About semen analysis

1. SEMEN
ANALYSIS

2. • Semen (or seminal fluid) is a fluid that is
emitted from the male genital tract and
contains sperms that are capable of fertilizing
female ova

4. • Testes:
– Male gametes or spermatozoa (sperms) are produced by
testes
– constitute 2-5% of semen volume.
• Epididymis:
sperms are stored in the epididymis where they
mature
Potassium, sodium, and glycerylphosphorylcholine
are secreted by epididymis.
• Vas deferens:
Sperms travel through the vas deferens to the ampulla
which is another storage area.
Ampulla secretes ergothioneine and fructose.

5. • Seminal vesicles:
During ejaculation, nutritive and lubricating fluids
secreted by seminal vesicles and prostate are added.
Fluid secreted by seminal vesicles consists of fructose,
amino acids, citric acid, phosphorous, potassium, and
prostaglandins.
Seminal vesicles contribute 50% to semen volume.
• Prostate:
Prostatic secretions comprise about 40% of semen
volume and consist of citric acid, acid phosphatase, calcium,
sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
• Bulbourethral glands of Cowper secrete mucus.

6. INDICATIONS FOR SEMEN ANALYSIS
1. Investigation of infertility
2. To check the effectiveness of vasectomy by
confirming absence of sperm.
3. To support or disprove a denial of paternity on the
grounds of sterility.
4. To examine vaginal secretions or clothing stains
for the presence of semen in medicolegal cases.
5. For selection of donors for artificial insemination.
6. For selection of assisted reproductive technology

7. COLLECTION OF SEMEN FOR
INVESTIGATION OF INFERTILITY
• Semen specimen is collected after about 3
days of sexual abstinence
– Longer abstinence - reduces motility of sperms.
– Shorter abstinence – reduces sperm count
• sample is obtained by masturbation

8. • collected in a clean, dry, sterile, and leakproof
wide-mouthed plastic container
• The entire ejaculate is collected, as the first
portion is the most concentrated and contains
the highest number of sperms.
• Ejaculate should be collected in privacy near
the laboratory
• It should be delivered to the laboratory within
1hr of collection

9. • Condom collection is not recommended as it
contains spermicidal agent
• Ejaculation after coitus interruptus leads to the
loss of the first portion of the ejaculate that is
most concentrated

10. SAMPLE COLLECTION -
PREPARATION
• Pass urine
• Wash hands and penis with soap
• Rinse away the soap
• Dry hands and penis with a fresh disposal towel
• Ejaculate into sterile container
• Precautions
– Lubricants should be avoided
– Collect the entire sample

11. STORAGE AND TRANSPORT
OF THE SPECIMEN
• Sample should be transported to the laboratory
within 30mts - 1 hour of collection
• Keep the sample warm under the clothing
while transportation
• Should not be stored in refrigerator

12. EXAMINATION OF SEMINAL
FLUID
Physical examination
Microscopic examination
 Immunologic analysis
Bacteriologic analysis
Biochemical analysis
Sperm function tests

13. PHYSICAL EXAMINATION

14. Volume:
• Normal - 2-5 ml
• Measure in a small graduated cylinder
• Hypospermia : <1.5ml
– Obstruction of the ejaculatory duct
– Androgen deficiency
– Incomplete collection
• Hyperspermia : >5.5ml
- Active exudation
• Aspermia : Absence of ejaculate

15. Colour and appearance
• Normal - viscous and opaque gray-white
– Less opaque – if sperm concentration is very low
– Slight yellow – prolonged abstinence
– Deep yellow – pyospermia, jaundice, or taking
certain vitamins or drugs
– Red – brown – haemospermia
– Rust colour – small bleeding in seminal vesicles
– Increased turbidity – inflammatory process

16. Viscosity
• Freshly ejaculated semen is highly viscous due
to substrate produced by seminal vesicles
• The viscosity of the sample is assessed by
filling a pipette with semen and allowing it to
flow back into the container.

17. • Normal semen will fall
drop by drop.
• If droplets form
‘threads’ more than 2
cm long, then viscosity
is increased.

18. Liquefaction
• Immediately following ejaculation, normal
semen is thick and viscous.
• Liquefaction is initiated by enzymes of
prostatic origin
• Liquefaction time – 15-30mts
• If liquefaction does not occur within 60
minutes, it is abnormal.
• Recognized both macroscopically and
microscopically

19. • Absence of initial coagulation
– Congenital absence of vas deferens & seminal
vesicles
– Obstruction of the ejaculatory duct
• Incomplete liquefaction
– Decreased production of prostatic enzymes

20. pH
• Normal : 7.2 – 8.0 (after 1 hour of ejaculation)
• pH paper
• The portion of semen contributed by seminal
vesicles is basic, while portion from prostate is
acidic.
• Low pH (< 7.0) with absence of sperms suggests
obstruction of ejaculatory ducts or absence of vas
deferens.
• Low pH is usually associated with low semen
volume

21. MICROSCOPIC
EXAMINATION

22. Motility
• The first laboratory assessment of sperm
function in a wet preparation
• Principle:
– All motile and non-motile sperms are counted in
randomly chosen fields in a wet preparation under
40× objective. Result is expressed as a percentage
of spermatozoa observed under each grade.

23. • Method
– A drop of semen is placed on a glass slide
– covered with a coverslip that is then ringed with
petroleum jelly to prevent dehydration
– examined under 40× objective.
– Atleast 200 spermatozoa are counted in several
different microscopic fields.

25. • Grading
– A Rapid forward progress
motility
– B Slow or sluggish progressive
motility
– C Non progressive motility
– D Immotility
 Asthenozoospermia: Reduced sperm motility

27. • Habitual factors affecting sperm motility
– High cosumption of tobacco
– Cosumption of coccaine
– Vaginal lubricants
– alcoholism

28. Sperm Viability or Vitality
Principle:
• A cell with intact cell membrane (a vital or viable cell)
will not take up the eosin Y and will not be stained
• while a non-viable or dead cell will have damaged cell
membrane, will take up the dye, and will be stained
pink-red
• Another stain (e.g. nigrosin) may be used to stain the
background material.
• The test is performed if motility is abnormal.

29. Method
• Mix one drop of semen with 1 drop of eosin-
nigrosin solution and incubate for 30 seconds.
• A smear is made from a drop placed on a glass
slide.
• The smear is air-dried and examined under oil
immersion objective.
White sperms - live or viable
Red sperms - dead or non-viable.

30. • At least 200 spermatozoa are examined.
• The result is expressed as a proportion of
viable sperms against non-viable as an integer
percentage.
• 58% or more of sperms are normally live or
viable

32. Method
1. Semen is diluted 1:20 with sodium bicarbonate
formalin diluting fluid
2. A coverslip is placed over the improved
Neubauer counting chamber
• The counting chamber is filled with the well-
mixed diluted semen sample.
• The chamber is then placed in a humid box for
10-15 minutes for spermatozoa to settle.
Sperm Count

33. 3. The chamber is placed on the microscope
stage.
• Number of spermatozoa is counted in 4 large
corner squares.

35. Sperm count /ml =
Sperms counted × correction factor
for dilution
× 1000 Number of × Volume of 1
square
squares counted

36. Sperms counted × 20
= × 1000
4 × 0.1
= Sperms counted × 50, 000
5. Normal sperm count is ≥ 20 million/ml
 Oligozoospermia: Sperm concentration <20 million/ml

37. • The staining techniques used are
– Papanicolaou
– Eosin nigrosin
– hematoxylin-eosin
– Rose Bengal-toluidine blue stain
• Percentages of normal and abnormal
spermatozoa should be recorded.
• Teratozoospermia: Spermatozoa with reduced
proportion of normal morphology
Sperm morphology

38. NORMAL MORPHOLOGY

42. ABNORMAL MORPHOLOGY

43. • WHO morphological classification of human spermatozoa
(1999):
1. Normal sperm
2. Defects in head:
• Large heads
• Small heads
• Tapered heads
• Pyriform heads
• Round heads
• Amorphous heads
• Vacuolated heads
• Small acrosomes
• Double heads

44. 3. Defects in neck:
• Bent neck and tail forming an angle >90° to the long
axis of head
4. Defects in middle piece:
• Asymmetric insertion of midpiece into head
• Thick or irregular midpiece
• Abnormally thin midpiece
5. Defects in tail:
• Bent tails
• Short tails
• Coiled tails
• Irregular tails

45. • Multiple tails
• Tails with
irregular width
6. Pin heads: Not to be
counted
7. Cytoplasmic droplets
• > 1/3rd the size of
the sperm head
8. Precursor cells:
Considered abnormal

46. ROUND CELLS
• Round cells
on
microscopic
examination
may be white
blood cells or
immature
sperm cells.

47. • Special stain is required to differentiate
between them.
• Leukocytospermia: >1 million white blood
cells/ml of semen
– indicate presence of infection.
• Presence of large number of immature sperm
cells indicates spermatogenesis dysfunction at
the testicular level.

48. WHITE BLOOD CELLS

49. EPITHELIAL CELLS

50. RED BLOOD CELLS

51. GERMINAL CELLS

52. PROTOZOAAND BACTERIA

54. IMMUNOLOGIC
ANALYSIS

55. • The immunological tests done on seminal fluid
include
–Mixed antiglobulin reaction (MAR test)
–Immunobead test.
• The antibodies can be tested in the serum,
seminal fluid, or cervical mucus.
Antisperm Antibodies

56. • If the antibodies are present bound to the head
of the sperm, they will prevent the penetration
of the egg by the sperm
• If antibodies are bound to the tail of the sperm,
they will retard motility

57.  SpermMARTM
test
• This test can detect IgG and IgA antibodies
against sperm surface in semen sample.
Direct Indirect

58.  Direct SpermMARTM
test

60. • a drop each of fresh unwashed semen and
of IgA-coated latex particles, are mixed
on a glass slide.
• The latex particles will bind to
spermatozoa if spermatozoa are coated
with IgA antibodies.
Direct SpermMARTM
IgA test

61. • Fluid without spermatozoa (e.g. serum) is
tested for the presence of antisperm antibodies.
• First bound antibodies to donor spermatozoa .
• These antibodies are then detected as for
direct tests.
Indirect SpermMARTM
test

62. • Antibodies bound to the
surface of the
spermatozoa can be
detected by antibodies
attached to immunobeads
• Percentage of motile
spermatozoa with
attached two or more
immunobeads are
counted amongst 200
motile spermatozoa.
Immunobead test

63. BIOCHEMICAL ANALYSIS

64. • Prostate gland function
– Zinc
– Citric acid
– Acid phosphatase
• Seminal vesicles
– Fructose
– Prostaglandins
• Epididymis
– Alpha glucosidase
– L – carnitine

65. Test for Fructose
• Resorcinol method
• 5 ml of resorcinol reagent + 0.5 ml of seminal
fluid.
• The mixture is heated and brought to boil.
• fructose is present red-colored
precipitate is formed within 30 seconds.

66. SPERM FUNCTION TESTS

67. Postcoital (Sims-Huhner) Test
• This is the examination of the cervical mucus after
coitus and assesses the ability of the sperm to
penetrate the cervical mucus
• Cervical mucus is aspirated with a syringe shortly
before the expected time of ovulation and 2-12 hours
after intercourse.
• Gross and microscopic examinations are carried out
to assess the quality of cervical mucus and to evaluate
the number and motility of sperms

68. • If ≥ 10 motile sperms are observed the test is
considered as normal.
• Abnormal test may result from:
– Poor quality of cervical mucus
– Absence of motile sperms due to
• ineffective technique of coitus
• lack of ejaculation
• poor semen quality
• use of coital lubricants that damage the sperm
• presence of antisperm antibodies.

69. Cervical Mucus Penetration Test
• Greatest distance traveled by the sperm in
seminal fluid placed and incubated in a
capillary tube containing bovine mucus is
measured.
• Fertile men show score >30 mm
• Infertile men show scores <20 mm.

70. Hamster Egg Penetration Assay
• Hamster oocytes are enzymatically treated to
remove the outer layers
• They are then incubated with sperms and
observed for penetration rate.
• This test detects
–sperm motility
–binding to oocyte
–penetration of oocyte

71. Hypo-osmotic Swelling of Flagella
• This test assesses the functional integrity of
the plasma membrane of the sperm by
observing curling of flagella in hypo-osmotic
conditions.
Computer-assisted Semen Analysis
• Computer software measures various
characteristics of the spermatozoa

72. • Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration < 20
million/ml
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility.

73. • Teratozoospermia: Spermatozoa with reduced
proportion of normal morphology
• Leukocytospermia: >1 million white blood
cells/ml of semen
• Oligoasthenoteratozoospermia: All sperm
variables are abnormal
• Necrozoospermia: All sperms are non-motile or
non-viable

74. THANK YOU…..