SEMINAL FLUID.

MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

SEMINAL FLUID 3) Seminal Vesicles (60-70%)

- Produces a viscous, yellowish
PHYSIOLOGY secretion rich in fructose, vitamin C,
- Sperm is the male gamete (male sex prostaglandin, and other substances,
cell) which has the capacity to fertilize which nourish and activate the sperm
an egg passing through the tract
- Sperm are produced in the
seminiferous tubes of the testes 4) Testis & Epididymis (5%)
- Germ cells for the production of - Spermatozoa are produced in the
spermatozoa testis under the influence of
- Specialized Sertoli cells provide testosterone, and then the epididymis
support and nutrients for the germ provides a temporary storage site for
cells as they undergo mitosis and the immature sperm that enter it from
meiosis (spermatogenesis) testis
- When spermatogenesis is complete,
the immature sperm (non-motile) enter
the epididymis
- In the epididymis, the sperm mature
and develop flagella
- The entire process takes
approximately 90 days
- They remain stored in the epididymis
until ejaculation
- The ejaculatory ducts receive both the
sperm from the ductus deferens and
fluid from the seminal vesicles FORMATION OF SPERM CELL

FLUID FRACTIONS Spermatogenesis is a cascade of cell

divisions:
1) Bulbourethral Glands (5%)
- are very small mucus secreting glands - Mitosis: spermatogonia to primary
- Produces alkaline mucus that spermatocytes
neutralizes the acidity of fluid coming - First meiotic division: secondary
from prostate spermatocytes
- Second meiotic division: haploid
2) Prostate Glands (20-30%) spermatids
- Produces acidic fluid - This process takes 70 ± 4 days in
- the secretion contains acid human
phosphatase and proteolytic enzymes - Spermiogenesis: differentiation of the
that act on the fluid from the seminal round spermatid into a spermatozoon
vesicles, resulting in the coagulation
and liquefaction of the semen

1
Psalm 56:3 When I am afraid, I put my trust in you.
 Transport.
SPERM TRANSPORT AND SEMINAL
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

PLASMA
- “Testicular sperm” need to undergo
more maturation steps before they are
ready to fertilize
- Transported from the testes to the
epididymis, where they mature, and
acquire the ability to swim
- Then moved to the vas deferens, for
storage
- At ejaculation, the sperm are
transported out of the vas and mix with
accessory gland secretions:
- prostatic fluid (pH slightly alkaline to
neutral; contains citric acid and zinc).
- seminal vesicle fluid (pH strongly
alkaline; contains fructose).
SUMMARY OF FUNCTION
PURPOSE OF TEST
- Seminiferous Tubules of Testes –
Spermatogenesis - Investigation of fertility
- Epididymis – Sperm maturation - Identify treatment options
- Ductus Deferens – Propel sperm to ◼ Surgical treatment
ejaculatory ducts ◼ Medical treatment
- Seminal Vesicles – Provide nutrients ◼ Assisted conception treatment
for sperm and fluid ◼ Determine the suitability of semen
- Prostate Gland – Provides enzymes for artificial insemination
and proteins for coagulation and
SAMPLE COLLECTION
liquefaction
- Bulbourethral Gland – Add alkaline - Specimen should be collected into pre-
mucus to neutralize prostatic acid and warmed (21oC), sterile, non-toxic,
vaginal acidity wide-mouth container, after abstaining
from sexual activity for 2-3 days to not
SPERMATOZOON
longer than 5 days
- is about 70 µm long - Specimens collected following
- The nucleus is in the head – contains prolonged abstinence tend to have
the 23 chromosomes higher volumes and decreased motility
- Head: binds to the egg at fertilization - When performing fertility testing, two
- Mid-piece: where energy for motility is or three samples are usually tested at
generated 2-week intervals, with two abnormal
- Tail: for motility; the beat is initiated samples considered significant
just behind the midpiece, then - The specimen should be delivered to
propagated along the tail the laboratory within 1 hour of
collection and the laboratory personnel
2
Psalm 56:3 When I am afraid, I put my trust in you.
 Methods.
must record the time of specimen -
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

The time interval from collection to

collection and specimen receipt analysis
- The sample must be kept at 37oC until
MACROSCOPIC EXAMINATION
analysis, which begins ideally within 30
min, but absolutely within 60 min, of Macroscopic evaluations which give
ejaculation useful diagnostic information about the
sample:
METHODS OF COLLECTION
- Appearance
1) Masturbation - the method of choice
- Odor
for all seminal fluid tests
- Liquefaction
2) By condom: not recommended for
- Volume
fertility testing because condoms may
- Viscosity
contain spermicidal agents
- pH
3) By coitus interrupts - withdrawal
method
4) TESE: Testicular sperm extraction
(Open Testicular Biopsy) – a highly
invasive, open surgical procedure
performed under general anaesthetic.
The scrotum and testes are cut open,
before testicular tissues are cut away
and examined for sperm, which, if
present can be extracted.

Color

- pearly white, opaque gray, grayish

white, yellow
LABELLING OF SAMPLE
- Yellow – prolonged abstinence (flavin
- Patient’s name causes yellow color)
- Log number - Red – bleeding
- Date and time - Increased white turbidity – due to
- Laboratory request form increase WBC
The following should be recorded on the Odor
laboratory analysis
- chlorox-bleach odor
- The period of abstinence (in days)
Specific gravity
- If sample collection was complete or
incomplete - 1.033
3
Psalm 56:3 When I am afraid, I put my trust in you.
 Liquefaction.
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

LIQUEFACTION - +2
- +3 = very viscous
- A fresh semen specimen is clotted and
should liquefy within 30 to 60 minutes MICROSCOPIC EXAMINATION
after collection; therefore, recording
- Motility
the time of collection is essential for
- Sperm aggregation (random
evaluation of semen liquefaction
clumping): “some” is normal, but large
- Analysis of the specimen cannot begin
clumps (each with hundreds of sperm)
until after liquefaction has occurred
is abnormal
- If after 2 hours the specimen has not
- Spermagglutination (between
liquefied, proteolytic enzymes such as
specific sites): could suggest the
alpha-chymotrypsin may be added to
presence of
allow the rest of the analysis to be
antisperm antibodies
performed
- Epithelial cells: usually present in
Volume small numbers
- Erythrocytes: should not be present
- normal is 2-5 mL - Bacteria and protozoa: presence
- Measured using disposable volumetric indicates infection
pipette
- WHO criteria specify that any volume NORMAL SEMEN ANALYSIS
greater than 2.0 mL is normal
- Low volume may indicate partial or 2mL or more
(usually 2-4
complete blockage of the seminal Semen volume
milliliters per
vesicles, or that the man was born ejaculation)
without seminal vesicles
Semen pH Semen pH of 7.2-8.0
pH
Liquefaction time 30 – 60 minutes
- Normal pH of semen is alkaline (7.0 to
8.0) 40 million
- Increased pH is indicative of infection spermatozoa per
ejaculate or more
within the reproductive tract Sperm count
(sperm
- A decreased pH is associated with
concentration x
increased prostatic fluid volume)
Viscosity
More than 30% of
- Estimated by aspirating semen into the sperm have
Sperm morphology
measuring pipette and allowing the normal shape and
semen to drop by gravity and will not structure.
appear clumped. Observe the length of More than 50% of
the thread. the sperm show
- Creamy, Gelatinous, Viscous progressive
Sperm motility movement or 25%
Grading
or more with rapid
- 0 = non viscous progressive
- +1 movement.
4
Psalm 56:3 When I am afraid, I put my trust in you.
 Microscopic.
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

SPERM CONCENTRATION
75% or more live,
Vitality
i.e., excluding dye HEMOCYTOMETER

Fewer than 1 million The number of squares assessed depends

White blood cells
WBCs/mL on the number of sperm counted in the first
large square:

MICROSCOPIC EXAMINATION - If < 10 counted, the whole grid is

assessed
- Normal values for sperm concentration - If 10-40 counted, 10 squares are
are commonly listed as 20-160 million assessed
sperm per milliliter, with - If > 40 counted, 5 squares are
concentrations between 10 and 20 assessed
million per milliliter considered
borderline
- Total sperm count for the ejaculate can
be calculated by multiplying the
sperm concentration by the
specimen volume
- Total sperm counts >40 million per
ejaculate are considered normal (20
million per milliliter 2 mL)
METHODS OF MEASURING SPERM CONC
HEMOCYTOMETER

- Sperm counted by making 1:20 dilution

in WBC pipette or by automatic pipette
(which is more accurate) with a
solution containing sodium bicarbonate
(5g) and formalin (1mL) (immobilize & Large central square
preserve the spermatozoa)
- tap water (100 mL) will suffice as a This square is ruled into 25 small squares,
diluent. each of which is further divided into 16

CALCULATIONS

- If the counts of the two chambers are

not within 5% of their average discard,
remix the sample, and set it up again
- If the two counts are in agreement,
then the sum of the two counts is
divided by the correction factor:
◼ If 2 × 25 squares counted, divide
their sum by 10
◼ If 2 × 10 squares counter, divide
their sum by 4
5
Psalm 56:3 When I am afraid, I put my trust in you.
 Interpretation.
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

◼ If 2 × 5 squares counted, divide cells per mL, but still have bad quality
their sum by 2 because only few of them are motile
- This gives the sperm concentration in - The other way around, a man can
millions per mL have a sperm count far less than 20
- Sperm count = concentration × total million sperm cells per mL and still
volume have good motility, if more than 60%
of those observed sperm cells show
Using a 1:20 dilution and four large WBC’s
good forward movement
squares counted:
SPERM MOTILITY GRADING (WHO)
- Sperm concentration/mL = No of
sperms counted x 50,000 1) Grade 4 – Rapid, straight-line motility.
Denoted as Motility A
Using a 1:20 dilution and five small RBC’s
2) Grade 3 – Slower speed, some lateral
squares counted:
movement. Denoted as Motility B
- Sperm concentration/mL = No of 3) Grade 2 – Slow forward progression,
sperms counted x 1,000,000 noticeable lateral movement. Denoted
as Motility B
INTERPRETATION 4) Grade 1 – No forward progression.
WHO Reference values: Denoted as Motility C
5) Grade 0 – No movement. Denoted as
- Sperm concentration is > 20×106 Motility D
sperm/mL
- Counts of less than 20 million per ALTERNATIVE SPERM MOTILITY
milliliter (<20 million/ml) are GRADING CRITERIA
considered sub-fertile 1) Progressive Motility (PM) – Sperm
- If a man has a sperm concentration moving linearly or in a large circle
<5×106 sperm/mL, WHO recommends 2) Nonprogressive Motility (NP) –
assessment for numerical and Sperm moving with an absence of
structural abnormalities of sex progression
chromosomes 3) Immotility (IM) – No movement
DIRECT SMEAR OR WET PREPARATION MOTILITY DETERMINATION
- Place 10µl of thoroughly mixed, 1) Hanging Drop Method
liquefied semen on a clean glass slide - 1st exam – 100% motile
- Lightly apply glass cover slip - 2nd exam after 1 hour – 50% motile
- Visualize sperm in a specimen of - 3 exam after 12 hour – complete
semen under HPF cessation
MOTILITY
2) Eosin – determines living and dead
- WHO reference value for motility is cells
50% and must be measured within 60 - 1:1 semen-eosin
minutes of collection - mix, then make a smear
- Man can have a total number of sperm - Dead cell – red (eosin is absorbed)
far over the limit of 20 million sperm - Living cell – colorless (eosin not
absorbed)
6
Psalm 56:3 When I am afraid, I put my trust in you.
 Morphology.
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

SPERM MORPHOLOGY
- Morphology is even more important
than motility and concentration
- Because of the small size of the
human sperm head, must use an air-
dried smear which has been stained
- Prepared samples are assessed using
a 100× oil-immersion objective under
bright field optics
- Stains – Wright, Giemsa, 1) Triple head sperm
Papanicolaou (stain of choice) 2) Acrosome reacted sperm
- WHO recommends that 200 3) Sperm with no acrosome
spermatozoa are counted per sample 4) Sperm with a tapering head and
- Fields for counting must be selected at swollen mid-piece
random
- Abnormal head – POOR OVUM
PENETRATION
- Abnormal tail – POOR MOTILITY
- Examine in 20 HPF
- Routine criteria: >50% normal
- KRUGER’S CRITERIA – measures
size, head, neck, tail with the use of
micrometer
◼ >30% should have normal
morphology
ABNORMAL MORPHOLOGY

SPERM VITALITY
- Usually performed using a vital stain,
such as eosin Y, with a counterstain
(nigrosin) to differentiate live
(unstained) and dead (stained red)
spermatozoa (BLOOM’S TEST)
- Normal: 75% alive

7
Psalm 56:3 When I am afraid, I put my trust in you.
 Agglutinate. MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

AGGLUTINATION than 20 million/mL. Sperm count 5-10

sperm/HPF.
- Agglutination means that motile
spermatozoa stick to each other, head
4) Severe Oligozoospermia: Sperm
to head, mid-piece to mid-piece, tail to
count 1-2 sperm/HPF.
tail, or mixed, e.g. mid-piece to tail
- The adherence of either immotile or
motile spermatozoa to mucus threads,
5) Polyzoospermia: denotes an
to cells other than spermatozoa, or to
increased number of spermatozoa in
debris is not considered agglutination
semen and is usually refers to a sperm
and should not be recorded as such
concentration in excess of 350
- The presence of agglutination is
million/mL
suggestive of an immunological factor
of infertility
6) Asthenozoospermia: refers to a man
OTHER CELLS IN SEMEN who produces a greater proportion of
sperm which are immotile or have
- Leukocytes: normally 1-4/HPF, reduced motility, compared to the
increase number (leukocytospermia) - WHO reference values
reproductive tract infection
- Epithelial cells: normally 1-2/HPF
- Spermatocytes: (Immature germ 7) Teratozoospermia: sperm carry more
cells) 1-2/HPF morphological defects than usual
- Erythrocytes: normally 1-2/HPF,
increased number --reproductive tract TZI
infection or damage to a small capillary
- Teratozoospermic Index is an
during sample production
expression of the average number of
Note: bacteria and protozoan (Trichomonas abnormalities per abnormal sperm
vaginalis) are uncommon in human semen, - Each sperm cell is assessed for
but their presence is indicative of possible abnormality in the head, neck/mid-
male reproductive tract infection and should piece, or tail, and for a cytoplasmic
be reported to the referring doctor for further droplet
evaluation - “Normal” - does not have any of
these abnormalities
TERMS - “Abnormal” - does have an
1) Aspermia: absence of semen abnormality; each abnormality is
scored. If a cell has an abnormal head
2) Azoospermia: total absence of and tail, it is counted as 1 cell, and 2
spermatozoa in semen (After abnormalities
centrifuge sperm count is zero/HPF). - TZI = (total # abnormalities) / (total #
sperm)
- TZI > 1.80 -- poor sperm fertilizing
3) Oligozoospermia: reduced number of ability
spermatozoa in semen and used to
describe a sperm concentration of less

8
Psalm 56:3 When I am afraid, I put my trust in you.
 Infertility.
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

INFERTILITY 7) Sexually transmitted infections (STIs)

8) Health problems that cause hormonal
- Infertility means not being able to get
changes, such as polycystic ovarian
pregnant after one year of trying. Or,
syndrome and primary ovarian
women who can get pregnant but are
insufficiency
unable to stay pregnant may also be
infertile. ASSISTED REPRODUCTIVE
TECHNOLOGY
Pregnancy - is the result of a process that
has many steps - all fertility treatments in which both
eggs and sperm are handled
1) A woman must release an egg from
- involve surgically removing eggs from
one of her ovaries (ovulation)
a woman’s ovaries, combining them
2) The egg must go through Fallopian
with sperm in the laboratory, and
tube toward the uterus (womb)
returning them to the woman’s body
3) A man's sperm must join with
- procedures in which a woman takes
(fertilize) the egg along the way
medicine only to stimulate egg
4) The fertilized egg must attach to
production without the intention of
the inside of the uterus
having eggs retrieved
(implantation)
5) Infertility can happen if there are TYPES
problems with any of these steps
1) In vitro fertilization (IVF) means
WHAT INCREASES MAN’S RISK OF fertilization outside of the body. IVF is
INFERTILITY the most effective ART. It is often used
when a woman's Fallopian tubes are
1) High Temperature of Testicles
blocked or when a man produces too
(Testes). Sperm are produced in the
few sperm.
testes which are in the scrotum
2) Smoking. Reduces optimum sperm
2) Zygote intrafallopian transfer (ZIFT):
production
is similar to IVF. Fertilization occurs in
3) Alcohol. Equivalent eight pints of
the laboratory. Then the very young
normal strength beer or sixteen small
embryo is transferred to the Fallopian
glasses of wine, may interfere with
tube instead of the uterus.
optimum fertility.
4) Medicines and Drugs. Some drugs
3) Gamete intrafallopian transfer
interfere with sperm production:
(GIFT) involves transferring eggs and
tetracyclines, colchicine, allopurinol
sperm into the woman's Fallopian
WHAT INCREASES WOMAN’S RISK OF tube. So fertilization occurs in the
INFERTILITY woman's body.

1) Age
2) Stress 4) Intracytoplasmic sperm injection
3) Poor diet (ICSI) is often used for couples in
4) Being overweight or underweight which there are serious problems with
5) Smoking the sperm. Sometimes it is also used
6) Excess alcohol use for older couples or for those with
9
Psalm 56:3 When I am afraid, I put my trust in you.
Rape.
MLSAUBFC
ANALYSIS
OF URINE
AND BODY
FLUIDS
[LEC]

failed IVF attempts. In ICSI, a single

sperm is injected into a mature egg.
Then the embryo is transferred to the
uterus or Fallopian tube.
IMPORTANCE IN MEDICO-LEGAL (FOR
ALLEGED RAPE CASE)
- Microscopic Examination – for the
presence of spermatozoa
◼ If specimen is fresh – get a piece
of cloth, place in centrifuge with
NSS, centrifuge, examine the
sediments
- Fluorescence test – test for choline in
semen
- ACP – most widely used exam
- Barbiero’s Test – test for spermine

10
Psalm 56:3 When I am afraid, I put my trust in you.