2. HISTORY
Antonie van Leeuwenhoek
Sperm first observed in 1677 by Antonie van Leeuwenhoek.
Sperm is the male reproductive cell .
“sperm “is derived from the Greek word sperma.
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3. SPERM
A sperm cell is 70 µm long, acrosomal cap include 40-70%
Tail is 45 µm, Head is 5×3 µm (length and width).
The middle section (body) is approximately the same length as
the head.
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7. Spermatogenesis is the process of forming motile
spermatozoa from spermatogonium.
Duration : 64 days (2 months)
Spermiogenesis is the production of mature, motile
spermatozoa from spermatids.
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10. SAMPLE COLLECTION
Methods :
Masturbation
Coitus interruptus : withdrawing the penis just before
ejaculation
Coitus with silicon condom
Ejaculation stimulated by electricity.
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11. Patient preparation :
Patient should be in a period of sexual abstinence for 3 days
but not more than 7 days.
Should not use any lubricants.
Avoid any hormonal medications.
Collect a complete semen specimen in a clean, sterile , wide
mouthed container .
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12. Specimen should collected in room temperature.
Avoid exposing the specimen to extreme hot or cold
climate.
Deliver it to the laboratory within 1 hr of collection.
The lab person should record the time of specimen
collection and specimen receipt.
Should not process the sample immediately. Wait for 30
minutes(liquefaction time).
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13. Safe handling of specimen :
Semen sample may contain dangerous infectious agents like
HIV, Hepatitis viruses or Herpes simplex virus etc.
Therefore handle the specimen as a biohazard.
Standard precautions must be taken during the analysis.
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15. COLOUR & CONSISTENCY :
Red-brown - Red blood cells are present.
Yellow - jaundice , taking certain vitamins or drugs.
Thick , lumpy or jelly like – male hormone deficiency.
Less opaque – sperm concentration is low
VOLUME :
Normal volume : 2-5 ml
Less than 1.5 ml : abnormal
Measured in a small graduated glass cylinder.
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16. LIQUEFACTION :
Definition : process when the gel formed by proteins from
the seminal vesicles is & the semen becomes more liquid
Normal Liquefaction time : 15-30 minutes after collection.
>60 minutes : abnormal, sperms may be trapped in
unliquefied jelly; maybe sign of prostatic infection, lack of
prostatic protease .
The semen has not liquefied after 2 hr, add alpha-
chymotrypsin (protease enzyme)
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17. VISCOSITY :
It is the consistency of the fluid.
Normal – smooth and watery.
Incompletely liquefied- highly viscous.
Normal semen droplets form a thin thread when released
from the pipette.
Threads >2 cm are considered highly viscous.
High viscosity impede sperm movements.
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18. pH :
The normal pH is alkaline (varies from 7.2 to 8)
Secretions from the prostate and seminal vesicles
contribute to seminal pH
Measured using litmus paper.
Increased pH- infection of reproductive tract.
Decreased pH-increased prostatic fluid.
Sperm die at < 6 pH
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21. Liquefied semen – up to 0.5 mark
Diluting fluid – up to 11 mark
o Examine under microscope and count the number of
spermatozoa in 4 corner squares.
o Number of spermatozoa/μl =N × 50
Normal count = 60-150 million/ml
Abnormal count = < 20 million/ml
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Diluting fluid :
Sodium bicarbonate - 5 g
(counteract with mucus)
Formalin - 1 ml
Diluting fluid
Sodium bicarbonate – 5g
Formalin – 1 ml
Distilled water – 100ml
23. Place a small, un-caliberated drop of liquefied well mixed
semen in the center of the chamber.
The number of spermatozoa counted in any strip of 10
squares indicates their concentration in millions per ml.
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24. c) Sperm counting chamber
10×10 Squares
Depth 0.01mm
Area 0.01mm2
No. of sperm counted in any strip of 10 Squares indicates
their concentration in millions/ml
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25. 2. Sperm motility
Should be performed on an Undiluted, well mixed, liquefied
semen within 1 hour of collection.
Place a drop of liquefied semen on a clean glass slide.
Put a cover slip over it and first examine under low power and
then under high power.
https://youtu.be/SMe_FvQifwU
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26. The microscopic slide screened systematically and the motility
of each spermatozoa encountered is graded as A,B,Cand D in %
according to whether it shows:
A- Rapid progressive motility
B- Slow progressive motility
C- Non-progressive motility
D- Immotility
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27. Normally within 2 hours of ejaculation :- > 60% of
spermatozoa are vigorously motile
After 6-8 hours :- 25-40% motile
If motility is less than 50%, a stain for viability such as
Eosin Y with Nigrosine as counter stain can be done.
Red dye accumulates in the heads of non-motile sperms
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29. 3. Sperm morphology
Make a thin smear using liquefied undiluted semen.
Air dried and stain with Leishman , Giemsa or H&E stain.
Examine under oil immersion objective.
Normally 80% of the spermatozoa are normal.
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30. A normal sperm has :
A smooth, oval-shaped head, 5-6 μm long and 2-3 μm wide
A well-defined cap (acrosome) that covers 40% to 60% of the
sperm head.
The mid-piece must be straight and slender, 0.5 µm in width
and 7-8µm long, straightly aligned to the head.
The tail must be straight and 45-50 µm long.
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Total of 400 sperms should be evaluated. Out of which
% of normal form is calculated as;
Normal Forms (%) = normal sperms x 100
the total number of sperms evaluated
Also seen for,
• Agglutination
• Pus cells/HPF
• Debris
32. Categories of defects :
Head defects, namely large, small, tapered, pyriform , globe,
amorphous heads, vacuolated heads, double heads and small
acrosome.
Neck and mid-piece defects namely bent neck, abnormal
mid-piece (thin or thick).
Tail defects namely short, multiple, hairpin, broken, bent tails,
coiled tail
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35. TERMS DISCRIPTIONS
Oligospermia Sperm concentration<15 millions/ml
Azoospermia No sperm in semen
Aspermia No semen volume
Pyospermia Leukocyte present in semen >
Necrozoospermia “Dead” sperm
Hematospermia Red cell present in the semen
Asthenozoospermia Reduced sperm motility
Teratozoospermia Sperms with abnormal morphology
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36. CHEMICAL EXAMINATION
FRUCTOSE TEST:
Fructose is produced by seminal vesicles and is released in
to the semen during ejaculation
Testing should be considered for patients with azoospermia
and low volume ejaculation
Normal seminal fructose level is 150-600 mg/dl
Fructose is measured qualitatively by resorcinol test/
Seliwanoff’s test
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37. Procedure
Take 5 ml of dilute HCl in a test tube.
Add 1 ml of semen.
Add 5 mg of resorcinol. Boil and cool it
Interpretation :
Cherry red colour –
presence of fructose
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38. IMMUNOLOGICAL ASSAY
The sperm antibody binding to the antigen present in the
head or tail is considered specific for immunologic infertility.
The antibodies are usually of IgA or IgG, and rarely of IgM
These are detected by direct or indirect mixed agglutination
reaction tests
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39. SPERM FUNCTION TEST
Sperm functional assay ,which indirectly measures the ability
of one spermatozoa to deliver the correct complement of
chromosomes to an ovum
This includes sperm penetration assay , sperm zona pellucida
binding test , acrosomal reaction test
Defective sperm function may affect various fertilizing
activities
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40. MICROBIOLOGICAL ASSAY
It includes the culture of semen in order to exclude
microbiological causes of infertility.
Culture aimed at the isolation of Neisseria gonorrhoeae ,
chlamydia trachomatis and ureaplasma species
Culture is done if there are pus cells in the semen.
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42. SQA-V Gold
It is a high performance analytical medical device that
combines technology in electro –optics, computer
algorithms and video microscopy.
Analysis time -75 seconds
Requires 0.5 ml of sample for testing
It archive up to 500 patient record
If the sample is viscous treat it with quick check
liquefaction kit
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44. SQUA-V Testing capillary tube
Disposable, capillary tube
Motility measured in the “motility section” which requires 20
micro liters of semen
Concentration is measured in the “cuvette section” which
requires 450 micro liters of semen
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45. SQUA-V Slide adaptor
It is a slide adaptor used for the visualization
compartment.
Use with a standard laboratory slide 76 x 25.6 mm and 22
x 22 mm cover-slip
A 10 micro liters sample placed 12 mm from the end of
the slide
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47. Step 1
Capillary is inserted in to the measurement compartment
Step 2
• Concentration- An optical density detector measures the
amount of light absorbed by the cells and convert it to
optical density
• The “OD” reading is translated in to sperm concentration
by a microprocessor
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48. Step 3
• Motility-The movement of motile sperm causes light
disturbances
• The light disturbances are converted in to electrical signals
with “peaks and valleys”
• It is analysed by a microprocessor and translated in to
motility parameters
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