SEMINALYSIS
semen and this fluid is the transport medium for the
Reasons for testing:
testing for males fertility
post-vasectomy examination
forensic examination
artificial insemination programs(donating
sperm)
Physiology
composed of four fractions:
testes and epididymis(spermatozoa)-5%
seminal vesicles(seminal fluid)-60%-70%
prostate gland(prostate fluid)-20%-30%
bulbourethral glands-5%
Each fraction is essential for the production of a normal semen
specimen
Testes-paired glands in the semineferous tubule for the
secretion of sperm
Semineferous tubule- spermatogenesis
Lower scrotum temperature-optimal for sperm
development
Germ cells-located in the epithelial cells of the
semineferous tubule;produces the spermatozoa
Specialized sertoli cells-provide support and nutrients
for the germ cells as they undergo mitosis and
meiosis(spermatogenesis)
Epididymisthe sperm mature and develop flagella.
(process takes approximately 90 days)
Ductus(vas) deferens-where ejaculated sperm is
propelled through to the ejaculatory ducts
Ejaculatory ducts-receive both the sperm from the
ductus deferens and fluid from the seminal vesicles
Seminal vesicle-produce most of the fluid present in
sperm. The fluid contains a high concentration of fructose
and flavin
Spermatozoa-metabolize the fructose for the
energy(motility) needed for the flagella to propel them
through the female reproductive tract.
Flavin-responsible for the gray appearance of semen
proteins-secreted by the seminal vesicles;involved in the
coagulation of the ejaculate
Prostate gland-located below the bldder, surrounds the
upper urethra;aids in propelling the sperm through the
urethra by contractions during ejaculation;produces
acidic fluid;provide enzymes and proteins for coagulation
and liquefaction
Milky acidic fluid-contains high concentrations of acid
phophatase, citric acid, zinc and proteolytic enzymes
responsible for both the coagulation and liquefaction of
the semen following ejaculation
Bulbourethral glands-located below the prostate;fluid
volume a thick,alkaline mucus that helps to neutralize
acidity from the prostate secretions and the vagina.
note: without neutralization, sperm motility would be
diminished
Specimen collection
Most of the sperm are contained in the first portion of
the ejaculates, making complete collection essential for
accurate testing of both fertility and postvasectomy
specimens.
A part of the first portion is missing:
sperm count will be decreased
pH falsely increased
specimen will not liquefy
A part of the last portion is missing:
 semen volume is decreased
sperm count is falsely increased
pH is falsely decreased
specimen will not clot/coagulate
atleast 2 days and not more than 7 days- sexual abstinence
Prolonged abstinence-tend to have higher volumes and
decreased motility
1. Warm sterile glass or plastic container
2. The specimen is collected in a room provided by a
laboratory.
3. If testing is done outside the laboratory, it should be kept
at room temp. and delivered to the lab within 1 hr.
4. Record the patients name and birth date, the period of
sexual abstinence, the completeness of the
sample,difficulties with collection and times of specimen
collection and specimen receipt.
5. Specimens awaiting analysis should be kept at 37
degreees celcius
METHODS OF COLLECTION:
masturbation
testicular sperm extraction
sperm aspiration
withdrawal system/coitus interruptus
note: not a reliable means of collection because the
first portion of the ejaculate, which contains the
highest number of spermatozoa, may be lost and the
low pH of the vaginal fluid may affect sperm motility
sexual intercourse using nonlubricant-containing
rubber or polyurethane condoms should be used
note: ordinary condoms contain spermicides
Specimen handling
1. Standard precautions should be observed all the
time since semen can be a potential reservoir for
HIV and hepatitis viruses
2. Specimens are discarded as biohazardous waste
3. Sterile culture and techniques should be used in
semen culture, bioassay, intra-uterine
insemination(IUI), or in vitro fertilization(IVF).
SEMEN ANALYSIS
Semen analysis for fertility evaluation consists of
both macroscopic and microscopic examination.
Appearance
N=gray-white color, translucent and has a musty odor
clear=very low sperm concentration
increased whit turnidity=presence of WBCs and
infection within the reproductive tract
note: during microscope exam, WBCs must be
differentiated from immature
sperm(spermatids)=use LE rgnt. strip
red coloration=presence of RBCs
yellow=caused by urine contamination and
medication(vit.C, B complex)
Liquefaction
30-60 mins. after collection=time of fresh semen
liquefaction
deficiency in prostatic enzymes=causes failure of
liquefaction to occur within 60 mins.
note:analysis of specimen cannot begin until
liquefaction has occurred
Physiologic Dulbeccos phosphate-buffered saline or
proteolytic enzymes such as alpha-chymotrypsin or
bromelain= added to the specimen to induce
liquefaction if it hasnt occurred for more than 2
hrs.;may affect biochemical tests, sperm motility and
sperm morphology
Jelly-like granules(gelatinous bodies)=may be
present in liquefied semen and have no clinical
significance
Mucus strands=interfere with the analysis
Volume
N=2-5ml
can be measured in a clean graduated cylinder
calibrated in 0.1 ml
increased volume=extended abstinence
decreased volume=associated with infertility;may
indicate dysfunction of one of the semen-producing
organs,primarily the seminal vesicles
Viscosity
refers to the consistency of the fluid and may be
related to specimen liquefaction
N=pours in droplet
highly viscous and clumped=incompletely liquefied
spec.;>2cm thread
reporting: ratings of 0 (watery) to 4 (gel-like); can
also be reported as low, normal or high
note:increased viscosity and incomplete liquefaction
impede testing for sperm motility, sperm
concentration,a ntisperm antibody detection and
measurement of biochemical markers
pH
indicates the balance between the pH values from the
acidic prostatic secretion and the alkaline seminal
vesicles secretion
should be measured within 1 hr. of ejaculation due to
the loss of CO2 that occurs
N=alkaline;7.2-8.0
increased pH=infection within repro. tract
decreased pH=increased prostatic fluid, ejaculatory
duct obstruction or poorly developed seminal
vesicles.
Sperm concentration and sperm count
Sperm motility

Sperm capability of forward, progressive movement

is critical for fertility=sperm must propel themselves
from cervix, uterus, fallopian tubes to ovum

N=50% w/ a rating of 2.0 after 1 hr.

Assessed using a well-mixed, liquefied semen within

1 hr. of collection

1. 10ul of sperm under a 22x22mm cover slip

2. allow it to settle for 1 min.
3. evaluation in approximately 20hpf/examine 200
sperm per slide and count the percentage
4. motility is evaluated by both speed and direction
Grade WHO criteria Sperm Motility
Action Sperm morphology
4.0 a Rapid,straight-line
motility Sperm morphology is evaluaeted with respect to the
3.0 b Slower speed,some structure of the head,neckpiece,midpiece,and tail.
lateral movement Abnormalities in this structures results affect
2.0 b Slow forward motility.
progression,lateral
movement N=oval-shaped head approximately 5um long and
1.0 c No forward 3um wide; long, flagellar tail approximately 45um
progression
long
0 d No movement
Acrosomal cap-enzyme-containing;critical to ovum
penetration;located at the tip of the head;should
encompass approximately half of the head and cover
approximately two thirds of the sperm nucleus
WHO Laboratory Manual for the Examination and
Processing of Human Semen currently recommends Neckpiece- attaches the head to the tail and the
a simpler system for grading motility. midpiece

Midpiece-approximately 7.0um long and is the

thickest part of the tail because it is surrounded by
Progressive Sperm moving
mitochondrial sheath
motility(PM) linearly or in a
large circle
Mitochondrial sheath-produces the energy required
Nonprogressive Sperm moving
motility(NP) with an absence by the tail for motility
of progression
Immotility(IM) No movement Sperm morphology is evaluated from a thinly
smeared, stained slide under oil immersion

10ul of semen spread at 45 degree angle;stain with

Computer-assisted semen analysis(CASA)-provides
objective determination of both sperm velocity and
trajectory;sperm concentration and morphology are
ilso included.
Wrights, Giemsa, Shorr or Papanicolaou stain
air-dried slide are stable for 24 hrs.
atleast 200 sperm should be evaluated and the
percentage of abnormal sperm reported

Routinely identified abnormalities in head:
double heads
giant and amorphous heads
pinheads
tapered heads
constricted heads
Abnormal sperm tails:
doubled
coiled or bent
Abnormally long neckpiece=cause the sperm head to
bend backward and interfere with motility
Aditional parameter in evaluating sperm
morphology:
measure: acrosome, head, neck, tail size
evaluate: for the presence of vacuoles
Inclusions to these parameters is referred to
Krugers strict criteria (requires the use of a stage
micrometer or morphometer;not routinely used but
WHO recommended)
Normal values depend on the evaluation method
used and vary from greater than 30% normal forms
when using routine criteria to greater than 14%
normal forms when using strict criteria
Calculating round cells
Differentiation and enumeration of round
cells(immature sperm and leukocytes) can also be
made during the morphology examination.
Peroxidase-positive granulocytes=predominant form
of leukocyte in semen and can be further
differentiated from spermatogenic cells and
lymphocytes using a peroxidase stain
1. count the no. of spermatids or leukocytes seen
with 100 mature sperm
2. calculate the amount per ml using this formula:
N xS
C=
100
N=# of spermatids or neutrophils per 100 mature
sperm
S=sperm concentration in millions/ml
N=<1.0 million/ml
>1.omillion/ml=inflammatory condition associated
with infection and poor sperm quality and may
impair sperm motility and DNA integrity
ADDITIONAL TESTING
Sperm vitality
Decreased sperm vitality=normal sperm
concentration;decreased motility
Eosin-nigrosin stain=stain used to count no. of dead cells in
100 sperm using a bright-field or phase-contrast
microscope
Living cells=remain bluish white
Dead cells= stain red against the purple background
N=>50% living cells
defective flagellum=large vitality but immobile
epididymal pathology=high no. of immotile and nonviable
cells
Seminal fluid fructose
Low sperm concentration=lack of the support medium
produced in the seminal vesicles;can be indicated by a low
to absent fructose level
Low fructose level=caused by the abnormalitites of the
seminal vesicles;bilateral congenial absence of the vas
deferens;obstruction of the ejaculatory duct;partial
retrograde ejaculation;androgen deficiency
Resorcinol test=produces an orange/orange-red color when
fructose is present
N=13umol/ejaculate
can be determined using spectrophotometric methods
fructose level testing should be done within 2 hrs. after
collection or frozen to prevent fructolysis
Antisperm antibodies
can be present in both men and women but male antisperm
antibodies are more frequently encountered
may be detected in semen, cervical mucosa or serum and
are considered a possible cause of infertility
blood-testes barrier=separates sperm from the male
immune system
barrier disruption=caused by
surgery(vasovasostomy/vasectomy reversal), trauma,
infection
antigen in the sperm= produces an immune response that
damages the sperm
damaged sperm= causes the production of antibodies in the
female partner
Clumps of sperm and decrease motility=presence of male
antibodies
sperm-agglutinating antibodies=cause sperm to stick to
each other in a head-to-head, head-to-tail, or tail-to-tail
pattern
agglutination is graded as few, moderate or many under
microscopic exam
female antisperm antibodies= normal semen analysis but
with continued infertility
presence of female antisperm antibodies may be
demonstrated by mixing the cervical mucosa or serum with
semen and observe the agglutination
Immunoassay kits are available for both semen and serum
testing
two(2) frequently used tests:
mixed agglutination reaction (MAR)- screening
procedure; detects the presence of IgG
 semen incubated with IgG antihuman
globulin(AHG) and a suspension of latex particles
or RBCs coated with IgG
result: visible clumps of sperm and particles or
cells(the bivalent AHG binds to both the antibody on the
sperm and the antibody on the latex particles or RBCs)

N=<10% of the motile sperm attached to the particles

immunobead test- more specific procedure ;can be
used to detect presence of IgG,IgM and IgA and
demonstrates what area of the sperm the
autoantibodies are affecting
sperm are mixed with polyacrylamide beads
coated with either anti-IgG, anti-IgM or anti-
IgA
results:(beads attached to sperm at particular areas)
head-directed Ab=interfere with penetration into
the cervical mucosa or ovum

tail-directed Ab= affect movement through the

cervical mucosa

Reporting: IgM tail antibodies, IgG head antibodies,

etc

N=<50% of sperm attached to beads