SEMEN ANALYSIS

ANATOMY AND PHYSIOLOGY

1. Testes are paired glands in the scrotum that contain the seminiferous
tubules
a. Germ cells located in the seminiferous tubules that produce
spermatozoa
b. Sertoli cells provide support and nutrients for the germ cells as they
undergo spermatogenesis
2. Epididymis storage reservoir for spermatozoa that become matured here
and competent to fertilized ova
3. Vas deferens propels sperm to ejaculatory duct
4. Seminal Vesicles secretes seminal fluid that composes up to 60-70% of
the total volume of semen volume
a. Seminal fluid is the transport medium of sperm, it contains:
Fructose provides energy for the motility of spermatozoa
Flavin responsible for the gray appearance of semen
5. Prostate gland aids in propelling the sperm through the urethra by
contractions during ejaculation and secretes milky acidic fluid.
a. Prostate Fluid milky and acidic fluid and it contains
Acid phosphatase
Citric acid
Zinc
Proteolytic enzymes responsible for coagulation and
liquefaction of the semen
6. Bulbourethral gland contributes 5% of the fluid volume that secretes
thick alkaline mucus that helps to neutralize the acidity of from the prostate
secretions and the vagina.
7. Penis male external organ that is used in sexual intercourse

Semen Production
Structure Function
Seminiferous tubules of testes Spermatogenesis
Epididymis Sperm Maturation
Ductus deferens Propel sperm to ejaculatory ducts
Seminal vesicles Provides sperm and fluid
Prostate gland Provide enzymes and proteins for
coagulation and liquefaction
Bulbourethral gland Add alkaline mucus to neutralized the
prostatic acid and vaginal acidity

Semen Composition
Spermatozoa 5%
Seminal Fluid 60%-70%
Prostate fluid 20%-30%
Bulbourethral 5%
glands SPERM PATHWAY
Testes Epididymis Vas deferens Prostate Urethra Vagina Cervix
Uterus Egg
WAYS TO COLLECT SEMEN
Masturbation most preferable way, directing the semen into a clean
sample cup without using lubricant
Coitus interruptus withdrawing the penis from the partner just before
ejaculating and follow by ejaculating into a clean sample cup
Coitus by using condom - A special (silicon) condom that does not contain
any substance that kills sperm (spermicide). After ejaculation, carefully
remove the condom from the penis. d
SPECIMEN COLLECTION
Abstinence days 2-6
Pass urine
Wash hands with soap, dry
Collect the entire sample into the wide mouth sterile container, 70% of
sperms is in the first part of the ejaculate
Keep the sample at body temperature (37OC), no sunlight
Deliver the sample within one hour of ejaculation

SEMEN ANALYSIS
A. Appearance
Normal - gray-white color, translucent and musty odor
Increased white turbidity presence of WBCs and infection within
reproductive tract
Red coloration presence of RBCs
Yellow coloration urine contamination
B. Liquefaction
A fresh semen is clotted and should liquefy within 30-60 minutes
after collection
If not liquefied after 2 hours, add physiologic Dulbeccos
phosphate-buffered saline
C. Volume
Normal 2-5 mL
Decreased volume indicates infertility
D. Viscosity
Normal small discrete droplets that do not appear clumped or
stringy
Abnormal droplets that form thread longer 2 cm
Reported by rating from 0 (watery) to 4 (gel-like)
Can also be reported low, normal or high
E. pH
Should be measured within 1 hour of ejaculation due to the loss of CO2
Normal 7.2-8.0
Increased pH infection within reproductive tract
 Decrease pH associated with increased prostatic fluid, ejaculatory
duct obstruction or poorly developed seminal vesicles
F. Sperm Concentration and Sperm Count
Various factors can affect sperm concentration, such as the days of
sexual abstinence before the collection, infection, or stress
Reference value for sperm concentration are commonly listed as
greater than 20 to 250 million sperm per milliliter
Concentration between 10 and 20 million per milliliter are consider
border line
The total sperm count for ejaculate can be calculated by multiplying
the sperm concentration by the specimen volume
Total sperm counts greater than 40 million per ejaculate are considered
normal (20 million per milliliter times 2 ml)
In the clinical laboratory, sperm concentrations is usually performed
using the Neubauer counting chamber
The most commonly used dilution is 1:20 prepared using a mechanical
pipette
Dilution of the semen is essential because it immobilizes the sperm
before counting
The traditional fluid contains sodium bicarbonate and formalin
Using Neubauer hemocytometer, sperm are usually counted in the four
corner and center squares of the large center square, similar to a
manual RBC count. Both sides of the hemocytometer are loaded and
allowed to settle for 3 to 5 minutes; then they are counted, and the
counts should agree within 10%. An average of the two counts is used
in the calculation. If the counts do not agree, both the dilution and the
counts are repeated
Only fully developed sperm should be counted. Immature sperm and
WBC, often referred to as round cell must not be included. However,
their presence can be significant and they may need to be identified
and counted separately
A count greater than 1 million leukocytes per milliliter is associated
with inflammation or infections of the reproductive organs that can
lead to infertility
G. Calculating Sperm concentration and Sperm count
The total sperm count is calculated by multiplying the number of
sperm per milliliter by the specimen volume
Several methods have been developed using specially designed and
disposable counting chambers that do not require specimen dilution
Comparison of these method and the standard neubauer counting
chamber method showed poor correlation with the neubauer method
and also among themselves.
H. Sperm motility
The presence of sperm capable of forward, progressive movement is
critical for fertility, because once presented to the cervix, the sperm
must propel themselves through the cervical mucosa to the uterus,
fallopian tubes, and ovum.
 Sperm motility should be assessed using a well-mixed, liquefied semen
specimen within 1 hour of specimen collection. The practice of
examining sperm motility at timed intervals over an extended period
has been shown to serve no useful purpose. Laboratories should place
a consistent amount of semen on a slide under the same size cover
slip, such as 10 uL under a 22x22 mm cover slip using a calibrated
positive-displacement pipette, and allow it to settle for 1 minute.
An alternate procedure is to examine 200 sperm per slide and count
the percentages of the different motile categories using a manual cell
counter.
Motility is evaluated by both speed and direction. Grading can be done
using a scale of 0 to 4, with 4 being rapid, straight-line movement and
0 indicating no movement. A minimum motility of 50% with a rating of
2.0 after 1 hour is considered normal.

SPERM MOTILITY GRADING

GRADE WHO SPERM MOTILITY ACTION
CRITERIA
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 d No forward progression
0 d No movement

ALTERNATIVE SPERM MOTILITY GRADING CRITERIA

Progressive motility (PM) Sperm moving linearly or in a large circle
Nonprogressive motility Sperm moving with an absence of progression
(NPM)
Immotility (IM) No movement

REFERENCE VALUES FOR SEMEN ANALYSIS

Volume 2-5 mL
Viscosity Pours in droplets
pH 7.2-8.0
Sperm >20 million/mL
Concentration
Sperm Count >40 million/ejaculate
Motility >50% within 1 hr
Quality >2.0
Morphology >14% normal forms (strict
criteria)
>30% normal forms (routine
criteria)
Round cells <1.0 million/mL
I. Sperm Morphology
An important part of any breeding soundness exam is an evaluation of
sperm morphology. In the most fundamental case, the size and shape
of the head, midpiece and tail are examined. Additional information
can be gained by evaluating integrity of the acrosome and sperm
membranes.
Sperm from different species vary in size and shape. Bull and human
sperm, for example, have paddle-shaped heads, rodent sperm have
hook-shaped heads, and the heads of chicken sperm are spindle-
shaped and almost difficult to distinguish from the midpiece. The
images shown below of rat, bull and chicken sperm are all at the same
magnification.
The results of a sperm morphology exam are reported as percent
normal. It is always the case that some sperm from an ejaculate are
morphologically abnormal, but when that fraction becomes excessive,
fertility may decrease. It is also useful to subclassify the abnormal
population into the types of abnormality observed. Two types of
classification schemes are commonly used: Abnormalities can be
classified as affecting the head, midpiece or tail. The most basic type
of classification scheme differentiates primary and secondary
abnormalities:
Anatomic site of the defect: The problem can involve
the head, midpiece or tail. Some abnormal sperm may have
defects in more than one site.
Primary versus secondary defects: Primary defects
are the more severe and are thought to originate while the
sperm was still within the semeniferous epithelium of the testis.
Secondary defects are less serious and thought to arise during
passage thought the epididymis or by mishandling after
ejaculation.
PREPARING SLIDES FOR A BASIC MORPHOLOGY EXAMINATION
A nigrosin-eosin stain is commonly used because it is effective, simple and, in
addition to allowing sperm to be readily visualized, it is a so-called "live-dead"
stain, allowing one to assess membrane integrity at the same time as
morphology. The technique for preparing an eosin-nigrosin-stained slide is as
follows:
1. Have microscope slides and nigrosin-eosin stain prewarmed to body
temperature.
2. Pipet a drop of stain onto the end of a slide, then pipet a small droplet
of semen next to the stain.
3. Place the edge of another slide into the drops of stain and semen - rock
that slide back and forth a few times to mix the sperm and stain, then
smear the second slide across the surface of the first.
4. Dry the slide rapidly by placing in on a warming plate or waving it back
and forth in the air.
5. Examine using a bright field microscope (typically using a 40-100X
objective lens).
ADDITIONAL TESTING
Sperm Vitality
Decreased motility with normal count
Should be assessed within 1 hour of ejaculation
Evaluated by mixing in eosin-nigrosin stain, preparing a smear and
counting the number of dead cells in 100 sperm using bright field
microscope
Normal vitality requires 50% of more living cells and should correspond
to the previously evaluated motility
Seminal Fluid Fructose
Low sperm concentration maybe caused by lack of seminal vesicle
support medium
Should be tested within 2 hours of collection
Specimens can be screened for the presence of fructose using the
resorcinol test
A normal quantitative level of fructose is equal to or greater than
13umol per ejaculate.
Antisperm Antibodies
Can be present in both men and women.
Can be detected in semen, cervical mucosa, or serum, and are
considered a possible cause of infertility
Vasectomy surgical removal of all or part of the vas deferens for the
purpose of male sterilization.
The presence of antibodies in male subject can be suspected when
clumps are observed during a routine senen analysis. While the female
subject result in a normal semen analysis accompanied by continued
infertility.
Two test to detect the presence of antibody-coated sperm:
1. Mixed agglutination reaction [MAC] - Screening procedure
used primary to detect the presence of immunoglobulin G [IgG]
antibodies.
2. Antihuman globulin [AHG] - Semen sample containing motile
sperm and a suspension of latex particles or treated RBCs
coated with IgG.
Immunobead test - is more specific procedure in that can be used to
detect the presence of IgG, IgM, and IgA antibodies and demonstrates
what area of the sperm [head, neckpiece, midpiece or tail] yhe
autoantibodies are affecting.
Microbial and Chemical Testing
Presence of more than 1 million leukocytes per millimeter indicates
within the reproduction system, frequently the prostate.
They are aerobic and anaerobic cultures and test:
1. Chlamydia trachomatis
2. Mycoplasma hominis
3. Ureaplasma urealyticum
Motile sperm can be detected for up to 24hours after the intercourse,
whereas non motile sperm can presist for 3 days.As the sperm die off,
 only the heads remain and may be present for 7 days after ther
intercourse.
Prostatic specific antigen [PSA] - Specific method is the detection of
seminal glucoprotein and is present even the absence of sperm.
Postvasectomy semen analysis
Is much less involved procedure when compared with infertility
analysis because the only concern is the presence or absence of
spermatozoa.
Sperm function test
Hamster egg penetration - sperm are involved with species- non
specific hamster eggs and penetration is observed microscopically.
Cervical mucus penetration - observation of sperms ability to
penetrate partners midcycle cervical mucus.
Hypo-osmotic swelling - sperm exposed to low-sodium
concentrations are evaluated for membrane integrity and sperm
viability.
In vitro acrosome reaction - evaluation of the acrosome to produce
enzymes essential for ovum penetration.

ADDITIONAL TESTING FOR ABNORMAL SEMEN ANALYSIS

Abnormal Result Possible Test
Abnormality
Decreased motility Viability Eosin-nigrosin
with normal count stain
Decreased count Lack of seminalvesicle Fructose level
support medium
Decreased motility with Male antisperm -Mixed agglutination reaction
clumping antibodies and immunobead tests
-Sperm agglutination with
male serum
Normal analysis with Female antisperm Sperm agglutination with
continued antibodies female serum
infertility

Semen Analysis Quality Control

A situation that has resulted from a lack of appropriate control
materials and the subjectivity of the motility and morphology analyses
 WRITTEN REPORT
OF
CHAPTER 10: SEMEN
ANALYSIS

SUBMITTED TO:
Maam Sheryl L. Sajulga, RMT

SUBMITTED BY:
M.C. Paglinawan
Catsteven C. Casio
Kris Jan D. Singco
Norol Ashiquin M. Mito-
on