may cause the sperm head to Figure 10–5. Spermatozoon with amorphous head,
bend backward and interfere with motility hematoxylin-eosin (×1000).
 Additional parameters in evaluating sperm morphology
o Measuring head, neck, and tail size, measuring
acrosome size, and evaluating for the presence of
vacuoles
o Inclusion of these parameters is referred to as Kruger’s
strict criteria
 Strict criteria evaluation requires use of stage micrometer
or morphometry
 Evaluation of sperm morphology using strict criteria is not
routinely performed in clinical laboratory but recommended
by WHO, in present times
 Strict criteria evaluation is an integral part of assisted Figure 10–6. Spermatozoon with double tail, hematoxylin-eosin
reproduction evaluations (×1000).
 Normal values for sperm morphology depend on evaluation
method used and vary from greater than 30% normal forms CALCULATING ROUND CELLS
when using routine criteria to greater than 14% normal  Differentiation and enumeration of round cells can be made
forms when using strict criteria during morphology examination
 Peroxidase-positive granulocyte: predominant form of
TECHNICAL TIP. Sperm motility can be evaluated at room leukocyte in semen and can further differentiated from
temperature
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 TRANS: SEMEN

spermatogenic cells and lymphocytes using a peroxidase SPERM VITALITY

stain
 By counting number of spermatids or leukocytes seen in
conjunction with 100 mature sperm, amount/milliliter can be
calculated using a formula
C=N×S
100
Where,
N = number of spermatids or neutrophils counted/100 mature
sperm
S = sperm concentration in millions/milliliter
 Such method can be used when counting can’t be
performed during hemocytometer count and to verify counts
performed by hemocytometer
 Greater than 1 million WBCs/milliliter/ejaculate indicates
inflammatory condition associated with infection and poor
sperm quality
o May impair sperm motility and DNA integrity
 Decreased sperm vitality may be suspected if specimen
has normal sperm concentration with markedly decreased
motility
 It must be assessed within 1 hour of ejaculation
 Vitality is evaluated by mixing specimen with eosin-nigrosin
stain, prepare smear then count the number of dead cells
in 100 sperm with brightfield or phase-contrast microscope
 Living cells aren’t infiltrated by dye and remain bluish white
o Dead cells stain red against normal background
 Normal vitality requires 50% or more living cells and should
correspond to previously evaluated motility
 Presence of large proportion of vital but immobile cells may
indicate defective flagellum
o High number of immotile and nonviable cells may
indicate epididymal pathology
SEMINAL FLUID FRUCTOSE
 Low sperm concentration maybe caused by lack of support
medium produced in seminal vesicles
o Indicative of low to absent fructose level in semen
 Low fructose levels: caused by abnormalities of seminal
vesicles, bilateral congenital absence of vas deferens,
obstruction of ejaculatory duct, partial retrograde
ejaculation and androgen deficiency
 Specimen can be screened for presence of fructose using
resorcinol test that produces orange color when fructose is
present
 Normal quantitative level of fructose is equal to or greater
than 13 µmol/ejaculate
o Can be determined using spectrophotometric methods
 Specimens for fructose levels should be tested within 2
hours of collection or frozen to prevent fructolysis

Table No. 10-5. Additional Testing for Abnormal Semen

Analysis
ABNORMAL POSSIBLE TEST
RESULT ABNORMALITY
Decreased motility Vitality Eosin-nigrosin stain
with normal count
Figure 10–7. Common abnormalities of sperm heads and tails Decreased count Lack of seminal vesicle Fructose level
support medium
ADDITIONAL TESTING Decreased motility Male antisperm Mixed agglutination
 When abnormalities are discovered in any routine with clumping antibodies reaction and
parameters, additional tests may be requested immunobead tests
o Most common are tests for sperm vitality, seminal fluid Sperm agglutination
fructose level, sperm agglutinins, and microbial with male serum
infection Normal analysis Female antisperm Sperm agglutination
with continued antibodies with female serum
infertility

Figure 10–8. Spermatozoon with bent neck and spermatid,

hematoxylin-eosin (×1000).

Figure 10–10. Nonviable spermatozoa demonstrated by the

eosinnigrosin stain (×1000)

PROCEDURE 10-3
SEMINAL FRUCTOSE SCREENING TEST

1. Prepare reagent (50 mg resorcinol in 33 mL concentrated HCl

Figure 10–9. Immature spermatozoa, hematoxylin-eosin diluted to 100 mL with water)
(×1000) 2. Mix 1 mL of semen with 9 mL of reagent
3. Boil
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4. Observe for orange-red color
ANTISPERM ANTIBODIES
 It can be present in men and women
 They may be detected in semen, cervical mucosa or serum
o Possible causes of infertility
 It is usual for both partners to demonstrate antibodies
o Male antisperm antibodies are more encountered
 Blood-testes barrier separates sperm from male immune
system under normal conditions
o When barrier is disrupted, antigen on sperm produce
immune response that damage the sperm
 Damaged sperm can cause production of antibodies in
female partner
 Presence of antibodies in males can be suspected when
clumps of sperm are observed during routine semen
analysis
 Sperm-agglutinating antibodies: causes sperm to stick to
each other in head-to-head, head-to-tail or tail-to-tail pattern
o Agglutination is graded as “few,” “moderate,” or
“many” on microscopic examination
 Presence of antisperm antibodies in females result in
normal semen analysis accompanied by continued fertility
o Its presence may be demonstrated by mixing semen
with female cervical mucosa or serum and observing
for aggulatination
 Mixed agglutination reaction (MAR) and Immunobead
Test: frequently used tests to detect presence of antibody-
coated sperm
o MAR test: screening procedure used primarily in
detection of presence of immunoglobulin G (IgG)
antibodies
 Semen sample containing motile sperm is
incubated with IgG antihuman globulin (AHG) and
suspension of latex particles or treated RBCs
coated with IgG, bivalent AHG binds to antibody
on sperm andantibody on the latex particles or
RBCs, forming microscopically visible clumps of
sperm and particles or cells, finding of less than
10% of motile sperm attached to particles is
normal
o Immunobead test: more specific procedure in that it
can be used to detect IgG, IgM and IgA antibodies
presence and demonstrates what are of sperm the
autoantibodies are affecting
 Sperm are mixed with polyacrylamide beads
known to be coated with anti-IgG, anti-IgM, or
antiIgA, microscopic examination of sperm shows
beads attached to sperm at particular areas,
depending on types of beads used, test could be
reported as IgM tail antibodies,” “IgG head
antibodies,” and so on, beads present on less than
50% of sperm is normal as defined by WHO
 Head-directed antibodies may interfere with penetration
into cervical mucosa or ovum
 Tail-directed antibodies affect movement through cervical
mucosa
MICROBIAL AND CHEMICAL TESTING
 Presence of more than 1 million leukocytes/milliliter:
infection w/in reproductive system, frequent on prostate
 Routine aerobic and anaerobic culture and tests for
Chlamydia trachomatis, Mycoplasma hominis, and
Ureaplasma urealyticum are frequently performed
 Additional chemical tests performed on semen
o Determine levels of neutral α -glucosidase, free L-
carnitine, glycerophosphocholine, zinc, citric acid,
glutamyl transpeptidase, and prostatic
phosphatase
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TRANS: SEMEN
 Decreased neutral α -glucosidase, glycerophosphocholine,
and L-carnitine suggest disorder of epididymis
 Decreased zinc, citric acid, glutamyl transpeptidase, and
acid phosphatase indicate lack of prostatic fluid
 Spectrophotometric methods: used to quantitate citric
acid and zinc
 Laboratory may be called to determine whether semen is
actually present in specimen on certain occasions
o Primary example: cases of alleged rape
 Microscopically examining specimen for presence of sperm
may be possible
o Best results obtained by enhancingspecimen with
xylene, examining under phase microscopy
 Motile sperm: detected for up to 24 hours after intercourse
o Nonmotile sperm can persist for 3 days
 As the sperm die off, only heads remain and may be present
for 7 days after intercourse
 Seminal fluid contains high concentration of prostatic acid
phosphatase
o Detecting this enzyme can aid in determining presence
of semen in specimen
 More specific method: detection of seminal glycoprotein
p30 (prostatic specific antigen [PSA])
o Present even in absence of sperm
 Further information are obtained by performing ABO blood
grouping and DNA analysis on specimen
Table No. 10-6. Reference Semen Chemical Values
Neutral α -glucosidase ≥ 20 mU/ejaculate
Zinc ≥ 2.4 µmol/ejaculate
Citric acid ≥ 52 µmol/ejaculate
Acid phosphatase ≥ 200 Units/ejaculate
POSTVASECTOMY SEMEN ANALYSIS
 Much less involved procedure when compared with
infertility analysis
o Only concern: presence or absence of spermatozoa
 Length of time required for complete sterilization vary
among patients and depends on time and number of
ejaculations
o Finding viable sperm in postvasectomy patient is
common and care should be taken not to overlook
even single sperm
 Specimens are routinely tested at monthly intervals
o Begins at 2 months postvasectomy and continuing until
two consecutive monthly specimen which show no
spermatozoa
 Recommended testing: examines a wet preparation using
phase microscopy for presence of motile and non motile
sperm
 Negative wet preparation is followed by specimen
centrifugation for 10 minutes and examination of sediment
SPERM FUNCTION TESTS
 Advances in assisted reproduction and IVF resulted in need
for sophisticated semen analysis to assess not only the
characteristic of sperm but also the functional ability
 Tests most commonly performed in specialized
andrology laboratories
o Hamster egg penetration assay, cervical mucus
penetration test, hypo-osmotic swelling test, and the in
vitro acrosome reaction
Table No. 10-7. Sperm Function Test
TEST DESCRIPTION
Hamster egg Sperm are incubated with species nonspecific
penetration hamster eggs and penetration is observed
acid
microscopically
CSU BS MLS 3RD YEAR 7
 TRANS: SEMEN

Cervical mucus Observation of sperm’s ability to penetrate

penetration partner’s midcycle cervical mucus
Hypo-osmotic Sperm exposed to low-sodium concentrations
swelling are evaluated for membrane integrity and sperm
viability
In vitro Evaluation of the acrosome to produce enzymes
acrosome essential for ovum penetration
reaction

SEMEN ANALYSIS QUALITY CONTROL

 Routine semen analysis has been subject to very little
quality control
o Situation which resulted from lack of appropriate
control materials and subjectivity of motility and
morphology analyses
 Analysis is rated as high complexity test under Clinical
Laboratory Improvement Amendments
o Testing personnel standards must be observed
 Increased interest in fertility testing promoted development
of quality control materials and in-depth training programs
 Standardized procedures developed by WHO provided
basis for laboratory testing and reporting
 CASA use aided in reducing subjectivity of analysis
o Even computerized analysis shown to vary among
operators
 Commercial quality control materials and training aids are
available and must be incorporated in laboratory protocols

TECHNICAL TIP. A single “motile” sperm on a wet preparation is

an indication of an unsuccessful vasectomy.

REFERENCES

Notes from the book by

Urinalysis and Body Fluids, Sixth Edition Strasinger (2014)

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