S E M E N A N A LY S I S -

CASA

Somanatha Sharma. S
II year Resident
Stanley Medical College
 Semen analysis

Plays a key role in evaluation of

men presenting with infertility.

Male factor – sole cause - 20%

infertile couple
• Normal semen is an admixture of spermatozoa suspended in secretions
(SEMINAL PLASMA ) from the glandular tissue of the male genital system.
• The ejaculate can be divided into four fractions:
• 1) PRE – EJACULATORY FRACTION : It is clear secretion of COWPER’S or LITTER’S
GLANDS & contains proteins with moderately viscous consistency, which may
possibly serve to neutralize residues of urine .
• 2)PRELIMNARY FRACTION : This originates from the prostate gland. It gives
SEMEN it’s characteristic ODOUR. It contain enzymes which liquefies the
spermatozoa coagulum.
• 3)MAIN FRACTION : It originates from the SEMINAL VESICLES, TESTES,
EPIDIDYMIS & partially from the prostate gland. The preliminary fraction & the
main fraction contain majority of spermatozoa.
• 4)TERMINAL FRACTION : Is formed by secretions of seminal vesicles & is entirely
gelatinous in consistency ,with large no. of immotile spermatozoa.
 FRACTION OF SEMEN CONTRIBUTED BY VARIOUS GLANDS

1. Urethral glands (2-5%) - small mucus secreting glands.

2. Prostate: 20-30% of the semen volume, acidic fluid produced by the

prostate gland, the secretion contains citrate, zinc, acid phosphatase and
proteolytic enzymes liquefaction of the semen.

3. Seminal vesicles: 46-80 % of the semen volume, alkaline viscous,

yellowish secretion is rich in fructose, vitamin C, prostaglandin, protein
kinase, and other substances, which nourish and activate the sperm.

4. Testis & Epididymis: (5%) spermatozoa.

 What is the purpose of the test?

 Investigation of infertility ( Primary or Secondary)

 Identify treatment options
 Surgical treatment.
 Medical treatment.
 Assisted conception treatment.
 Determine the suitability of semen for ICSI/IVF
 Pre and Post vasectomy – Confirmation.
 Following vasectomy reversal.
 Human sperm cell is about
70
 The head size: 4-5µm
µm long.
Nucleus - contains the 23 chromosomes.
Acrosome
 Mid-piece: 4-5µm
The energy for motility is generated.
 Tail: 55µm
Motility -Propagated along the tail.
 Standard guidelines for the
collection of semen
 There should be 2 to 7 days of sexual abstinence before collection.
 Two separate samples at least 7 days apart should be analyzed.
 The duration of abstinence should be constant
 Masturbation in a clinical setting is the recommended procedure.
 Collection - Private room in the same centre where the semen will be analyzed.
 Pre warmed (21oC), sterile, non-toxic, wide-mouth container.

2 to 7 days 7 days
 PRECAUTIONS
 Pass urine.
 Wash hands with soap and dry.
 Glans and the penis should be cleaned with a wet paper towel (avoid soap).
 Lubricants should be avoided - interfere with motility.
 Collect the entire sample -70% of sperms is in the first part of the ejaculate.

Other methods of collection

Coitus interrupts
Condom collection - Polyurethane - Latex
 Assistance - unable to achieve adequate
erection and ejaculation.
Phosphodiesterase type 5 inhibitors - 30 to 60 min before collection.
Cavernosal and subcutaneous injections of prostaglandins

Vacuum erection devices

Vibratory stimulation - spinal cord injury.

Rectal probe electro - stimulation induces ejaculation by stimulation of the efferent fibers of
the hypo gastric plexus.
 LABELLING OF SAMPLE

 Patient name
 Age
 Clinic or Doctor name

Laboratory analysis form:

 The period of abstinence (in days).
 Date &Time of collection.
 Mode of collection.
 Complete or incomplete.
 The time interval from collection to analysis.
 TIMING OF
ANALYSIS
Semen is placed in a 37° C gently shaking incubator for 30 minutes.

The semen sample should be examined,

Ideally within 30 mins
Absolutely within 1 hour of collection.

Motility decreases significantly after 2

hours
 WHO 2010
Parameter 1992 Lower Reference Limit 2010
Semen volume 2 ml 1.5 ml
Sperm concentration 20 M 15 x 106/ml
Total sperm number 39 x106/ejaculate
Progressive motility >50 % 32 % A
Total motility 40 % A+B
Vitality (live sperms) 58 %
Sperm morphology >15 % 4 %
pH >/=7.2 >/=7.2
Leucocyte <1M <1 x106/ml
MAR/Immunobead test <10 % <50 %
 Terminologies in SA (WHO)
 Normospermia - Normal semen volume
 Aspermia - No semen volume
 Hypospermia - Semen volume < 1.5 ml
 Hyperspermia - Semen volume > 6.0 ml
 Azoospermia - No spermatozoa in semen
 Oligospermia - Sperm concentration <15 M/ml
 Polyzoospermia - High sperm concentration, >200M/ml
 Asthenozoospermia - <40% grade (A&B) or < 32 PR%
 Teratozoospermia - <4% spermatozoa
 Leukospermia - Leukocytes present in semen, >1M/ml
 Hematospermia - Red blood cell present in semen
 Necrozoospermia - “dead” sperm
 OAT =Oligo-astheno-teratozoospermia
 WET SMEAR
PREPARATION
Normally 10 ul semen to 190 ul water = 20x dilution.
In cases of very low sperm count = 4x dilution
In cases of azoospermia = no dilution

 Add 10 ul of mixture to the chamber

 Cover slip
 Wait 2-3 min to settle
 20x magnification
 Sperm density = Sum of 5 squares x 106/ml
The semen analysis characteristics can be classified into two groups.

Macroscopic

Microscopic
 Macroscopic Examination
Volume Normal: 1.5 ml per ejaculation
Low volume (<1ml) reflect a problem
Seminal vesicles and prostate,
Retrograde ejaculation,
Infection or lack of androgen.
pH Normal: =/>7.2
(alkaline)
Acidic pH (<7.0) in a low volume
indicates
Congenital bilateral absence of vas deferens (in which seminal
vesicles are also poorly developed)
Ejaculatory duct obstruction.
 Macroscopic Examination
WHO criteria 2010 Description
Appearance Normal: Whitish to grey opalescent
Yellow (urine, jaundice)
Pink/Reddish/Brown (RBCs)
Liquefaction Normal: 15–30 minutes after collection
>60 min
Lack of prostatic protease, maybe sign of prostatic infection
Viscosity Normal Smooth and watery
Abnormal thick with long threads.
• Semen is ejaculated in liquid state.
• It gets coagulated due to enzyme PROTEIN KINASE from seminal vesicles.
• Absence of coagulation indicates CONGENITAL ABSENCE OF VAS
DEFERENS,SEMINAL VESICLES OR OBSTRUCTION OF THE EJACULATORY
DUCT
• LIQUEFACTION
• AT ROOM TEMPERATURE ,NORMAL SEMEN GETS LIQUEFIED WITHIN 30 MINUTES
AFTER COLLECTION.
• Presence of MUCOUS STREAKS indicate incomplete liquefaction.
• Sometimes the sample may not liquefy
• in this situation a treatment with PLASMIN 0.35 – 0.50 UNITS/ ML or CHYMOTRYPSIN
150 UNITS / ML may be needed to make the sample fit for analysis.
• Incomplete liquefaction is indicative of dysfunctional accessory reproductive organs
like prostate which leads to decreased production of prostatic enzymes.
FRUCTOLY
SIS
• Fructose is main sugar present in seminal plasma & is imp. nutrient for the sperms.
• The quality of semen can be assessed by measuring the rate of utilization of fructose.
• FRUCTOLYTIC INDEX is the amount of fructose used or lactic acid formed by spermatozoa
per hour at 37 deg.
• The semen sample should be well buffered otherwise the fructolysis will stop at certain
stage & result will be erroneous.
• The normal fructose value is 13 mol or more per ejaculate.
• In case of azoospermia caused by congenital absence of vas deferens , low fructose level
may indicate an assoc. dysgenesis of seminal vesicles.
• Fructose determination is also useful in rare cases of ejaculatory duct obstruction.
• There is positive correlation between rate of anaerobic fructolysis & deg. of motility.
 MICROSCOPIC ASSESSMENT
OF SEMEN
 Sperm agglutination
 Count and concentration
 Motility
 Morphology
 Viability
 Non sperm cells
 SPERM
AGGLUTINATION
Wet smear
Sperm form clumps within semen

Sperm-to-non sperm elements (nonspecific agglutination) - accessory gland

infection.
Sperm-to-sperm agglutination (site-specific agglutination) – anti sperm antibodies.

When agglutination is observed - semen cultures and antibody assessment.

 COUNT AND
CONCENTRATION.
Sperm concentration (number of sperm per milliliter)
Sperm count (number of sperm per ejaculate)

Azoospermia (absence of sperm)

Abnormal spermatogenesis, ejaculatory dysfunction,
or obstruction.

Oligospermia (abnormally lower sperm

concentration)

Polyzoospermia (abnormally elevated sperm

concentration) - rare.
May be caused by a long period of abstinence -
associated with sperm of poor quality.
 MOTILITY

Most important predictor of the functional aspect of spermatozoa.

Sperm motility is a reflection of the normal development of the axoneme.

Sperm motility is a reflection of the normal maturation within the
epididymis.

The sperm motility is graded according to the WHO as

follows: A—Rapid forward progress motility;
B—Slow or sluggish progressive motility;
C—Non progressive motility; D—
Immotility.

The cutoff value for normal

32% grade A
motility 40% grade
 Limitation of sperm motility
assessment
The method most commonly employed is the simple estimation of the motility of sperm
on several fields.

 Assessment of this parameter is subjective - potential for technical mistakes.

In-vitro motility of sperm may not reflect the true motility within the
female reproductive tract.
 Causes of
asthenospermia
 Inherent defects of sperm,
 Artifactual - Spermicides, Lubricants, Or Rubber Condoms.
 Prolonged Abstinence Periods,
 Genital Tract Infection,
 Varicocele.
 ASA - peculiar shaking pattern – preventing penetration through cervical
mucus.

> 10% to 15% of clumping of spermatozoa is indicative of antisperm antibodies

 HABITUAL FACTORS AFFECTING SPERM DENSITY / MOTILITY

 High intake of soya – decrease sperm density.

 High consumption of tobacco – decrease sperm density / motility.
 Consumption of cocaine / Marijuana – decrease sperm motility.
 Vaginal lubricants – decrease sperm motility.
 Alcoholism – affects all semen parameters.
MORPHOLOGY
 Viability
When the
motility is
reported as less
than 5% to 10%
To differentiate immotile from dead sperm

 Staining method (commonly used)

 Hypo-osmotic swelling test (HOST) (alternative)

Staining method (commonly used)

Eosin Y followed by counter staining with Nigrosin.
Principle is that viable sperm have intact cell
membranes. Do not take up the dye and will remain
unstained.
 Hypo-osmotic swelling test (HOST) (alternative)

Exposure of the sperm to hypo osmotic fluid.

Principle is that viable sperm have intact cell membranes.
Cause swelling of the cytoplasmic space and curling of the sperm
tail. Nonviable sperm - will not exhibit this effect.

Reproducible and relatively inexpensive test

Helps in selection of viable sperm - IVF or ICSI.
 NON SPERM CELLS
 Leukocytes: normally (1-4/HPF)
Leukocytospermia as levels above 1 × 106 WBC/mL - infection

 Epithelial cells: normally (1-2/HPF)

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

Erythrocytes: (1-2/HPF). Increased number may indicate a reproductive tract

infection or damage to a small capillary during sample production.

Bacteria and protozoan such as Trichomonas vaginalis are uncommon in

human semen but their presence is indicative of possible male reproductive
tract infection
 COMPUTER - ASSISTED SPERM ANALYSIS
Computer-assisted sperm analysis (CASA) is a semiautomated
technique that provides data on
Sperm density, Motility (straightline and curvilinear velocity,
linearity, average path velocity, amplitude of lateral head
displacement, flagellar beat frequency, and hyper activation)

Advantages:
High precision
Quantitative assessment of sperm kinetics.

Disadvantages:
Expensive equipment and still requires the subjective
participation of a technician.

Hence not used for routine semen analysis

Commonly done in high volume andrology
labs.

Emerging use of ICSI - diminished the role of

motility assessment
ISAS (Integrated Semen Analysis System)
SCA (Sperm Class Analyzer)
IVOS (Integrated Visual Optical System )
SQA-V (Sperm Quality Analyzer)
 ISAS (Integrated Semen Analysis System)

 ISAS is a CASA system based on image analysis.

 ISAS analyzes motility and concentration in more than 17 sperm parameters

 ISAS also do DNA fragmentation analysis

 SCA (Sperm Class
Analyzer)
SCA provides fast, accurate and
repeatable results.
 SCA Motility & Concentration
 SCA DNA Fragmentation
 Morphology
 SCA Vitality
 IVOS (Integrated Visual Optical
System )
The IVOS is unique in that it is the only CASA system that
integrates the optical system within the unit, so that an
external microscope is not needed.

 Able to analyze sperm of multiple species (rat)

(Research institutes, IVF clinics, pharmaceutical

companies, reproductive toxicology labs, veterinary and animal
breeding centres)

 A single field - analyzed in just 0.5 second.

 SQA-V (Sperm Quality
Analyzer)
 Fully automated
 SQA-V semen analysis eliminates inter-operator variation.
 Electro-optics, computer algorithms and video microscopy
 Provide a precise and accurate - 75 second

The SQA-V ( 16 clinical

parameters )
 Limitation of semen analysis
Clinical research has shown,

 Normal semen analysis may not reflect the true fertility status of an individual.
 Men with poor sperm parameters can cause spontaneous pregnancies.
 Men with good sperm parameters are still subfertile
 Only 50% of subfertile men have recognizable causes detectable by semen analysis.

Semen analysis is only a surrogate test to measure the man’s fertility potential.
 SPERM FUNCTION ASSESSMENT
 Sperm- mucus interaction assay
 Acrosome reaction testing
 Sperm penetration assay
 SPERM-MUCUS
INTERACTION/POSTCOITAL TEST
Assess cervical environment as a cause of infertility.
Cervical mucus - heterogenous fluid - cyclical changes in consistency

Postcoital test (PCT)

Conducted when the cervical mucus is thin and clear just before
ovulation. Examined 2 to 8 hours after normal intercourse.
Progressively motile sperm > 10 to 20 per HPF is designated as
normal.

Abnormal test - advised to proceed with IUI.

 Inappropriate timing testing / intercourse,
 Anatomic abnormalities,
 Semen or cervical mucus antisperm antibodies,
 Abnormal sperm.
 ACROSOME REACTION
The Acrosome is a membrane-bound organelle covers the anterior 2/3 of the sperm
head.
 Acrosome reaction is an important prerequisite for successful fertilization.
 ZP3
 Involves fusion of acrosomal membrane and plasma membrane.
 Acrosin and Hyaluronidase – required to digest the oocyte cumulus cells and
ZP

Acrosome reaction testing - not widely practiced in laboratories - research interest.

 Profound abnormalities of head morphology

 Unexplained infertility
 SPERM PENETRATION ASSAYS
The sperm penetration assay (SPA) or the hamster egg
penetration assay (HEPT) It address the functional
ability.
Unexplained infertility / IVF failure

Principle - a normal spermatozoa can bind and penetrate the oocyte membrane.

 Incubating zona-free hamster oocytes in sperm droplets for 1 to 2 hours.

 The oocytes are examined microscopically for sperm penetration.
 Penetrations are indicated by swollen sperm heads within the oocyte cytoplasm.
 Normally, 10% to 30% of ova are penetrated (WHO, 1999).

Oligozoospermic and severely teratospermic men – negative testing

Sperm capacitation index (SCI) is a variant of the SPA test, assessing the mean number of
penetrations per ovum. ICSI has been recommended - SCI less than 5 instead of standard IVF
procedures.
 ADVANCED SPERM TESTING

 Antisperm antibody testing

 Electron microscopy
 Sperm DNA damage assay
 Antisperm Antibody Testing
AB Against sperm
IgG, IgA

 Sperm agglutinating,
 Sperm immobilizing,
 Spermotoxic.

Normally the tight Sertoli-cell junctions provide the testis with a barrier that prevents
the immune system from coming in contact with the post-meiotic germ cells.

This unique barrier can be violated,

Testicular torsion, Vasectomy, Testicular trauma, testicular surgeries
Sperm agglutinating AB:
Agglutination of spermatozoa, which reduces
the availability of motile spermatozoa
penetrating the cervical mucus.

Sperm immobilizing AB:

Induce loss in motility of the sperm -
Characteristic “shaking” pattern in motility on
postcoital test.

Spermotoxic AB: Complement-dependent loss

in viability of spermatozoa.
Testing of ASA

Direct ASA test detects sperm-bound immunoglobulins. (preferred)

Indirect testing detects the biologic activity of circulating ASA.

Sperm MAR(mixed antiglobulin reaction ) are recommended screening tests that are
economical and readily available.

Immunobead Test (IBT), which measures IgG, IgA, and IgM, may be additionally
recommended when the previous tests gives a positive result.

Acceptable normal values by WHO (1992) standards

Less than 10% (IgG MAR)
Less than 20% (IBT).
Less than 50% WHO
 Clinical implications of ASA on male infertility.

 10% of sub fertile men.

 2% of fertile men.
 ASA are present in 34% to 74% of vasectomized men.
 Persist in 38% to 60% after vasectomy reversal.
 Does not affect the decision to do a vasectomy reversal.

Routine testing is not recommended

(IVF versus ICSI) in immunologic infertility

Inability for ZP binding, ICSI is the procedure of choice.

•A viable sperm still can be defective.
ELECTRON
•Ultra structural details MICROSCOPY
of the sperm can only be seen under the electron
microscope (EM).

•Candidates:
•Low sperm motility (<5% to 10%) with high viability & density.

•Findings,
Less intact acrosome membrane,
More droplets attached to the acrosome membrane.
Mitochondrial & Micro tubular defects- not visible under the usual
Papanicolaou smear can be detected.
 SPERM DNA DAMAGE
DNA fragmentation was initially
described in 1993
Chromatin -Tightly packed.
Disulfide cross linkages between protamines.

DNA damage is multifactorial.

 Protamine deficiency.
 Mutations - affect DNA packaging or compaction during spermiogenesis.
 Tobacco use, chemotherapy, testicular carcinoma, and other systemic cancers.

DNA damage is correlated positively with poor semen parameters.

Selection of sperm for ICSI

 Genetic
evaluation
Men with infertility of unknown Y chromosome microdeletion
etiology and sperm concentrations, and G-band karyotyping
10 million/mL

Non-obstructive azoospermia in a male considering Y chromosome micro deletion

testicular sperm retrieval for ART and G-band karyotyping

Azoospermic or oligozoospermic men CFTR gene mutation analysis

with the absence of at least one vas
deferens at physical examination

Azoospermic men with signs of normal CFTR gene mutation analysis

spermatogenesis (e.g., obstructive
azoospermia of unknown origin)

History of recurrent miscarriage or G-band karyotyping

personal/familiar history of
genetic syndromes
• Thank you