Introduction to Semen Analysis

 Seminal fluid is a composite solution formed by:

 The testes (5% of the entire volume) as well as the accessory male reproductive organs such as

o the seminal vesicles (60% of the entire volume),

o the prostate gland (20% of the entire volume)

 The epididymis, vasa differentia, bulbourethral glands (Cowper’s glands), and urethral glands

o 10 – 15% of the total volume

 It consist of sperm cells suspended in seminal plasma.

 Plasma provide the nutritive medium of proper osmolity and volume as well as contribute to the
fertilization process by activating the motility of the sperm cells to go through the end cervical mucus.

Importance

• To investigate the causes of infertility in marriage

 The chief points to be observed are the number and the motility of the spermatozoa)

• To check the effectiveness of vasectomy

• To determine if a male can be a father

• Support or disapprove denial paternity

• For medico-legal

Physiology

Semen:

• Thick, viscous fluid secreted by male and their ejaculation during the height of sexual
intercourse.

Examination of seminal fluid is usually performed as a part of comprehensive infertility investigation

involving both partners of barren marriage.

Methods of Collection

Self-production or Masturbation  Best method of collection because there is no use of

any lubricant which may cause contamination and of course the entire volume can be collected.
Condom Collection  Amount is reduced and contaminated

 Rubber tubing is used for prophylactic purposes.

 If to be used for collection, it should be washed first and dried before using. This is to eliminate
the spermicidal and bactericidal powder/substances in the condom.

Interrupted Coitus or Coitus Interruptus  Interrupt sexual contact

 Reduced amount with contamination

Aspiration from Vaginal Vault  Erroneous

Semen Production

Testes (spermatozoa) • Volume: 5%

• Contain the seminiferous tubules

• Seminiferous tubules of testes Spermatogenesis.

• 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

Epididymis • Sperm maturation and develop flagella.

• They remain stored (Storage) in the epididymis until ejaculation

Ductus deferens (vas deferens) • Propel sperm to ejaculatory ducts.

Seminal vesicles • Volume: 60 – 70% produce the majority of the fluid present in semen

• Provide nutrients for sperm and fluid.

• Spermatozoa metabolize the fructose for the energy needed for the flagella to propel them
through the female reproductive tract. In the absence of fructose, sperm do not display motility in the
semen analysis

Bulbourethral glands (Cowper’s glands) • Volume: 5%

• Located: below the prostate

• Add alkaline mucus to neutralize prostatic acid and vaginal acidity.

Prostate gland • Volume: 20 – 30%

• Located: below the bladder, surrounds the upper urethra

• Provide enzymes and proteins for coagulation and liquefaction.

• Aids in propelling the sperm through the urethra by contractions during ejaculation.
Fluids

FLUIDS from SEMINAL VESICLES • Fructose

• Citric acid

• Potassium

• Ascorbic acid

• Phosphorylcholine

• Ergothionine

 It is the fluid that contains a high concentration of fructose.

FLUIDS from PROSTATE GLANDS• Citric acid

• Acid phosphatase

• Proteolytic enzymes

 An enzyme chiefly responsible for the coagulation & liquefaction of semen

Macroscopic Examination: Semen Analysis

Volume

• Should follow a weak sexual abstinence for a reliable result

• Varies from a few drops to 10 ml per ejaculation

• Normal value

 1.5 – 5 ml per ejaculation

 With an average of 3.5 ml per ejaculation

• Less than 1.5 mL

 Abnormal

• 15 mL per volume per ejaculation

 Consider as Normal

Volume:

 extended abstinence.

Volume
 associated with infertility and may indicate improper functioning of one of the semen-producing
organs, primarily the seminal vesicles.

• Volume is affected by:

 Psychological – attraction, response of women.

 Physiological – abnormalities in genitalia.

Color

Gray-white/ Pearly white • Appears translucent

• Slightly yellow tinge due to flavin content.

Red coloration • Presence of red blood cells (RBCs) in sperm.

Yellow coloration • Caused by urine contamination, prolonged abstinence, and medications.

Odor

• Musty

• Distinct

• Spermatic

• Clorox-like

• Acrid

Viscosity

• The consistency of the fluid and may be related to specimen liquefaction.

• The normal semen specimen should be easily drawn into a pipette and form droplets that do not
appear clumped or stringy when discharged from the pipette.

• Incompletely liquefied specimens = clumped & highly viscous.

• Normal droplets form a thin thread when released from the pipette.

• Highly viscous

 Droplets with threads longer that 2 cm.

• Ratings of 0 (watery) to 4 (gel-like) can be assigned to the viscosity report.

• Viscosity can also be reported as low, normal, and high.

• Affect sperm motility

• Increased viscosity and incomplete liquefaction.

Reaction

• Alkaline, range of 7.2 to 8.0.

• Average of 7.8 pH

 Increase pH

o Infection within the reproductive tract.

 Decrease pH

o associated with  prostatic fluid

Specific Gravity

• 1.033 Specific gravity

Liquefaction

• A fresh semen specimen is clotted

• Should liquefy within 30 to 60 minutes after collection

• Analysis of the specimen cannot begin until after liquefaction has occurred.

• If after 2 hours the specimen has not liquified, proteolytic enzymes such as alpha-chymotrypsin
may be added to allow the rest of the analysis to be performed.

• Failure of liquefaction to occur may be caused by a deficiency in prostatic enzymes and should
be reported.

Sperm Concentration

• Normal > 20 million/Ml

• Borderline = bet. 10 and 20 million per milliliter

• Performed using the Neubauer counting chamber.

Microscopic Examination: Semen Analysis

Motility

Motility is necessary for the spermatozoa in order to penetrate the cervical mucus and subsequently
migrate to fertilize the ovum in the fallopian tube.

Procedure:

a) Place a drop of semen on a clean slide.

b) Cover with a cover glass and seal the rim with petroleum jelly or cover with cover glass after
ringed with Vaseline.

c) Under low illumination, examine the preparation for the motility.

After examination, the preparation is kept in darkness for 6-8 hours and the motility is rechecked or
reneated.

Normally 2 hours are vigorously motile and show a progressive activity, in 6-8 hours, 25-40% are still
motile, and in 12 hours there is complete cessation of motility.

Normal

• Minimum motility of 50% with a rating of 2.0 after 1hr.

• Grading can be done using a scale of 0 to 4, with 4 indicating rapid, straight-line movement and
0 indicating no movement.

Interpretation states:

• Within 1 hour, 50% or more sperm should be motile in categories a, b, and c, or 25% or more
should show progressive motility (a and b).

Additional Notes:

 High Motile

 Low Motile

- Delikado sa acidic environment.

 non-Motile
 Fructose, one key component for the motility.

 70-80% normal value for motility.

Instrumentation

Computer-Assisted Semen Analysis (CASA) • Provides objective determination of both sperm

velocity and trajectory (direction of motion).

• Specialize in andrology and perform a high volume of semen analysis.

Methods: Bloom’s Method

BLOOM'S METHOD:

a) Place a drop of semen on a clean slide.

b) Add 2 drops of 5% eosin (twice the amount of semen).

c) Add 4 drops of nitrogen and mix.

d) Make a thin even smear and fix with heat.

e) Examine under a microscope.

Originally dead sperm cells will take up the stain, so it will appear RED;

While those living will resist the stain, so it will be unstained against the BLACK background.

Sperm Motility Grading

GRADE WHO 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 C No forward progression

1 D No movement
Sperm Morphology

• The presence of sperm that are morphologically incapable of fertilization also results in
infertility.

• Sperm morphology is evaluated with respect to the structure of the head, neckpiece, midpiece,
and tail.

Abnormalities

Abnormal Head

• Associated with poor ovum penetration.

Abnormal Neck, Middle piece Tail

• Affect motility.

Normal Sperm

• Oval-shaped head approximately 5m long & 3m wide & a long

• Flagellar tail approximately 45 m long.

Acrosomal Cap • Enzyme-containing located at the tip of the head.

• Encompass approximately half of the head and covers approximately two thirds (2/3) of the
sperm nucleus.

Neckpiece • Attaches the head to the tail and the midpiece.

Midpiece • Thickest part of the tail because it is surrounded by a mitochondrial sheath that
produces the energy required by the tail for motility.

Stained slide under oil immersion.

Stained used:

1. Wright’s stain

2. Giemsa stain, or

3. Papanicolaou stain
 Air-dried slides are stable for 24 hrs.

 At least 200 sperm should be evaluated and the percentage of abnormal sperm reported.

 Long Neckpiece = cause the sperm head to bend backward.

Kruger’s strict criteria

 Requires the use of a stage micrometer or morphometry.

 Not routinely performed in the clinical laboratory but is recommended by the WHO.

 An integral part of assisted reproduction evaluations.

 >14% normal forms (strict criteria)

 >30% normal forms (routine criteria)

Method A: Williams-McGugan-Carpenter

The smear is prepared similar with a blood smear, dried in the air, fixed with moderate heat, and treated
with 0.5% chlorazene solution for 2-4 mins. to remove the mucus film that overlies the spermatozoa.

Wash slides gently with distilled water and then with 95% alcohol. Stain as follows:

Method I - Crystal violet and rose Bengal:

1. Cover the film with 0.25% aqueous solution of crystal gentian violet for 2-3 minutes.

2. Wash with water.

3. Decolorize with 95% alcohol for ½ to 2 mins.

4. Wash with distilled water.

5. Counterstain with 1% rose beugal (aqueous) for 8 seconds.

6. Wash in water and examine under microscope.

 If the spermatozoa have not taken up sufficient rose Bengal to impart a distinct reddish color to the
ancrosome and the nuclear membrane, repeat the staining interval with rose Bengal until this is
accomplished. Care should be taken not to overstain with rose Bengal, if overstained the contrast
between the nucleus and the acrosome will be last.

 Normally, 80% have a normal morphological appearance and 20% having an abnormal appearance.
Additional Testing for Abnormal Semen Analysis

Abnormal Result Possible

Abnormalities Test

Decreased motility with normal count Viability Eosin-nigrosin stain

Decreased count Lack of seminal

vesicle support

medium Fructose level

Decreased motility

with clumping Male antisperm

antibodies Mixed agglutination

reaction and

immunobead tests Sperm agglutination

with male serum

Normal analysis with

continued infertility Female antisperm

antibodies Sperm agglutination

with female serum

Method B: Meaker Method

1) Make a smear and fix with heat

2) Wash with 0.5% chloral-zene to remove mucus

3) Wash with water

4) Wash with 95% alcohol and dry

5) Stain with carbolfuchsin and bluish eosin (carbolfuchsin 2 parts, filtered bluish eosin 1 part,
alcohol 1 part)

6) Wash with water

7) Stain with methylene blue (methylene blue I part and water 4 parts)
8) Wash with water, dry and examine under microscope.

Method C: Holbert’s Method (Similar to Williams)

1) Make a thin smear and shake until dry

2) Flame gently and flood it with 0.5% chloral-zene for 2 minutes

3) Wash gently with running water

4) Flood with 95% alcohol and let stand for 1 minute

5) Tip off excess alcohol and allow to dry

6) Stain with 0.5% aqueous gentian violet for 3 minutes

7) Rinse with water and rinse with 95% alcohol, then with water

8) Counterstain with 1% aqueous rose Bengal for 1 minute

9) Wash with water and allow to dry.

Seminal Fluid Fructose

1. FRUCTOSE 173.3% (Sweetest sugar)

2. SUCROSE 100%

3. GLUCOSE 74%

4. MALTOSE 32%

5. LACTOSE 16%

 Low sperm concentration may be caused by lack of the support medium produced in the
seminal vesicles. This can be indicated by a low to absent fructose level in the semen.

 Specimens can be screened for the presence of fructose using the resorcinol test that produces
an orange color when fructose is present.

 Specimens for fructose levels should be tested within 2 hours or frozen to prevent FRUCTOLYSIS.

Anti-sperm Antibodies

• Anti-sperm antibodies can be present in both men and women.

• They may be detected in:

 (To determine the presence of Anti-sperm antibodies; and are considered a possible cause of
infertility)
 Semen

 Cervical mucosa

 Serum

 Seminal fluid

 Under normal conditions, the blood-testes barrier separates sperm from the male immune
system. When this barrier is disrupted, as can occur following surgery, vasectomy reversal
(vasovasostomy), trauma, and infection, the antigens on the sperm produce an immune response that
damages the sperm. The damaged sperm may cause the production of antibodies in the female partner.

The presence of antibodies:

• MALE subject

 Can be suspected when clumps of sperm are observed during a routine semen analysis.

• FEMALE subject

 Results in a normal semen analysis accompanied by continued infertility.

• WOMEN

 May be demonstrated by mixing the semen with the female cervical mucosa or serum and
observing for agglutination.

TESTS for Anti-sperm antibodies:

A. Mixed Agglutination Reaction (MAR) Test

 Screening procedure used primarily to detect the presence of immunoglobulin G (IgG)

antibodies.

B. Immunobead Test

 Used to detect the presence of IgG, IgM, & IgA antibodies.

 Demonstrates what area of the sperm (head, neckpiece, midpiece, or tail) the autoantibodies
are affecting.

Sperm Function Tests

Hamster egg

penetration Sperm are incubated with species nonspecific hamster

eggs and penetration is observed microscopically

Cervical mucus
penetration Observation of sperm penetration ability of partner’s 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