URINALYSIS AND BODY FLUIDS

CHAPTER 11:
SEMEN
BY: ANTIGUA, MONTALLANA, AND TIU
 INTRODUCTION
Semen is composed of four fractions
that are contributed by the testes,
epididymis, seminal vesicles, prostate
gland, and bulbourethral glands. Each
fraction differs in its composition,
and the mixing of all four fractions
during ejaculation is essential for the
production of a normal semen
specimen.
 PHYSIOLOGY
The Testes are paired glands in the scrotum that contain the
seminiferous tubules for the secretion of sperm. The external
location of the scrotum contributes to a lower scrotum
temperature that is optimal for sperm development. Germ
cells for the production of spermatozoa are 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). When
spermatogenesis is complete, the immature sperm
(nonmotile) enter the epididymis. In the epididymis,
the sperm mature and develop flagella. The entire
process takes approximately 90 days. The sperm
remain stored in the epididymis until ejaculation, at
which time they are propelled through the ductus
deferens (vas deferens) to the ejaculatory ducts.
The ejaculatory ducts receive both the sperm from
the ductus deferens and fluid from the seminal
vesicles. The seminal vesicles produce most of the
fluid present in semen (60% to 70%), and this
fluid is the transport medium for the sperm. The
fluid contains a high concentration of fructose
and flavin.
 FRUCTOSE Fructose - metabolized for the
energy needed for the flagella to
AND FLAVIN propel them through the female
reproductive tract.
IN SEMEN
Flavin - responsible for the gray
appearance of semen, as well as
the blue to yellow fluorescence
when semen is visualized under
ultraviolet light (Wood’s lamp).
 FRUCTOSE Additional notes:

AND FLAVIN In the absence of fructose,

sperm do not display motility
IN SEMEN in the semen analysis.

Various proteins secreted by

the seminal vesicles are
involved in the coagulation of
the ejaculate.
PHYSIOLOGY (CONT’D)
The muscular prostate gland, located just below the bladder,
surrounds the upper urethra and aids in propelling the sperm through
the urethra by contractions during ejaculation.
Approximately 20% to 30% of the semen volume is acidic fluid
produced by the prostate gland. The milky acidic fluid contains high
concentrations of the following:
acid phosphatase
citric acid
zinc
proteolytic enzymes (responsible for both the coagulation
and liquefaction of the semen after ejaculation)
PHYSIOLOGY (CONT’D)
The bulbourethral glands, located below the prostate,
contribute about 5% of the fluid volume in the form of a
thick, alkaline mucus that helps 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.
 SEMEN
PRODUCTION
THE MALE GENITALIA
 SPECIMEN COLLECTION
Most of the sperm are contained in the first portion of the
ejaculate, making complete collection essential for accurate
testing of both fertility and postvasectomy specimens.

When a part of the first portion of the ejaculate is missing:

sperm count- decreased;
pH- falsely increased; and
specimen- will not liquefy.

Likewise, when part of the last portion of ejaculate is missing:

semen volume- decreased;
sperm count- falsely increased;
pH- falsely decreased; and
specimen- will not clot.
 SPECIMEN COLLECTION
Specimens are collected after a period of sexual abstinence of
at least 2 days to not more than 7 days. Specimens collected
after prolonged abstinence tend to have higher volumes and
decreased motility.

When performing fertility testing, the World Health

Organization (WHO) recommends that two or three specimens
be collected not less than 7 days or more than 3 weeks apart,
with two abnormal specimens considered significant.
SEMEN COMPOSITION
 SEMEN ANALYSIS

Semen analysis for fertility evaluation consists of

both macroscopic and microscopic examination.
Parameters reported include the following:
appearance
volume
viscosity
pH
sperm concentration and count
motility
morphology
REFERENCE
VALUES FOR
SEMEN
ANALYSIS
 APPEARANCE

Normal semen has a gray-white color, appears

translucent, and has a characteristic musty odor.
When the sperm concentration is very low, the
specimen may appear almost clear. Increased white
turbidity indicates the presence of WBCs and
infection within the reproductive tract.
 APPEARANCE
During microscopic examination, WBCs must be differentiated
from immature sperm (spermatids). The leukocyte esterase
reagent strip may be useful to screen for the presence of
WBCs.

red coloration - presence of RBCs; is abnormal

yellow coloration - may be caused by urine contamination,
specimen collection after prolonged abstinence, and
medications
 LIQUEFACTION
Analysis of the specimen
A fresh semen is clotted cannot begin until
liquefaction has occurred. If
and should liquefy within
after 2 hours the specimen
30 to 60 minutes after
has not liquefied, an equal
collection. Failure of volume of Dulbecco’s
liquefaction to occur phosphate-buffered saline
within 60 minutes may be (DPBS) or proteolytic
caused by a deficiency in enzymes, such as alpha-
prostatic enzymes and chymotrypsin or bromelain,
should be reported. may be added to induce
liquefaction.
Jelly-like granules
(gelatinous bodies) may
be present in liquefied
semen specimens and
have no clinical
significance. Mucous
strands, if present, may
interfere with semen
analysis.
VOLUME
Normal semen volume ranges between 2 and 5 mL.
Increased volume may be seen after periods of
extended abstinence.
Decreased volume is associated more frequently with
infertility and may indicate improper functioning of
one of the semen-producing organs, primarily the
seminal vesicles. Incomplete specimen collection must
also be considered.
VISCOSITY
Specimen viscosity refers to the consistency of the fluid and
may be related to specimen liquefaction. Specimens that are
liquefied incompletely are clumped and highly viscous.

Normal semen specimen - drawn into a pipette easily and

form small discrete droplets that do not appear clumped
or stringy when falling by gravity from the pipette

Abnormal semen specimen - droplets that form threads

longer than 2cm; considered highly viscous
 pH
The pH of semen indicates the balance between the pH values from
the acidic prostatic secretion and the alkaline seminal vesicles
secretion. The pH should be measured within 1 hour of ejaculation
due to the loss of CO2 that occurs. The normal pH of semen is
alkaline with a range of 7.2 to 8.0.

Increased pH - indicates infection within the reproductive tract

Decreased pH - may be associated with increased prostatic
fluid, obstruction of the ejaculatory duct, or poorly developed
seminal vesicles
SPERM CONCENTRATION AND
SPERM COUNT
The actual number of sperm present in a semen specimen is a
valid measurement of fertility .
Various factors can affect sperm concentration:
days of sexual abstinence before the collection
infection
stress
>20 to 250 million sperm/ml
>40 million per ejaculate
NEUBAUER COUNTING
CHAMBER
1:20 - using a mechanical
(positive-displacement) pipette.
The traditional diluting fluid
contains sodium bicarbonate and
formalin, however, good results
also can be achieved using saline
and distilled water.
Using the Neubauer
hemocytometer, sperm usually
are counted in the fourth corner
and center squares of the large
center square, similar to manual
RBC count.
The addition of a stain, such as
crystal violet, aids in visualization
when using brightfield
microscope.
Only fully developed sperm
should be counted. However, the
presence of immature sperm and
WBCs can be significant and
they need to be counted
separately.
A count > 1 million leukocytes/mL
is associated with inflammation
or infection of the reproductive
organs that can lead to
infertility.
CALCULATION OF SPERM CONCENTRATION AND SPERM COUNT

Calculation of sperm concentration

depends on the dilution used and the size
and number of squares counted.

Example:
Using a 1:20 dilution, an avarage of 60
sperm are counted in the five RBC counting
squares on both sides of the
hemocytometer. Calculate the sperm
concentration per mL and the total sperm
count in a specimen with a volume of 4
mL.

60 sperm counted x 1,000,000 =

60,000,000 sperm/mL x 4mL =
240,000,000 sperm/ejaculate
 SPERM MOTILITY

Sperm motility should be assessed using a well-

mixed, liquefied semen specimen within 1 hour of
specimen collection.
SPERM MOTILITY
CASA provides objective determination of both sperm
velocity and trajectory (direction of motion).
Automated system provide faster results withy higher
precision and standardization, as well as reduced
potential for human error.
Sperm Class Analyzer (SCA, Microptic, Spain)
CEROS CASA systems (Hamilton Thorne, Beverly,
MA)
Automated Sperm Quality Analyzer (SQA-Vision,
Medical Electronic Systems, Los Angeles, CA)
SPERM MORPHOLOGY
The presence of sperm that are morphologically
incapable of fertilization results in infertility.
Sperm morphology is evaluated with respect to the
structure of the head, neckpiece, midpiece, and tail.
Abnormalities in head morphology are associated with
poor ovum penetration, whereas abnormalities in the
neckpiece, midpiece, and tail affect motility.
The normal sperm has an oval-shaped head
approximately 5µm long and 3µm wide, with a long,
flagellar tail approximately 45µm long.
SPERM MORPHOLOGY
SPERM MORPHOLOGY

Critical to ovum penetration is the enzyme-containing

acrosomal cap located at the tip of the head.
The neckpiece attaches the head to the tail and the
midpiece.
The midpiece is approximately 7µm long and is the
thickest part of the tail because it is surrounded by a
mitochondrial sheath that produces the energy required
for motility.
SPERM MORPHOLOGY
Abnormalities in head structure that are identified routinely include double
heads, giant and amorphous heads, pinheads, tapered heads, and constricted
heads.
Abnormal sperm tails are frequently doubled, coiled, or bent. A neckpiece
that is abnormally long may cause the sperm head to bend backward and
interfere with motility.
Additional parameters in evaluating sperm morphology include measuring
head, neck, and tail size; measuring acrosome size; and evaluating for the
presence of vacuoles.
Inclusion of these parameters is referred to as Kruger’s strict criteria. Strict
criteria evaluation requires the use of a stage micrometer or morphometry.
Calculating Round Cells

By counting the number of spermatids or leukocytes seen in

conjunction with 100 mature sperm, the amount per mL can be
calculated using the following formula:
ADDITIONAL
TESTING

SEMINAL FLUID FRUCTOSE LEVEL

SPERM AGGLUTININS
MICROBIAL INFECTION
CHEMICAL ANALYSIS
SPERM VITALITY

Decreased sperm vitality may be suspected when a specimen has a

normal sperm concentration with a markedly decreased motility

Normal vitality requires 50% or more living cells and should

correspond to the motility evaluated previously

Large proportion of vital but immobile cells may inidicate a

defective flagellum , where as a high number of immotile and
nonviable cells may indicate epididymal pathology
SEMINAL FLUID FRUCTOSE

low sperm concentration may be caused by lack of the support

medium produced in the seminal vesicles , which can be indcated by
a fructose that is low to absent in the semen
low fructose level are caused by abnormalities of the seminal
vesicles , bilateral congenital absence of the vas deference ,
obstruction of the ejaculatory duct , partial retrograde ejaculation
and androgen deficiency
resorcinol test produces as an orange color when frucdtose is
present
ANTISPERM ANTIBODIES
can be present in both men and women
may be detected in semen , cervical mocusa
or serum and are considered a possible cause of
infertility .Under normal conditions , as the blood
barrier separates sperm from the male immune
system .
sperm agglutinating antibodies cause to stick
to each other in a head to head , head to tail ,
tail to tail pattern
graded as “ few “ “moderate” “ many “
The presence of antibodies in a male subject can
be suspected when clumps of sperm are observed
during a routine semen analysis .
The presence of antisperm antibodies in as
female subject results in a normal semen
analysis accompanied by continued infertility .
The presence of antisperm antibodies in women
may be demonstrated by mixing the semen with
the female cervical mocusa or serum and
observing for agglutination .
The MAR test is a screening procedure used
primariliy to detect the presence of
immunoglobulin G (IGG) antibodies
The immunobead test is more specific
procedure in that it can be used to detect
the presence of IGG, IGM and IGA antiodies
and demonstrate the area of the sperm
 MICROBIAL AND routine aerobic and anaerobic cultures and test for
Chlamydia trachomatis , Mycoplasma hominis and
CHEMICAL TESTING Ureaplasma urealyticum
determining the levels of neutral @-glucosidase ,
free L-carnitine , glycerophosphocholine , zinc, citric
acid , glutamyltranspeptidase and prostatic acid
phosphatase

Motile sperm can be detected for up to 24 hrs

after intercourse , whereas nonmotile sperm can
persist for 3 days . AS the sperm die off , only the
heads remain msy be present for 7 days after
intercourse
POSTVASECTOMY SEMEN
ANALYSIS only concern is the presence of abscence of
spermatozoa
finding viable sperm in a postvasectomy patient
is not uncommon and cxare should be taken not
to overlook even a single sperm
monthly intervals , beginning at 2 months
popstvasectomy and continuing until two
consecutive monthly specimen shows no
spermatozoa
 SPERM FUNCTION TEST

The test are performed most commonly in specialized andrology

laboratories and include the hamster egg penetration assay ,
cervical mucus penetration test , hypo-osmotic , swelling test
and the in vitro acrosome reaction
SEMEN ANALYSIS QUALITY
CONTROL the analysis is rated as a high complexity test

increased interest in fertility testing has a promoted the development

of quality control materials and in depth training programs
The use of CASA has aided in reducing the subjectivity of the analysis
College of American Pathologist and American Association of
Bioanalyst that include sperm concentration , vitality and morphology