SEMEN ANALYSIS, physical chemical and microscopy

SEMEN ANALYSIS
MITHUN VENUGOPAL
HOD, ASST. PROFESSOR
DEPT. OF PATHOLOGY

INTRODUCTION
• Problems with male semen account for 40% of all infertility.
• Semen analysis is the best way to test male infertility.
• It gives important information about the quality and quantity of
the sperm.

PRODUCTION AND COMPOSITION
• Semen consists of four fractions contributed by the testis, bulbourethral
gland, seminal vesicle and the prostate.
• Spermatozoa or sperms are produced in the testis and mature in the
epididymis, where they are stored until ejaculation.
• Sperms account for about 1-2% of the total volume of ejaculate.
• The secretory product of the bulbourethral gland functions to lubricate
the urethra.

PRODUCTION AND COMPOSITION
• The majority of the semen is contributed by the seminal vesicle in the
form of an alkaline viscous coagulum which contains prostaglandins and
fructose.
• The prostatic fluid in semen is acidic, contain acid phosphatase and
proteolytic enzymes which acts on the coagulum and helps in
liquefaction of the semen.

INDICATIONS
• Evaluation of infertility.
• To evaluate the effectiveness of surgical therapy.
• To select appropriate donor for therapeutic insemination.
• Screening for exposure to reproductive toxins.
• To evaluate the effectiveness of vasectomy.
• In suspected cases of rape, paternity issues and other medico legal
cases.

PATIENT INSTRUCTIONS
• Patient should be given clear and simple instructions explaining the
need for semen analysis and what is required for specimen collection.
• Patient should be informed about the importance of abstinence time.
Ejaculate must be collected after 3-5 days (but not more than 7 days) of
abstinence.
• Prior to sample collection, the patient must void and wash hands and
genitals to minimize the chances of contamination.
• Samples should be obtained by masturbation and collected in a sterile,
nontoxic plastic or glass wide-mouth container.
• All sample containers are labelled with adequate information to
eliminate any chances of error.

PATIENT INSTRUCTIONS
• Ideally, the samples must be collected close to the laboratory.
• If the specimen cannot be produced close to the laboratory, it must be
delivered to the laboratory as soon as possible, certainly within 1 hour of
collection.
• During this period, the sample has to be kept warm under the clothing
and temperature extremes must be avoided.
• The WHO recommends obtaining two samples for initial evaluation at
an interval of not less than 7 days or more than 3 weeks.
• If the results from the two samples are distinctly different, additional
samples have to be collected and examined.

INFORMATION ON THE SAMPLE
CONTAINER AND PATIENT SHEET
• Once the patient has collected the specimen, some preliminary
information about the specimen should be obtained.
Patient’s complete name & Hospital number.
Collection date and time.
Abstinence time in days.
Time the sample is received at the laboratory.

PHYSICAL EXAMINATION
LIQUEFACTION TIME:
• A normal sample usually liquefies within 60 minutes at room
temperature although usually this occurs within 15-20 minutes.
• It is determined by the time required for the gelatinous mass to liquify.
• A normal sample might contain gel-like gelatinous corpuscles that do
not liquefy.
• Exact liquefaction time is of no diagnostic importance unless >2 hours
elapse without any change. This may indicate poor prostatic secretion
since the liquefying enzymes are derived from the prostate gland.
• On the other hand, absence of coagulation may indicate ejaculatory
duct obstruction or congenital absence of seminal vesicles.

VOLUME:
• Volume of the ejaculate should be measured by transferring the
liquefied sample into a graduated 15 ml conical centrifuge tube.
• The normal volume of ejaculate after 2-5 days of sexual abstinence is
about 2-6 ml.
• Retrograde ejaculation, obstruction of lower urinary tract (urethra,
congenital absence of vas deferens, seminal vesicles) may yield low
volume.

• According to the WHO, the reference value for semen volume is ≥ 2.0ml;
however, for clinical purposes; semen volume is differentiated into three
categories to facilitate interpretation and diagnosis:
1. Aspermia: No semen produced after orgasm.
2. Hypospermia: <0.5 ml of semen ejaculated (partial or complete
retrograde flow of semen, accessory glands impairment).
3. Hyperspermia: > 6 ml of semen ejaculated (long period of sexual
abstinence or overproduction of fluids from the accessory sex
glands).
• If the volume is <1 ml it is important to determine if the sample is
complete.
• The highest sperm concentration is seen in the initial ejaculate.

COLOUR AND APPEARANCE:
• Normal – homogenously opaque, whitish grey or pearly white.
• Yellow – pyospermia.
• Blood tinged – bleeding in the seminal vesicle.

VISCOSITY:
• Viscosity measures the seminal fluid’s resistance to flowing.
• It is measured by the length of the ‘thread-lines.’
• It can be estimated by using a glass rod and observing the length of
thread that forms on withdrawal and using a Pasteur pipette, expelling
the semen drop by drop.
• A normal sample leaves small, discrete drops; abnormal samples will
form threads more than 2 cm long.
• High viscosity may interfere with determinations of sperm motility.
• Viscosity can be categorized as normal, moderate or high.

PH:
• Normal semen pH is in the range of 7.2 - 8.2, and it does tend to
increase with time after ejaculation.
• Any change in the normal range of pH may be caused by inflammation
of the prostate or seminal vesicles.
• The pH of liquefied semen is determined by using pH test strips.

MICROSCOPIC EXAMINATION
MOTILITY OF THE SPERM:
• Place a small drop of semen on a glass slide and cover it with a
coverslip.
• Examine under high power objective under reduced light.
• Count atleast 200 sperms and grade the motility of each of the sperm.
• Sperm motility is the ratio of the number of motile sperm to total
number of sperm in a given volume and is expressed as a percentage.

Grade – 0: No motility
Grade – 1: Slow forward progression and mild lateral movement.
Grade – 2: Slow forward progression with substantial yaw.
Grade – 3: Slightly faster forward progression with little yaw.
Grade – 4: Spermatozoa moving rapidly in a straight line with little yaw
and no lateral movement.

• The number of sperms belonging to each grade is expressed in
percentage.
• Normally the individual should have >25% of sperms belonging to
grade 3 or 4 and >50% of sperms in grade 2, 3 or 4.
• The sperm motility is recorded at the end of 2, 3, 6 hours. The number of
motile sperms decreases by 5% per hour after four hour of collection.
• Necrozoospermia: Total absence of motility.
• Asthenozoospermia: Decreased sperm motility.

SPERM COUNT:
• Mix the specimen after liquefaction.
• Draw the semen up to 0.5 in a WBC pipette.
• Draw the semen diluting fluid (sodium bicarbonate, formalin, distilled
water) up to mark 11 and mix well.
• Charge the improved Neubauer counting chamber and allow the sperm
to settle for 5 minute.
• Count all 4 squares of WBC area.
• Normal: 40-300 millions/ml.

• Oligozoospermia: Sperm count <20 million/ml.
• Azoospermia: Absence of sperms.
• Causes of low sperm count:
• Infections (mumps, orchitis, prostatitis, etc.)
• Obstruction,
• Endocrinopathies

SPERM MORPHOLOGY:
• After liquefaction make a thin smear on a glass slide.
• Allow the smear to dry and then heat fix.
• If necessary remove the mucous by dipping the slide in semen diluting
fluid and then into buffered distilled water.
• Stain the smear using Leishman’s stain.

MORPHOLOGY OF NORMAL SPERM:
• It has a head which measures 4um in length and 3um in
diameter.
• It has a cap called acrosomal cap which stain slight blue in
color.
• The nucleus of the sperm stains dark blue.
• The middle piece measures 7um in length and 1um in
diameter.
• The neck measures 0.3um which is situated between the
head and the middle piece.
• The tail measures 45-50um in length and stains red or pink.

ABNORMAL SPERM FORMS:
• Morphological abnormalities can be found in head, body or tail.
• Abnormality of the head: Too large (macrocephaly) or too small
(microcephaly) head, double heads, pointed heads, ragged heads,
vacuoles in the chromatin, etc.
• Abnormality of the middle piece: Absence of middle piece, bifurcated or
swollen middle piece. If the middle piece defects are >25% it is
pathological.
• Abnormalities of the tail: double, triple, quadruple tails, rudimentary or
absence of tail, cytoplasmic vacuole along the tail indicate an immature
sperm. If the defects are >25% it is pathological.

SPERM VIABILITY TESTING:
• Assessing the ability of the sperm plasma membrane to exclude
extracellular substances.
• The cytologically intact ‘live’ cells can be determined using several vital
staining techniques such as eosin Y and trypan blue.

EOSIN-NIGROSIN STAIN:
• An Eosin-Nigrosin stain must be done on all specimens having a motility
of 30% or less. The stain must be performed immediately following the
initial motility examination.
• Eosin-Nigrosin stains the dead sperms and live sperms are unstained.
• Add 2 drops of 1% aqueous eosin Y and 3 drops of 10% aqueous
Nigrosin. Mix well.
• Immediately make two thin smears from this mixture and air dry.
• Count 100 sperm on each slide in duplicate using high power (×40).
• Calculate percentage of viable (unstained) and non-viable (stained)
sperm.

CHEMICAL EXAMINATION
QUANTITATION OF SEMEN FRUCTOSE:
• Fructose reacts with resorcinol in a strong acidic medium to give a red
colored complex.
• Normal value: 150-300 mg/dl
• High fructose level is associated with low sperm count and vice versa.
• Low fructose levels are associated with decreased testosterone.

MICROBIOLOGICAL EXAMINATION
ORGANISMS FOUND IN SEMEN
• Ureaplasma
• Mycoplasma
• Chlamydia trachomatis
• Neisseria gonorrhoeae
• Trachomonas vaginalis
• E-Coli
• HIV
• HPV
• CMV

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