Introduction

• Semen analysis measures the amount of semen a man

produces and determines the number and quality of
sperm in the semen sample
•

• It is usually one of the first tests done to help determine

whether a man has a problem fathering a child
(infertility)
•

• A problem with the semen or sperm affects more than

one-third of the couples who are unable to have
children (infertile)
 Physiology
• Sperm is the male gamete (male sex cell) which has the capacity
to fertilize an egg
• Sperm are produced in the seminiferous tubes of the testes
• Germ cells for the production of spermatozoa
• 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 (non-
motile) enter the epididymis
• In the epididymis, the sperm mature and develop flagella
• 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
 Fluid Fractions
1. Bulbourethral Glands (2-5%)
2.

• are very small mucus secreting glands

• Produces alkaline mucus that neutralizes the acidity
of fluid coming from prostate
3.

4. Prostate Glands (approx 20-30%)

5.

• Produces acidic fluid

• the secretion contains acid phosphatase and
proteolytic enzymes that act on the fluid from the
seminal vesicles, resulting in the coagulation and
liquefaction of the semen
1. Seminal Vesicles (46-80%)
2.

• Produces a viscous, yellowish secretion rich in

fructose, vitamin C, prostaglandin, and other
substances, which nourish and activate the sperm
passing through the tract
3.

4. Testis & Epididymis (5%)

5.

• Spermatozoa are produced in the testis under the

influence of testosterone, and then the epididymis
provides a temporary storage site for the immature
sperm that enter it from testis
 Formation of the Sperm Cell
• Spermatogenesis is a cascade of cell divisions:
•

• Mitosis: spermatogonia to primary spermatocytes

• First meiotic division: secondary spermatocytes
• Second meiotic division: haploid spermatids
• This process takes 70 ± 4 days in human
•

• Spermiogenesis: differentiation of the round spermatid

into a spermatozoon
 Sperm Transport and Seminal 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).
 Spermatozoon
• is about 70 µm long
• The nucleus is in the head –
contains the 23 chromosomes
• Head: binds to the egg at
fertilization
• Mid-piece: where energy for
motility is generated
• Tail: for motility; the beat is
initiated just behind the mid-piece,
then propagated along the tail
 What the Purpose of the Test?
• Investigation of fertility
• Identify treatment options
•

• Surgical treatment
• Medical treatment
• Assisted conception treatment
• Determine the suitability of semen for artificial
insemination
 Sample Collection
•
Specimen should be collected into pre-warmed (21oC), sterile,
non-toxic, wide-mouth container, after abstaining from sexual
activity for 2-3 days to not longer than 5 days
• Specimens collected following prolonged abstinence tend to
have higher volumes and decreased motility
• When performing fertility testing, two or three samples are
usually tested at 2-week intervals, with two abnormal samples
considered significant
• The specimen should be delivered to the laboratory within 1
hour of collection and the laboratory personnel must record the
time of specimen collection and specimen receipt
• The sample must be kept at 37oC until analysis, which begins
ideally within 30 min, but absolutely within 60 min, of
ejaculation
 Methods of Collection
1. Masturbation - the method of choice for all seminal
fluid tests
2. By condom: not recommended for fertility testing
because condoms may contain spermicidal agents
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.
 Labeling of Sample
• Patient’s name
• Log number
• Date and time
• Laboratory request form
•

• The following should be recorded on the laboratory

analysis form:
•

• The period of abstinence (in days)

• If sample collection was complete or incomplete
• The time interval from collection to analysis
 Macroscopic Examination
• Macroscopic evaluations which give useful diagnostic
information about the sample:
•

• Appearance
• Odor
• Liquefaction
• Volume
• Viscosity
• pH
 Liquefaction
• A fresh semen specimen is clotted and should liquefy
within 30 to 60 minutes after collection; therefore,
recording the time of collection is essential for
evaluation of semen liquefaction
•

• Analysis of the specimen cannot begin until after

liquefaction has occurred
•

• If after 2 hours the specimen has not liquefied,

proteolytic enzymes such as alpha-chymotrypsin may
be added to allow the rest of the analysis to be
performed
 Macroscopic Examination
• Volume:
•

• normal is 2-5 mL
• Measured using disposable volumetric pipette
• WHO criteria specify that any volume greater than
2.0 mL is normal
• Low volume may indicate partial or complete
blockage of the seminal vesicles, or that the man
was born without seminal vesicles

volumetric pipette
 Macroscopic Examination
• Viscosity
•

– Estimated by aspirating semen into measuring

pipette and allowing the semen to drop by gravity
and will not appear clumped. Observe the length
of the thread.
– Creamy, Gelatinous, Viscous
Grading:
0 = non viscous
+1
+2
+3 = very viscous
 pH
• Normal pH of semen is alkaline (7.0 to 8.0)
•

• Increased pH is indicative of infection within the

reproductive tract
•

• A decreased pH is associated with increased prostatic

fluid
 Macroscopic Examination
• Color
•

− pearly white, opaque gray, grayish white, yellow

•

Yellow – prolonged abstinence (flavin causes yellow

color)
Red – bleeding
Increased white turbidity – due to increase WBC

• Odor: chlorox-bleach odor

•

• Specific Gravity: 1.033

 Microscopic Examination
• The characteristics assessed are:
•

• Motility
• Sperm aggregation (random clumping): “some” is normal,
but large clumps (each with hundreds of sperm) is abnormal
• Spermagglutination (between specific sites): could suggest
the presence of antisperm antibodies
• Epithelial cells: usually present in small numbers
• Erythrocytes: should not be present
• Bacteria and protozoa: presence indicates infection
Microscopic
Examination
 Microscopic Examination
• Normal values for sperm concentration are commonly
listed as 20-160 million sperm per milliliter, with
concentrations between 10 and 20 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 Concentration

• Hemacytometer
•

• 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 & preserve the spermatozoa), tap water
(100 mL) will suffice as a diluent.
 Sperm Concentration - Hemocytometer

• The number of squares assessed First large square counted

depends on the number of
sperm counted in the first large
square:
•

• If < 10 counted, the whole

grid is assessed
• If 10-40 counted, 10
squares are assessed
• If > 40 counted, 5 squares
are assessed
 Sperm Concentration - 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
• If 2 × 5 squares counted, divide their sum by 2
• This gives the sperm concentration in millions per mL
• Sperm count = concentration × total volume
 Calculations
•

• Using a 1:20 dilution and four large WBC’s squares

counted:
•

• Sperm concentration/mL = No of sperms

counted x 50,000
•

• Using a 1:20 dilution and five small RBC’s squares

counted:
•

• Sperm concentration/mL = No of sperms

counted x 1,000,000
 Sperm Concentration – Interpretation
• WHO Reference values:
•

Sperm concentration is > 20×106 sperm/mL

• Counts of less than 20 million per milliliter (<20
million/ml) are considered sub-fertile
• If a man has a sperm concentration <5×106 sperm/mL,
WHO recommends assessment for numerical and
structural abnormalities of sex chromosomes
 Direct Smear or Wet Preparation
• Place 10µl of thoroughly mixed, liquefied semen on
a clean glass slide
• Lightly apply glass cover slip
• Visualize sperm in a specimen of semen under HPF
 Motility
•
WHO reference value for motility is 50% and must be
measured within 60 minutes of collection
•

•
Man can have a total number of sperm far over the
limit of 20 million sperm cells per mL, but still have
bad quality because only few of them are motile
•

•
The other way around, a man can have a sperm count
far less than 20 million sperm cells per mL and still
have good motility, if more than 60% of those
observed sperm cells show good forward movement
 Grading of Motility
•
•
Grade 4 – Progressive motility
•
•
Swim fast in a straight line. Denoted as Motility A
•
•
Grade 3 – Non-linear motility
•
Move forward but tend to travel in a curved or
crooked motion. Denoted motility B
•

•
•
Grade 2 – Non-progressive motility
•
Do not move forward despite the fact that they
move their tails
•

•
•
Grade 1 – Immotile
•
fail to move at all
 Motility Determination
• Hanging Drop Method
•

- 1st exam – 100% motile

- 2nd exam after 1 hour – 50% motile
- 3 exam after 12 hour – complete cessation
•

•
Eosin – determines living and dead cells
•

- 1:1 semen-eosin
- mix, then make a smear
- Dead cell – red (eosin is absorbed)
- Living cell – colorless (eosin not absorbed)
 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, Papaniculoa (stain of choice)
• WHO recommends that 200 spermatozoa are counted
per sample
• Fields for counting must be selected at 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
1. Triple head
sperm
2. Acrosome
reacted sperm
3. Sperm with
no acrosome
4. Sperm with
a tapering head
and swollen
mid-piece
 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
 Agglutination
• Agglutination means that motile spermatozoa stick to
each other, head to head, mid-piece to mid-piece, tail to
tail, or mixed, e.g. mid-piece to tail

• The adherence of either immotile or motile

spermatozoa to mucus threads, to cells other than
spermatozoa, or to debris is not considered
agglutination and should not be recorded as such
•

• The presence of agglutination is suggestive of an

immunological factor of infertility
 Other Cells in Semen
•
Leukocytes: normally 1-4/HPF, increase number
(leukocytospermia) -- reproductive tract infection
•

•
Epithelial cells: normally 1-2/HPF
•

•
Spermatocytes: (Immature germ cells) 1-2/HPF
•

•
Erythrocytes: normally 1-2/HPF, increased number
--reproductive tract infection or damage to a small
capillary during sample production
•

•
Note: bacteria and protozoan (Trichomonas vaginalis) are
uncommon in human semen but their presence is indicative
of possible male reproductive tract infection and should be
reported to the referring doctor for further evaluation
 Semen Biochemistry
• Acid phosphatase: marker for prostatic function
• Citric acid: can indicate prostatic function – low
levels may indicate dysfunction or a prostatic duct
obstruction
• Zinc: marker for prostatic function – colorimetric
assay (WHO)
• Fructose: marker for seminal vesicle function,
and is a substrate for sperm metabolism –
spectrophotometric assay (WHO)
 Terms
• Aspermia: absence of semen
• Azoospermia: total absence of spermatozoa in semen
(After centrifuge sperm count is zero/HPF).
• Oligozoospermia: reduced number of spermatozoa in
semen and used to describe a sperm concentration of
less than 20 million/mL. Sperm count 5-10 sperm/HPF.
• Severe Oligozoospermia: Sperm count 1-2 sperm/HPF.
• Polyzoospermia: denotes an increased number of
spermatozoa in semen and is usually refers to a sperm
concentration in excess of 350 million/mL
• Asthenozoospermia: refers to a man who produces a
greater proportion of sperm which are immotile or
have reduced motility, compared to the WHO
reference values
• Teratozoospermia: sperm carry more morphological
defects than usual
 TZI
• Teratozoospermic Index is an expression of the
average number of abnormalities per abnormal sperm
• Each sperm cell is assessed for abnormality in the head,
neck/mid-piece, or tail, and for a cytoplasmic droplet
• “Normal” - does not have any of these abnormalities
• “Abnormal” - does have an abnormality; each
abnormality is scored. If a cell has an abnormal head
and tail, it is counted as 1 cell, and 2 abnormalities
• TZI = (total # abnormalities) / (total # sperm)
• TZI > 1.80 -- poor sperm fertilizing ability
infertility
 What is Infertility?
• Infertility means not being able to get pregnant after one year of
trying. Or, women who can get pregnant but are unable to stay
pregnant may also be infertile.
• Pregnancy: is the result of a process that has many steps. To get
pregnant:
•

• A woman must release an egg from one of her ovaries

(ovulation)
• The egg must go through Fallopian tube toward the uterus
(womb)
• A man's sperm must join with (fertilize) the egg along the way
• The fertilized egg must attach to the inside of the uterus
(implantation)
• Infertility can happen if there are problems with any of these
steps
 What Increases a Man's Risk of Infertility?
• High Temperature of Testicles (Testes). Sperm are
produced in the testes which are in the scrotum
• Smoking. Reduces optimum sperm production
• Alcohol. Equivalent eight pints of normal strength
beer or sixteen small glasses of wine, may interfere
with optimum fertility.
• Medicines and Drugs. Some drugs interfere with
sperm production: tetracyclines, colchicine, allopurinol
 What Things Increase a Woman's Risk of
Infertility?
• Age
• Stress
• Poor diet
• Being overweight or underweight
• Smoking
• Excess alcohol use
• Sexually transmitted infections (STIs)
• Health problems that cause hormonal changes, such as
polycystic ovarian syndrome and primary ovarian
insufficiency
Assisted Reproduction Techniques
(ART)
ASSISTED REPRODUCTIVE TECHNOLOGY
•

• all fertility treatments in which both eggs and sperm are

handled
•

• involve surgically removing eggs from a woman’s

ovaries, combining them with sperm in the laboratory,
and returning them to the woman’s body
•

• procedures in which a woman takes medicine only to

stimulate egg production without the intention of having
eggs retrieved
Types of Assisted Reproductive Technology (ART)

1. In vitro fertilization (IVF) means fertilization outside of

the body. IVF is the most effective ART. It is often used
when a woman's Fallopian tubes are blocked or when a
man produces too few sperm. 
2.

3. Zygote intrafallopian transfer (ZIFT): is similar to

IVF. Fertilization occurs in the laboratory. Then the very
young embryo is transferred to the Fallopian tube
instead of the uterus.
1. Gamete intrafallopian transfer (GIFT) involves
transferring eggs and sperm into the woman's
Fallopian tube. So fertilization occurs in the woman's
body.
2.

3. Intracytoplasmic sperm injection (ICSI) is often used

for couples in which there are serious problems with
the sperm. Sometimes it is also used for older couples
or for those with 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.
 Sperm Preparation

• Discontinuous Density Gradient: A technique in

which all sperm in the seminal fluid are separated by
centrifugation
• Swim up: a technique in which the most motile sperm
are selected from the ejaculate. Fresh semen is
covered with a medium and placed in a cylinder which
is laid at a 45oC angle. Motile sperm in the ejaculate
will then swim up to the top of the tube, leaving
immotile sperm and debris in the lower part of the
cylinder.