Semen analysis examines the constituents, volume, pH, motility, count, morphology, and function of sperm in semen. It is used to investigate infertility, check vasectomy effectiveness, and for other purposes. The test involves examining semen parameters like volume (>1.5mL), pH (>=7.2), motility (>32% progressively motile sperm), sperm count (>15 million/mL), and normal sperm morphology (>4%). Additional analyses include assessing sperm viability, antibodies, and function through tests like examining sperm's ability to penetrate cervical mucus or hamster eggs. Semen is collected and analyzed according to standardized WHO guidelines to evaluate male fertility potential.

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)