Muscular Prostate Gland

MLS 065: ANALYSIS OF URINE AND BODY FLUIDS ➔ located just below the bladder, surrounds the upper urethra and aids in
the propelling the sperm through the urethra by contractions during
LESSON TITLE: SEMEN ejaculation

○ Approximately 20% to 30% of the semen volume is acidic fluid

PHYSIOLOGY produced by the prostate gland
○ Milky acidic fluid contains high concentrations of:

SEMEN → composed of four fractions that are attributed by the:

1. Testes
2. Epididymis
3. Seminal vesicles
4. Prostate gland ➔ responsible for both the coagulation and liquefaction of
5. Bulbourethral glands the semen following ejaculation
○ Each fraction differs in its composition, and the mixing
of all four fractions during ejaculation is essential for the Bulbourethral glands
production of a normal semen specimen ➔ located below the prostate, contribute about 5% of the fluid volume in the
form of a thick, alkaline mucus that helps to neutralize acidity from the
prostate secretions and the vagina

○ It is important for semen to be alkaline to neutralize the vaginal

acidity present as a result of normal bacterial vaginal flora
○ Without this neutralization, sperm motility would be diminished

Testes
➔ are paired glands in the scrotum that contain seminiferous tubules
for the secretion of sperm

External location of the scrotum = lower scrotum temperature

(optimal for sperm development)

Germ cells for the production of spermatozoa

➔ located in the epithelial cells of the seminiferous tubules

Specialized Sertoli cells

➔ provide support and nutrients for the germ cells as they undergo
mitosis and meiosis (spermatogenesis) SPECIMEN COLLECTION

○ When spermatogenesis is complete, the immature sperm (nonmotile) ○ The variety in the composition of the semen fractions makes proper
enter the epididymis collection of a complete specimen essential for accurate evaluation of
○ In the epididymis, the sperm mature and develop flagella male fertility
○ The entire process takes approximately 90 days ○ Most of the sperm are contained in the first portion of the ejaculate,
○ The sperm remain stored in the epididymis until ejaculation, at which making complete collection essential for accurate testing of both
time they are propelled through the ductus deferens (vas deferens) to the fertility and post vasectomy specimens
ejaculatory ducts

Ejaculatory ducts When a part of the first portion of the ejaculate is missing:
➔ receive both the sperm from the ductus deferens and fluid from the ➢ sperm count will be decreased
seminal vesicles ➢ pH falsely increased
➢ specimen will not liquefy
Seminal vesicles When part of the last portion of ejaculate is missing:
➔ produce most of the fluid present in semen (60%-70%) ➢ semen volume is decreased
➔ Various proteins secreted are involved in the: ➢ sperm count is falsely increased
➢ coagulation of the ejaculate ➢ pH is falsely decreased
➢ specimen will not clot

Fluid → transport medium for the sperm

→ contains a high concentration of fructose and flavin
SPECIMENS COLLECTED:

○ In the absence of fructose, sperm do not display motility in Period of sexual abstinence: Prolonged abstinence:
semen analysis ➢ at least 2 days 1. Higher volumes
○ Flavin is responsible for the gray appearance of semen ➢ not more than 7 days 2. Decreased motility

If possible: If not appropriate:

Spermatozoa
➢ Specimen is collected in a ➢ Specimen should be kept
➔ metabolize the fructose for the energy needed for the flagella to propel
room provided by the at room temperature and
them through the female reproductive tract
laboratory delivered to the laboratory
within 1 hour of collection
 Specimen should be collected by: If this is not possible: APPEARANCE
➢ masturbation ➢ non lubricant-containing
rubber
➢ polyurethane condoms Normal semen - gray-white color
- translucent
Ordinary condoms - musty odor
- NOT ACCEPTABLE
- contain spermicides Sperm concentration is very - almost clear
low

○ Patients should receive detailed instructions for specimen collection Increased white turbidity - indicates the presence of
○ When performing fertility testing, the World Health Organization (WHO) WBCs and infection within
recommends: the reproductive tract
➢ two or three samples be collected
If required:
➢ not less than 7 days or more than 3 weeks apart ➢ Specimen culturing is
➢ two abnormal samples considered significant performed prior to continuing
○ The laboratory should provide the patient with warm sterile glass or with the semen analysis
plastic containers ➢ During microscopic
○ Laboratory personnel must record the: examination, WBCs must be
differentiated from immature
➢ patient’s name and birth date
sperm (spermatids)
➢ period of sexual abstinence ➢ Leukocyte esterase reagent
➢ completeness of the sample strip - useful to screen for the
➢ difficulties with collection presence of WBCs
➢ times of specimen collection
➢ specimen receipt Abnormal - associated with the presence
○ Specimens awaiting analysis should be kept at 37°C (varying amounts of red of RBCs
coloration)

SPECIMEN HANDLING Yellow coloration - urine contamination

- specimen collection following
prolonged abstinence
○ All semen specimen are potential reservoirs for HIV and hepatitis - medications
viruses, and standard precautions must be observed at all times during
analysis Urine
○ Specimens are discarded as biohazardous waste - TOXIC TO SPERM
○ Sterile materials and techniques must be used when specimen is to be - affecting evaluation of motility
performed or processed for:
➢ semen culture
➢ bioassay LIQUEFACTION
➢ intrauterine insemination (IUI)
➢ in vitro fertilization (IVF)
Fresh semen specimen - clotted
- should liquefy within 30 to 60
SEMEN ANALYSIS minutes after collection

Semen analysis for fertility evaluation ➢ Recording the time of collection is essential for evaluating
semen liquefaction
→ macroscopic and microscopic examination

Failure of liquefaction to - caused by a deficiency in

Parameters reported include:
occur within 60 minutes prostatic enzymes and should be
➢ appearance reported
➢ volume
➢ viscosity ➢ Analysis of the specimen cannot begin until liquefaction has
➢ pH occurred
➢ sperm concentration and count
➢ motility After 2 hours the - equal volume of physiologic
➢ morphology specimen has not Dulbecco’s phosphate-buffered
liquefied saline or proteolytic enzymes
such as alpha chymotrypsin or
bromelain may be added to
induce liquefaction and allow the
rest of the analysis to be
performed

➢ These treatments may affect biochemical tests, sperm

motility, and sperm morphology, so their use must be
documented

Dilution of semen with bromelain → calculating sperm concentration

Jelly-like granules (gelatinous bodies) → present in liquefied semen

→ no clinical significance

Mucus strands → if present, interfere with semen analysis

 ➢ Semen for pH testing can be applied to the pH pad of a
VOLUME urinalysis reagent strip and the color compared with the
manufacturer’s chart
➢ Dedicated pH testing paper also can be used
Normal semen volume - ranges between 2 and 5 mL

➢ It can be measured by pouring the specimen into a clean SPERM CONCENTRATION AND SPERM COUNT
graduated cylinder calibrated in 0.1-mL increments
○ Even though fertilization is accomplished by one spermatozoon, the
Increased volume - seen following periods of extended actual number of sperm present in a semen specimen is a valid
abstinence
measurement of fertility
○ Various factors can affect sperm concentration:
Decreased volume - more frequently associated with
infertility ➢ days of sexual abstinence before the collection
- indicate improper functioning of ➢ infection
one of the semen producing organs, ➢ stress; therefore, more than one semen specimen should be
primarily the seminal vesicles evaluated for infertility studies

➢ Incomplete specimen collection must also be considered Reference values for sperm concentration:
greater than 20 to 250 million sperm per milliliter

Borderline:
VISCOSITY concentrations between 10 and 20 million per milliliter

Specimen viscosity
➔ refers to the consistency of the fluid and may be related to specimen Total sperm count for the ejaculate can be calculated:
liquefaction
Total Sperm Count = Sperm concentration x Specimen volume

NOTE:
Normal semen specimen - easily drawn into a pipette
Total sperm counts greater than 40 million per ejaculate are
- form small discrete droplets
considered normal (20 million per milliliter × 2 mL)
- do not appear clumped or stringy
when falling by gravity from the
pipette
○ In the clinical laboratory, sperm concentration is usually performed
Incompletely liquefied - clumped using the Neubauer counting chamber
specimens - highly viscous
○ The sperm are counted in the same manner as cells in the
cerebrospinal fluid cell count:
Abnormal - droplets that form threads longer
➢ diluting the specimen
than 2 cm
- highly viscous ➢ counting the cells in the Neubauer chamber
○ The amount of the dilution and the number of squares counted vary
among laboratories
Ratings of 0 (watery) to 4 (gel-like) → viscosity report ○ Most commonly used dilution → 1:20 prepared using a mechanical
(positive-displacement) pipette
Viscosity can also be reported as: ○ Dilution of the semen is essential because it immobilizes the sperm
➢ low before counting
➢ normal ○ The traditional diluting fluid contains:
➢ high ➢ sodium bicarbonate and formalin
- immobilize and preserve the cells;
○ Increased viscosity and incomplete liquefaction impede testing for ➢ saline and distilled water
sperm motility, sperm concentration, antisperm antibody detection, - good results can also be achieved
and measurement of biochemical markers ○ Using the Neubauer hemocytometer, sperm are usually counted in
the four corner and center squares of the large center square,
pH similar to a manual RBC count

pH of semen
➔ indicates the balance between the pH values from the acidic prostatic
secretion and the alkaline seminal vesicles secretion

➢ pH should be measured within 1 hour of ejaculation due to the

loss of CO2 that occurs

Normal pH of - alkaline with a range of 7.2 to 8.0

semen

Increased pH - indicates infection within the reproductive

tract

Decreased pH - associated with increased prostatic fluid,

ejaculation duct obstruction, or poorly
developed seminal vesicles
 ○ Both sides of the hemocytometer are loaded and allowed to settle for 3 ○ Grading can be done using a scale of 0 to 4
to 5 minutes; then they are counted, and the counts should agree ➢ 4 - rapid, straight-line movement
within 10% ➢ 0 - no movement
○ An average of the two counts is used in the calculation ○ Minimum motility of 50% with a rating of 2.0 after 1 hour → normal
○ If the counts do not agree, both the dilution and the counts are
repeated
○ Counts are performed using either phase or bright-field microscopy
○ The addition of stain, such as crystal violet, to the diluting fluid aids in
visualization when using bright-field microscopy

COUNTED NOT COUNTED

- only fully developed - immature sperm and WBCs
sperm (“round” cells)

➢ can be significant, and

they may need to be
identified and counted
separately

WHO → uses a rating scale of a, b, c, d

○ Stain included in the diluting fluid aids in differentiating between
immature sperm cells (spermatids) and leukocytes, and they can be
Interpretation states that within 1 hour:
counted in the same manner as mature sperm
● 50% or more of the sperm
➢ Motile
count greater than - associated with inflammation or ➢ Categories a, b, and c
1 million leukocytes infection of the reproductive
per milliliter organs that can lead to infertility ● 25% or more of the sperm
➢ Progressive motility
presence of more - indicates disruption of ➢ Categories a and b
than 1 million spermatogenesis
spermatids per - caused by viral infections,
milliliter exposure to toxic chemicals, and WHO Laboratory Manual for the Examination and Processing of Human
genetic disorders Semen (2010)
➔ currently recommends a simpler system for grading motility that does not
include speed because of the difficulty in standardized reporting
SPERM MOTILITY ➔ Motility is graded as:
➢ progressive motility (PM)
➢ nonprogressive motility (NP)
Presence of sperm - critical for fertility ➢ immotility (IM)
capable of forward, (sperm must propel themselves through the cervical
progressive movement mucosa to the uterus, fallopian tubes, and ovum)

Traditionally, clinical laboratory reporting of sperm motility has been a

subjective evaluation performed by:
➢ examining an undiluted specimen
➢ determining the percentage of motile sperm
➢ quality of the motility

Sperm motility - Assessed using a well-mixed, liquefied

semen specimen within 1 hour of
specimen collection
Presence of a high - requires further evaluation to
Sperm motility at timed - No useful purpose percentage of immobile determine sperm vitality or the
intervals over an sperm and clumps of sperm presence of sperm agglutinins
extended period

Computer-Assisted Semen Analysis (CASA)

○ To provide continuity in reporting, laboratories should place a consistent ➔ provides objective determination of both sperm velocity and trajectory
amount of semen on a slide under the same size cover slip, such as (direction of motion)
10 μL under a 22×22 mm coverslip using a calibrated ➔ Sperm concentration and morphology are also included in the analysis
positive-displacement pipette, and allow it to settle for 1 minute ➔ found primarily in laboratories that specialize in andrology and perform a
○ This procedure should be done in duplicate for accuracy high volume of semen analysis

Percentage of sperm showing actual forward movement: SPERM MORPHOLOGY

Estimated after evaluating approximately 20 high-power fields

Alternate procedure: Sperm morphology

➢ Examine 200 sperm per slide ➔ is evaluated with respect to the structure of the head, neckpiece,
➢ Count the percentage of the different motile categories midpiece, and tail
using a manual cell counter

Normal sperm → oval-shaped head (5 μm long and 3 μm wide)

○ Motility is evaluated by both speed and direction
→ long, flagellar tail (45 μm long)
Acrosomal cap Additional parameters in evaluating sperm morphology include:
➔ critical to ovum penetration and the enzyme- ➢ Measuring head, neck, and tail size
containing ➢ Measuring acrosome size
located at the tip of the head ➢ Evaluating for the presence of vacuoles
➔ should encompass approximately half of the ➔ Kruger’s strict criteria
head and cover approximately two thirds of - requires the use of a stage micrometer or morphometry
the sperm nucleus - NOT ROUTINELY PERFORMED in the clinical
laboratory but is RECOMMENDED by the WHO
Neckpiece - integral part of assisted reproduction evaluations
➔ attaches the head to the tail and the midpiece

Normal values for sperm morphology depend on the evaluation

Midpiece
method used:
➔ approximately 7.0 μm long
➔ thickest part of the tail ● Routine criteria → greater than 30% normal forms
(surrounded by a mitochondrial sheath that produces the energy required ● Strict criteria → greater than 14% normal forms
by the tail for motility)

ADDITIONAL TESTING
Presence of normal number of
sperm that are non-motile
- infertility
Presence of sperm that are
morphologically incapable of
fertilization

Abnormalities in head - associated with poor ovum

morphology penetration

Abnormalities in neckpiece, - affect motility

midpiece, and tail

SPERM MORPHOLOGY
➢ evaluated from a thinly smeared, stained slide under oil
immersion

PROCEDURE:
1. Smears are made by placing approximately 10 μL of semen near the
frosted end of a clean microscope slide
2. Place a second slide with a clean, smooth edge in front of the
semen drop at a 45° angle and draw the slide back to the edge of MOST COMMON ADDITIONAL TESTS
the drop of semen, allowing the semen to spread across the end 1. Sperm vitality
3. When the semen is evenly distributed across the spreader slide, 2. Seminal fluid fructose level
lightly pull the spreader slide forward with a continuous movement 3. Sperm agglutinins
across the first slide to produce a smear 4. Microbial infection
4. Staining can be performed using Wright’s, Giemsa, Shorr, or
Papanicolaou stain and is a matter of laboratory preference
5. Air-dried slides are stable for 24 hours SPERM VITALITY
6. At least 200 sperm should be evaluated and the percentage of
abnormal sperm reported
7. Routinely identified abnormalities in head structure include:
➢ double heads Decreased sperm - normal sperm concentration with markedly
➢ giant and amorphous heads vitality decreased motility
➢ pinheads
➢ tapered heads ➢ Sperm vitality should be assessed within 1 hour of ejaculation
➢ constricted heads
Abnormal sperm tails: Vitality is evaluated by:
➢ frequently doubled 1. mixing the specimen with an eosin-nigrosin stain
➢ coiled 2. preparing a smear
➢ bent 3. counting the number of dead cells in 100 sperm using a brightfield or
Abnormally long neck piece: phase-contrast microscope
➢ cause the sperm head to bend backward
and interfere with motility Living cells → not infiltrated by the dye and remain bluish white
Dead cells → stain red against the purple background

Normal vitality - requires 50% or more living cells

- should correspond to the previously evaluated
motility

Presence of a large - indicate a defective flagellum

proportion of vital
but immobile cells

High number of - indicate epididymal pathology

immotile and
nonviable cells
 SEMINAL FLUID FRUCTOSE MAR test
➔ is a screening procedure used primarily to detect the
presence of immunoglobulin G (IgG) antibodies
Low sperm concentration - caused by lack of the support medium
○ Semen sample containing motile sperm is incubated with:
produced in the seminal vesicles
→ IgG antihuman globulin (AHG)
- indicated by a low to absent fructose
→ suspension of latex particles/treated RBCs coated with IgG
level in the semen
○ bivalent AHG binds simultaneously to both the antibody on the
Low fructose level - caused by abnormalities of the sperm and the antibody on the latex particles or RBCs
seminal vesicles, bilateral congenital → visible clumps of sperm and particles or cells
absence of the vas deferens,
obstruction of the ejaculatory duct, ○ finding of less than 10% of the motile sperm attached to the
partial retrograde ejaculation, and particles → normal
androgen deficiency

➢ Specimens can be screened for the presence of fructose using: Immunobead test
○ Resorcinol Test - produces an orange color when fructose is ➔ more specific procedure
present ➔ can be used to detect the presence of IgG, IgM, and IgA
antibodies and demonstrates what area of the sperm (head,
Normal quantitative level of fructose: neckpiece, midpiece, or tail) the autoantibodies are affecting
equal to or greater than 13μmol per ejaculate
○ can be determined using spectrophotometric methods Head-directed antibodies
- interfere with penetration into the cervical mucosa or ovum
Tail-directed antibodies
➢ Specimens for fructose levels should be tested within 2 hours of - affect movement through the cervical mucosa
collection or frozen to prevent fructolysis
○ Sperm are mixed with polyacrylamide beads known to be
coated with either anti-IgG, anti-IgM, or anti-IgA
ANTISPERM ANTIBODIES
○ Microscopic examination of the sperm:
→ shows the beads attached to sperm at particular areas
Antisperm antibodies
➔ can be present in both men and women ○ Depending on the type of beads used, the test could be
➔ detected in semen, cervical mucosa, or serum, and are considered a reported as:
possible cause of infertility ➢ IgM tail antibodies
➢ IgG head antibodies
➔ not unusual for both partners to demonstrate antibodies, although male
antisperm antibodies are more frequently encountered ○ Presence of beads on less than 50% of the sperm → normal
(defined by the WHO)
○ Under normal conditions, the blood–testes barrier separates sperm
from the male immune system
○ When this barrier is disrupted, as can occur following surgery, MICROBIAL AND CHEMICAL TESTING
vasectomy reversal (vasovasostomy), trauma, and infection, the
antigens on the sperm produce an immune response that damages
the sperm Presence of more - indicates infection within the reproductive
○ The damaged sperm may cause the production of antibodies in the than 1 million system, frequently the prostate
female partner leukocytes per
millimeter

Presence of antibodies in - suspected when clumps of sperm are

a male subject observed during a routine semen Routine aerobic and anaerobic cultures and tests for:
analysis ➢ Chlamydia trachomatis
➢ Mycoplasma hominis
➢ Ureaplasma urealyticum
Presence of antisperm - results in a normal semen analysis
➔ most frequently performed
antibodies in a female accompanied by continued infertility
subject
Additional chemical testing performed on semen:
1. Determining the levels of neutral α-glucosidase
Presence of antisperm - demonstrated by mixing the semen with 2. Free L-carnitine
antibodies in women the female cervical mucosa or serum 3. Glycerophosphocholine
and observing for agglutination 4. Zinc
5. Citric acid
6. Glutamyl transpeptidase
Sperm-agglutinating antibodies
7. Prostatic acid phosphatase
➔ cause sperm to stick to each other in a head-to-head, head-to-tail, or
tail-to-tail pattern
Decreased fructose - associated with a lack of seminal fluid,
Agglutination is graded on microscopic examination as: levels decreased neutral a -glucosidase,
glycerophosphocholine, and L-carnitine
➢ few
- suggest disorder of the epididymis
➢ moderate
➢ many
Decreased zinc, - indicate a lack of prostatic fluid
citric acid, glutamyl
Two frequently used tests to detect the presence of antibody- transpeptidase, and
coated sperm: acid phosphatase
1. Mixed Agglutination Reaction (MAR) test
2. Immunobead test
Spectrophotometric methods
➔ used to quantitate citric acid and zinc
 ○ On certain occasions, the laboratory may be called on to determine
whether semen is actually present in a specimen
○ Primary example → cases of alleged rape
1. Microscopically examining the specimen for the presence of sperm
may be possible
➢ Best results: obtained by enhancing the specimen with xylene
and examining under phase microscopy
2. Motile sperm → detected for up to 24 hours after intercourse
Nonmotile sperm → persist for 3 days
3. As the sperm die off, only the heads remain and may be present
for 7 days after intercourse
4. Seminal fluid contains a high concentration of prostatic acid
SEMEN ANALYSIS QUALITY CONTROL
phosphatase, so detecting this enzyme can aid in determining
the presence of semen in a specimen
○ Prostatic Specific Antigen (PSA) ○ Traditionally, routine semen analysis has been subject to very little
➔ more specific method quality control, a situation that has resulted from a lack of appropriate
➔ detection of seminal glycoprotein p30 control materials and the subjectivity of the motility and morphology
➔ which is present even in the absence of sperm analyses
○ ABO Blood Grouping and DNA Analysis → rated as a high complexity test under the Clinical Laboratory
➔ obtaining further information on the specimen Improvement Amendments, and testing personnel standards
must be observed
○ Increased interest in fertility testing has promoted the development
POST VASECTOMY SEMEN ANALYSIS
of quality control materials and in-depth training programs
○ The standardized procedures developed by the WHO have provided
Postvasectomy semen analysis a basis for laboratory testing and reporting
➔ much less involved when compared with infertility analysis because the ○ The use of CASA has aided in reducing the subjectivity of the
only concern is the presence or absence of spermatozoa analysis
○ However, even computerized analysis has been shown to vary among
○ Length of time required for complete sterilization: operators
➢ vary greatly among patients ○ Laboratories can now participate in proficiency testing programs
➢ depends on both time and number of ejaculations offered by the College of American Pathologists and the American
○ Finding viable sperm in a postvasectomy patient is not uncommon, Association of Bioanalysts that include:
and care should be taken not to overlook even a single sperm ➢ sperm concentration
○ Specimens are routinely tested at monthly intervals, beginning at 2 ➢ vitality
months postvasectomy and continuing until two consecutive monthly ➢ morphology
specimens show no spermatozoa ○ Commercial quality control materials and training aids are available
○ Recommended testing includes: and should be incorporated into laboratory protocols
➢ examining a wet preparation using phase microscopy
- presence of motile and nonmotile sperm
➢ specimen centrifugation for 10 minutes and examination of
the sediment

SPERM FUNCTION TESTS

○ Advances in assisted reproduction and IVF have resulted in a need for

more sophisticated semen analysis to assess not only the
characteristics of sperm but also the functional ability
○ The tests are most commonly performed in specialized andrology
laboratories
1. Hamster egg penetration assay
2. Cervical mucus penetration test
3. Hypo-osmotic swelling test
4. In vitro acrosome reaction