semen analysis based on who guidelines

1. Semen Analysis
(based on WHO guidelines 2010)

2. Constituents of Semen
● Normal semen is an admixture of spermatozoa
suspended in secretions (seminal plasma) from
glandular tissues of male genital system.

3. ● Testes produces spermatozoa and constitutes
5% of the semen volume.
● Vas deferens produces ergothionine
● Epididymis ( maturation/ storage of sperm)
produces:
– Choline - energy source of sperms.
– Alpha glucosidase
– Carnitine

4. ● Seminal vesicle – nutritive fluid containing
fructose, and is secreted during ejaculation.
(50% of semen volume)
● Prostate produces (40% of semen volume)
– Citric acid
– Acid phosphatase
– Proteolytic enzyme
– Zinc
● Bulbourethral glands of Cowper produces
mucous. ( constitutes 5% of semen volume)

5. ● Indications of Semen Analysis
● Investigation of infertility
● Check effectiveness of vasectomy
● Paternity testing
● Rape cases
● Selection of donors for artificial insemination/
assisted reproductive technology.

6. ● Sample collection
– Sample should be collected after 48 hrs of
abstinence. Higher abstinence → decreased
motility. Lesser abstinence → decreased count.
– Collection is done by masturbation.
Not recommended: condom collection, coitus
interruptus. (loss of initial portion of the ejaculate)
– Collection should be done in a clean, wide mouth,
leak proof container.

7. ● Transport
– Should be done within one hour to the laboratory.
– Temperature should be maintained as close to the
body temperature as possible (inside pocket)
● Two specimens should be examined at least 2
to 3 weeks apart.

8. Examination of Seminal fluid in
Infertility
● Physical examination
– Visual appearance : opaque to grey – white, slightly
yellow after abstinence.
● Inflammation of male accessory organs → yellow color of
semen → pyospermia
● White clear semen → azoospermia
● Brown or red color → hemospermia

9. – Viscosity
● Assessed by filling a pipette with semen and allowing it to
flow back to the container
● Normal semen fall drop by drop
● If droplet form threads > 2 cm long → increased viscosity
● Normal semen liquefies in 30 min. If liquefaction does not
occur in 60 min → abnormal increase in viscosity. This
decreases sperm motility.
● If sample does not liquefy → treat with plasmin or
chymotrypsin.

10. ● Volume : more than 1.5 ml
– If the sample volume is less than 1 ml spillage or
incomplete collection must be ruled out
– Conditions leading to low semen volume
(hypospermia)
● Disorders of seminal vesicles or prostate
● Retrograde ejaculation
● Congenital absence of prostate or seminal vesicle

11. ● PH : normal >= 7.2
– Seminal vesicle secretion is basic
– Prostatic secretion is acidic
– If pH = 7 with absence of sperm → indicates either
obstruction of ejaculatory duct or absence of vas
deferens.

12. Microscopic examination
● Motility
– Ability of the sperm to move
– 3 types of motility
● Rapidly progressive – moving fast and forward in a
straight line
● Slowly progressive – crooked, curved, slow forward
movement
● Non progressive – movement of tail only

13. – Only those sperms with rapid progressive
movement are capable of fertilizing an ovum.
– Method
● A drop of semen is placed on a slide, covered with
coverslip and sealed with petroleum jelly.
● Examination is done under 40x
● Count at least 200 spermatozoa
● Find the percentage of rapidly progressive, slowly
progressive, non progressive and non motile sperm.
● Normal values
– > 32 % progressive motility
– > 40 % progressive + non progressive motility

14. ● Vitality
– Number of live sperms are called viable
– A viable sperm will have intact cell membrane and
will not take up eosin Y
– Method
● 1 drop of semen + 1 drop of eosin – nigrosin
● Wait for 30 sec
● Put a drop on a slide
● Air dry
● Examine under oil immersion and count 200 sperms
● Red sperms not viable; white sperm viable
● Normal viable count > 58%

15. ● Count
– Wait for liquefaction
– Mix 1ml semen with 20 ml diluting fluid(sodium
bicarbonate – formalin)
– Charge Neubauer’s chamber with pateur’s pipette
– Place chamber in humid conditions for 10 – 15 min
– Count in 4 large chambers

16. – Calculation
count = sperm counted x correction for dil. Fluid x1000
–--------------------------------------------------
No. of squares counted x vol of 1 square
= N x 20 x1000
-------------
4 x 0.1
= N x 50,000
– Normal count > 15 million/ ml

17. ● Morphology
– Drop of seminal fluid on the slide
– Stain with pap/eosin-nigrosin/rose bengal-toludine
blue
– Examine the morphology of at least 200 sperms
– Normal > 4 % of sperm should have normal
morphology.

18. ● Normal morphology of spermatozoa
– Head : consists of nucleus with condensed chromatin
and some nuclear vacuoles.
– Acrosome: anterior 2/3rd of the head shows an
acrosom cap, secrets enzymes that dissolve the cells
of corona radiata and zona pellucida of the ovum
during fertilization.
– Middle piece contains mitochondria → provides
energy.
– The tail used for motility.

21. Immunological analysis (antisperm antibody
determination )
● Sperm Mar Test
– Direct SMT
● For detection of sperms coated with IgG/IgA
– Indirect SMT
● For detections of antisperm IgG/A antibodies in serum.

22. ● Immunobead test
– Similar to sperm mar test but uses plastic beads
instead of latex particles to detect
antigen/antibodies
● Normal
– <50 % motile spermatozoa with bound particles.

23. Biochemical Analysis
● Seminal vesicle marker (Fructose)
– 50 mg of resorcinol in 33ml of conc. Hcl then diluted
with 100 ml of Distilled water.
– 0.5 ml of seminal fluid is added
– The mixture is heated → produces red precipitate in
30 seconds
– Presence of red precipitate indicates presence of
Fructose
– Absence of fructose → no seminal vesicle present
● D/t obstructed vas deferens or absent seminal vesicle.

24. Sperm function test
● Post coital (Sims-Huhner) test
– Principle
● Examination of the quality of cervical mucus post coitus can
give an idea about the quality of cervical mucus and the ability
of the sperm to penetrate it.
● Normally in proliferative phase (estrogen phase), mucus is
watery and sperm can penetrate easily.
● During secretory phase (progesterone phase), mucus is viscus.
● Hence testing mucus is scheduled just before ovulation.

25. – Method
● Cervical mucus is aspirated 2 – 12 hrs after intercourse.
– Gross examination
● Normal – mucus stretches at least 2 inches, dries in fern
like pattern.
● Abnormal – can not stretch 2 in. No fern like pattern on
drying.
– Microscopic examination
● Normal – more than or equal to 10 motile sperms
● Abnormal – less than 10 motile sperms
– Causes – antisperm antibodies, cervicitis, wrong judgement of
date.

26. ● Cervical mucus penetration test
– Evaluation of the distance traveled by sperm in bovine mucus.
– Fertile sperm travel > 30mm
– Infertile sperm travel <20 mm
● Hamster egg penetration assay
– Hamster egg → enzymatically treated → removal of outer coat
– Incubate with sperm
– Look for number of eggs penetrated ( <15% indicate low fertility ) and
number of sperm penetrating the egg (normal > = 5%)

27. ● Hypoosmotic swelling of flagella
– If sperm is exposed to hypoosmotic solution, sperm
curls up if plasma membrane is abnormal.
– Assessment of functional integrity of plasma
membrane.
● Computer assisted semen analysis
– All above parameters are measured by automated
machines.

28. Summary of normal values
(WHO 2010)