This document provides information about performing a semen analysis, including definitions of key terms, procedures, and normal ranges. It discusses: 1. The components and normal appearance of semen after liquefaction. 2. Procedures for assessing semen volume, viscosity, pH, sperm motility, morphology, concentration, and vitality under a microscope. 3. Factors that can affect the results, such as abstinence time, sample handling, and medical conditions. The document is intended as a reference for medical professionals performing semen analyses to evaluate male fertility and identify potential issues.

1. Semen analysis
Dr. Mohd Daud Akhtar
MD, PATHOLOGY

2. BASIC TERMINOLOGY RELATED TO SPERM AND
SEMEN
• SPERMATOZOAN = single sperm cell
• ASPERMIA = absence of semen upon ejaculation
• HYPOSPERMIA = insufficient semen (< 1.5 ml)
• HYPERSPERMIA = too much semen (> 6 ml)
• HEMOSPERMIA = blood present in the semen
• PYOSPERMIA = pus present in the semen
• AZOOSPERMIA = absence of sperms in the semen
• OLIGOSPERMIA = < 15 million sperms /ml

3. • POLYSPERMIA = > 200 millions sperms / ml
• ASTHENOSPERMIA = reduced motility of sperms
• TERATOSPERMIA = > 4 % abnormally formed sperms
• NECROSPERMIA = presence of dead sperms
• CRYPTOSPERMIA = very few sperms detectable
• GLOBOSPERMIA = only round headed sperms detected

4. SEMEN
• Concentrated suspension of spermatozoa in seminal plasma.
• Ejaculate contains 4 part:
1. Pre-ejaculatory fraction : Cowper’s or Litter’s gland.
2. Prelimnary fraction : Prostate gland.
3. Main fraction : Seminal vesicles, Testis and Epidydmis.
4. Terminal fraction : Seminal vesicles.

5. FRACTION OF SEMEN CONTRIBUTED
BY VARIOUS GLANDS
• Seminal vesicles : 45-50%, alkaline viscous, yellowish secretion
which is rich in fructose, vitamin C, prostaglandin and prostakinase.
• Prostate : 35-40%, acidic fluid containing zinc, citrate, acid
phosphatase and proteolytic enzymes.
• Testis and epidydmis : 5-10%, alpha-glucosidase iso-enzyme.
• Urethral glands : 2-4%

7. Length : 65 μm
Head : 4-5 μm, contain nucleus and acrosome
Middle piece : 4-5 μm, energy for motility is generated
Tail : 55 μm, motility propagated along the tail

8. INDICATIONS FOR SEMEN ANALYSIS
1. Investigation of Infertility.
2. Post-vasectomy by confirming absence of sperm.
3. To support or disprove a denial of paternity on the ground 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 techniques. Eg. In vitro
fertilization, Gamete intrafallopian transfer technique.
7. After reversal of vasectomy to confirm the success of procedure.

9. EQUIPMENTS AND REAGENTS REQUIRED FOR
SEMEN ANALYSIS
• SEMEN DILUTING FLUID
- Sodium bicarbonate 5 gm
- Formalin neutral 1 ml
- Distilled water 100 ml

10. Eosin 1% and Nigrosin 10% solution
stain for assessing sperm vitality
Romanoswky stain (Leishman stain)
for assessing sperm morphology

11. Wide mouthed sterile container for sperm collection

12. pH paper strip for measuring pH

13. Pasteur pipette
Graduated tube

14. Micropipettes with microtips Glass slides Cover slip

15. Neubauer’s counting chamber

16. SAMPLE COLLECTION - PREPARATION
• Sexual abstinence – 3 to 7 days.
• Ideally two separate samples 2-3 weeks apart should be analyzed.
• Sample collection – Private room in the same laboratory.
• Recommended procedure – Masturbation.
• Pre-warmed, sterile, non-toxic, wide mouthed container should be used.
• Bladder should be evacuated prior to ejaculation.

17. • Wash hands and penis with soap.
• Dry hands and soap with a fresh disposable towel.
• Ejaculate into the wide mouthed sterile container.
• Precautions :
- Lubricants should be avoided as it can interfere with motility.
- Sample should not be collected in condoms as the lubricant applied in
condoms can be spermicidal.
- Entire sample should be collected. The first part of the ejaculate contains
70% of sperms.

18. • If retrograde ejaculation is suspected, a post ejaculation urine sample is
collected and examined for presence of sperms.
• Other methods of collection:
- Coitus interruptus
- Condom collection
• Longer period of abstinence – Increased volume and reduced motility.
• Shorter period of abstinence – Lower sperm count.

19. • If the sample is collected at home it should be delivered to the laboratory within
1 hour of collection.
• During transport the sample should be kept between 20 to 370 C.
• Semen samples may contain infectious agents and should be handled carefully
as they may be potential biohazards -
oHIV
oHepatitis
oHerpes

20. LABELLING OF SAMPLE
• Patient name
• Age
• Doctor name
• Laboratory analysis form
The period of abstinence in days
Date and time for collection
Place of collection
Method of collection
Time interval from collection to analysis

21. TIMING OF ANALYSIS
• Semen sample should be examined ideally within 30 min and absolutely
within 1 hour of collection.
• Motility decreases significantly after 2 hours.
SAMPLE REJECTION
• Unlabelled samples.
• Incomplete ejaculate.
• Samples received after 1 hour of ejaculation.
• Inadequately transported samples should not be accepted.

22. MACROSCOPIC
EXAMINATION

23. LIQUEFACTION
• Immediately after ejaculation – Semen is typically a semisolid coagulated
mass due to enzyme protein kinase from seminal vesicles.
• Enzymes of prostate (PSA) initiate liquefaction.
• Protein fragments are degraded into free amino acids and ammonia.
• FAILURE TO LIQUEFY INDICATES INADEQUATE PROSTATIC
SECRETION.

24. • Within a few minutes – the semen begins to liquefy (become thinner) at
which time a heterogenous mixture of lumps will be seen in the fluid.
• As liquefaction continues, the semen becomes more homogenous and quite
watery.
• In the final stages - only small areas of coagulation remain.
• Normal time – 15 to 30 min after collection.
• If the sample does not liquefy within 30 min then wait for another 30 min as
rarely it may take upto 60 min.

25. • Normal liquefied semen samples may contain jelly like granules (gelatinous
bodies) which do not liquefy – No clinical significance.
• Presence of mucin strands – Indicate incomplete liquefaction.
• Immobilized spermatozoa gain the ability to move as the semen liquefies.
• So if immobilized spermatozoa is seen in the microscopic examination – More
time must be allowed for the liquefaction process to be completed.
• During liquefaction – Continuous gentle mixing or rotation of the sample
container on a two dimensional shaker – either at room temperature or in an
incubator at 37 0C – helps to produce a homogenous sample.

26. DELAYED LIQUEFACTION
• Additional treatment may be required for liquefaction.
• Treatment with PLASMIN (0.35 - 0.50 units/ml) or CHYMOTRYPSIN (150
units/ml) or BROMELAIN (1 ml semen + 10 μL reagent X 30 min).
• Addition of an equal volume of physiological medium.
(Ex. Dulbecco’s phosphate buffered saline).
• Inhomogenenousity can be reduced by repeated (6 to 10 times) gentle passage
through a blunt 18 gauge needle attached to a syringe.

27.  Absence of initial coagulation : Congenital absence of vas deferens,
seminal vesicles or obstruction of the ejaculatory duct.
 Incomplete liquefaction : Dysfunctional accessory reproductive organs
like prostate – decreased production of prostatic enzymes.

28. SEMEN VISCOSITY
• Freshly ejaculated semen – Highly viscous due to substrate produced by
seminal vesicles.
• The coagulum liquefies spontaneously to form a translucent viscous
fluid by the action of enzymes of prostatic origin.
• Procedure : Gently aspirate sample into a wide bore 5 ml plastic disposable
pipette – allow the semen to drop by gravity – observe the length of any thread.
• A normal sample leaves the pipette in small discrete drops.
• If the viscosity is abnormal – the drop will form a thread more than 2 cm long.

29. • High viscosity –
- Poor penetration of cervical mucus.
- Interfere with the determination of sperm motility, sperm concentration,
detection of antibody coated spermatozoa and measurement of biochemical
markers.
• Low or Absence of viscosity points to reduced cell content and may be
due to mixing of urine with semen.
• The semen from males with bilateral congenital absence of vas deferens
and seminal vesicles fails to coagulate due to absence of substrate.

30. APPEARANCE OF THE EJACULATE
• Assessed after liquefaction
• Normal - Homogeneous, grey-opalescent appearance.
• The colour may also be different –
 Slightly yellow – Prolonged abstinence
 Deep yellow – Pyospermia, jaundice or taking certain vitamins or drugs
 Red-brown - Red blood cells are present (hemospermia)
 Rust colour - Small bleedings in seminal vesicles
 Increased turbidity - Inflammatory process in some part of the reproductive tract.

31. SEMEN VOLUME
• Normal : ≥ 1.5ml
• Sample is measured
using a Pasteur’s pipette
to transfer the entire
amount into a graduated
tube and readings are
taken from the markings.

32. • Low semen volume causes:
– Obstruction of the ejaculatory duct
– Congenital bilateral absence of the vas deferens (CBAVD)
– Partial retrograde ejaculation
– Androgen deficiency
– Incomplete collection
• High semen volume
– may reflect active exudation in cases of active inflammation of the
accessory organs.

33. Semen pH
• Reference value - ≥ 7.2
• pH of the semen reflects the balance between
the pH values of the different accessory gland
secretions.
• Measured after liquefaction preferably after 30
minutes.
• Should be measured within 1 hour of
ejaculation since it is influenced by the loss of
CO2 that occurs after production.

34. • Seminal vesicles : alkaline secretion
• Prostate : acidic secretion
Procedure:
• Mix the semen sample well.
• Spread a drop on pH paper.
• Wait for 30 seconds to note the color change.
• Match the color change with the color strip provided and note the
corresponding pH value.

35. • High pH
- Acute infections of prostate, seminal vesicles and epidydmis.
• Low pH
- Contamination with urine.
- Obstruction of ejaculatory ducts.
- Specimen mainly consists of prostatic fluid.

36. MICROSCOPIC
EXAMINATION

37. SPERM MOTILITY
• Reference range:
– Total motility (PR + NP) ≥ 40%
– Progressive motility (PR) ≥ 32%
• Also look for:
– Mucus strand formation.
– Sperm aggregation or agglutination (clumping).
– Cells other than spermatozoa. Eg. Epithelial cells, Round cells
(Leukocytes and Immature germ cells) and Isolated sperm heads or tails.

38. • Mix the semen sample well.
• Remove an aliquot of semen immediately after mixing.
• Place a standard volume of semen. Eg. 10 μl onto a clean glass slide.
• Cover it with a coverslip. Eg. 22 mm × 22 mm for 10 μl to provide a
chamber approximately 20 μm deep (D= V/A).
• Wait for the sample to stop drifting (within 60 seconds) and then assess the
slide.
• For motility assess at least 200 spermatozoa.
MAKING A WET MOUNT PREPARATION
FOR MOTILITY

39. Categories of sperm movement
• Progressive motility (PR): Spermatozoa moving actively either linearly or
in a large circle regardless of speed.
• Non-progressive motility (NP): All other patterns of motility with an
absence of progression.
– e.g. swimming in small circles or when only a flagellar beat can be observed.
• Immotility (IM): No movement.
• Slowly progressive : crooked, curved, slow forward movement.

40. How to assess motility?
Where to look for?
• Wait for the sample to stop drifting and the
under high power look for spermatozoa in an
area 5 mm away from the edges of the
coverslip.
• Systematically scan the slide to avoid
counting in the same field.

41. • Assess the motility of all spermatozoa within a
defined area of the field.
• First count PR cells.
• Next count NP spermatozoa.
• Finally IM spermatozoa.
• With experience, it may be possible to score all three categories of
sperm movement at one time.

42. HABITUAL FACTORS AFFECTING SPERM DENSITY /
MOTILITY
• High intake of Soya – Decrease sperm density
• High consumption of Tobacco – Decrease sperm density / motility
• Consumption of Cocaine / Marijuana – Decrease sperm motility
• Vaginal lubricants – Decrease sperm motility
• Alcoholism – Affects all semen parameters.

45. Aggregation of spermatozoa
• The adherence of immotile spermatozoa to each other.
OR
• Adherence of motile spermatozoa to non sperm elements like
mucus strands, epithelial cells or debris.

47. Agglutination of spermatozoa
• Adherence of motile spermatozoa to each other - head to head, tail to tail or in a
mixed way.
• The presence of agglutination - suggestive of the presence of anti-sperm antibodies
– indicates immunological infertility.
• Severe agglutination can affect the assessment of sperm motility and concentration.

49. IMMATURE GERM CELLS
• Round cells with relatively large
amount of cytoplasm and highly
condensed nucleus.
• Few immature cells are normally
present - 4 to 6 / hpf.
• Indicates spermatogenesis
dysfunction at the testicular level.

50. PUS CELLS
• 3-4 /hpf leucocytes are normally
present in the semen.
• Normal range - Fewer than 1
million WBC’s per mL
• Leukocytospermia or
Pyospermia if levels above
1 × 106 WBC/mL.
• Seen in infection due to
inflammation of the accessory
glands.

51. RBCs
• Normally absent.
• Presence indicates –
infection, inflammation or
trauma of the reproductive
tract.

52. • Bacteria, Yeasts
and Trichomonas
can be present
denoting specific
pathologies.
• May be
responsible for
male infertility.
Trichomonas

53. SPERM VITALITY
• Mandatory - If the percentage of motile cells is low.
• Estimated by assessing the membrane integrity of the cells.
• Assessed after liquefaction - at 30 mins.
• Normal - ≥ 58% live forms
• High percentage of immotile and non-viable cells - Necrozoospermia –
may indicate :
- Epididymal pathology
- Immotile live sperms - Immotile cilia syndrome (structural defects in the
flagellum )

54. 2 methods :
1) The dye exclusion method - (using eosin-nigrosin)
• Most commonly used
• Principle: Damaged plasma membranes of non-vital (dead) cells allow
entry of membrane-impermeant stains.
2) The hypo-osmotic swelling test (HOS)
• Alternative method
• Principle : Only cells with intact membranes (live cells) will swell in
hypotonic solutions

55. Vitality test using Eosin–Nigrosin
• Eosin-Nigrosin is a staining technique that assesses the vitality of a sperm sample
when the initial motility is less than 25 %.
• Nigrosin increases the contrast between the background and sperm heads, making
sperm easier to visualize.
• Eosin stains only the dead sperm, turning them a dark pink, whereas live sperm
appears white.

56. – Mix the semen sample well.
– Remove a 50μl aliquot of semen and mix with an equal volume of
eosin–nigrosine suspension in a test tube and wait for 30 seconds.
• Make a smear on a glass slide and allow it to dry in air.
• Examine immediately after drying or later after mounting.
• Count the number of stained (dead) or unstained (vital) cells.
METHOD :

57. Eosin-nigrosin staining: After the sperm
die, the permeability of membrane
increases, and the Eosin-Y staining then
can penetrate and stain the sperm red,
while the live spermatozoa will not be
stained. The black background formed by
nigrosin would make the staining
difference between dead and live
spermatozoa much more observable.

59. Vitality test using hypo-osmotic swelling
• Spermatozoa with intact membranes swell within 5 minutes in hypo-osmotic
medium and all flagellar shapes are stabilized by 30 minutes
Procedure :
• Incubate 1 ml hypo-osmotic saline for 15 mins.
• Add 100 μl of semen to hypo osmotic saline and incubated at 370 C for 5 or
30 mins.
• Transfer a 10 μl aliquot to a clean slide and cover with a 22 mm × 22 mm
coverslip.
• Swelling of the sperm tail is examined under phase contrast microscope.
• Sperms which have active membrane swell after 30mins.

60. Scoring :
• Live spermatozoa are identified by changes in the shape of the cell as
indicated by coiling of the tail- Swollen spermatozoa
• Dead spermatozoa - no change.

62. Response of spermatozoa
for Hypo-Osmotic Swelling
(HOS) test.
'A' indicates positive
response by spermatozoa
which show coiled tails.
'B' indicates negative
response by spermatozoa
which show uncoiled tails.

63. • Microscopic observation of different grades of sperm swelling. The different sperm
swelling grades (A–G) as observed using (A) phase-contrast microscopy of fresh
samples and (B) fixed and stained samples using normal light microscopy.

64. SPERM CONCENTRATION AND SPERM COUNT
“TOTAL SPERM COUNT” and “SPERM CONCENTRATION” are not
synonymous.
• Sperm concentration - Number of spermatozoa per unit volume of semen
• Normal - ≥ 15 x 106/ml
• It is a function of the number of spermatozoa emitted and the volume of
fluid diluting them.
• Total sperm number/count - Total number of spermatozoa in the entire
ejaculate and is obtained by multiplying the sperm concentration by the
semen volume.
• Normal - ≥ 39 x 106/ml

65. TOTAL NUMBER OF SPERMATOZOA
• Reflects sperm production by the testes.
• Patency of the duct.
TOTAL FLUID VOLUME (ACCESSORY GLANDS)
• Reflects the secretory activity of the glands which is important for
proper sperm function.

66. How to do total sperm count ?
• Improved Neubauer chamber used.
Principle : The sperm count is done after
liquefaction in a counting chamber following
dilution and total number of spermatozoa is
reported in millions / mL. (106/mL)
Method :
• Semen is diluted 1:20 with sodium
bicarbonate formalin diluting fluid.
• Take 1 ml liquefied semen in a graduated
tube and fill with diluting fluid to 20 ml
mark. Mix well.

67. • A coverslip is placed over the Neubauer
counting chamber and the counting chamber
is filled with well mixed diluting semen
sample using a Pasteur pipette.
• The chamber is then placed in a humid box
for 10-15 min for spermatozoa to settle.
• The chamber is placed on the microscope
stage. Using the 20X or 40X objective and iris
diaphragm lowered sufficiently to give
sufficient contrast.
• Number of spermatozoa is counted in 4 large
corner WBC squares.

68. • Count the sperms in all 4 WBC squares.

70. • Sperm count = Sperms counted X
Correction factor for dilution
No.of squares counted X Volume of 1 square
X 1000
• Sperm count =
Sperm counted X 20
4 X 0.1
X 1000
• Sperms counted in 4 squares ╳ 50000 = Count in millions / mL of ejaculate
• Total sperm count / ejaculate = Sperm count in millions/mL╳ Volume of the ejaculate
in mL
• Oligospermia or Azoospermia can result from many endocrine and obstructive
causes and need to be interpreted along with basal testosterone , LH and FSH
levels.

71. Normal morphology of spermatozoa
• Head : consists of nucleus with condensed chromatin and some
nuclear vacuoles.
• Acrosome: anterior 2/3rd of the head shows an acrosome cap.
– 40- 70% of head.
– secretes 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.

72. Preparation of semen smears
• Ramonowsky stain (Leishman stain) is used to stain and study the
sperm morphology.
• Take a clean glass slide and place 5-10 μL of well mixed semen
sample at one end.
• Now take another slide at an angle of 450 and pull the drop preparing a
smear.
• Allow it to air dry and stain using Leishman stain.
• Examine the smear under low, high and oil immersion objectives to
assess the morphology and note the defects in head, midpiece or tail
region.

73. • Sperm morphology can predict fertility.
• The lower reference normal limit for normal form – 4%.
• Many sperms can have multiple defects.
• Average number of defects per sperm is called Teratozoospermic Index – is a
significant predictor of sperm function.

74. ABNORMAL FORMS
• Head defects:
• Large or small
• Tapered
• Pyriform
• Round
• Amorphous
• Vacuolated (more than two vacuoles or >20% of the head area occupied by
unstained
• Vacuolar areas)
• Vacuoles in the post-acrosomal region
• Double heads or any combination of these

75. Neck and midpiece defects:
• Asymmetrical insertion of the midpiece into the head
• Thick or irregular
• Sharply bent
• Abnormally thin or any combination of these.
Principal piece defects:
• Short
• Multiple
• Broken
• Smooth hairpin bends
• Sharply angulated bends
• Irregular width
• Coiled or any combination of these.

76. • Excess residual cytoplasm (ERC):
• Spermatozoa characterized by large amounts of irregular stained cytoplasm, one
third or more of the sperm head size, often associated with defective midpieces are
abnormal.
• This abnormal excess cytoplasm should not be called a cytoplasmic droplet.
• Cytoplasmic droplets – membrane bound vesicles on the midpiece at the head–neck
junction are normal components.
• If swollen, they may extend along the length of the midpiece.

77. CD vs. ERC Structure. Illustration comparing (A)
spermatozoa with typical cytoplasmic droplets (CD) and
(B) spermatozoa presenting with excess residual
cytoplasm (ERC). Also listed are specific causes of
spermiogenesis arrest that can lead to ERC.
CD and ERC under Microscope. True examples of
cytoplasmic droplets (A, B) and excess residual
cytoplasm (C, D) in human spermatozoa as examined by
microscopy. © Oxford Journals.

80. Abnormal shape of the head of spermatozoa

81. Neck and midpiece defects

82. Tail defects

83. ANTI-SPERM ANTIBODIES
• Immune response can be generated against sperm antigens.
• Since Spermatogenesis begins during puberty. So normally the tight
Sertoli-cell junctions provide the testis with a barrier that prevents the
immune system from coming in contact with the post-meiotic germ
cells.

84. • This unique barrier can be violated there by allowing the immune system to
be exposed to a large number of sperm, facilitating the production of anti
sperm antibodies.
- Testicular torsion
- Vasectomy
- Testicular trauma
- Testicular surgeries
• Sperms are exposed to immune system - lead formation of anti-sperm
antibodies.
• 60% of candidates for vasectomy reversal may have circulating anti sperm
antibodies.

85. • It is also known that men with primary infertility have a higher incidence
of anti sperm antibodies in their serum and semen than do age-matched
fertile controls.
• There is considerable evidence that sperm antibodies impair fertility by
blocking penetration of cervical mucus by spermatozoa, by interfering in
the fertilization process or possibly by exerting an embryo- toxic effect.
• The extent to which some of these effects are expressed is related to the
antibody level, immunoglobulin class, and regional specificity of these
antibodies.

86. • If spermatozoa demonstrate agglutination the presence of
sperm antibodies may be the cause.
• Sperm antibodies can be present without sperm
agglutination.
• Anti-sperm antibodies (ASAs) in semen belong almost
exclusively to two immunoglobulin classes: IgA and
IgG.
• Normal - < 50%

87. TESTS TO DETECT ANTIBODIES
• Direct test : Tests for antibodies on spermatozoa.
• Indirect Test : Tests for anti-sperm antibodies in sperm-free fluids i.e. seminal
plasma, blood serum and solubilized cervical mucus.
2 TESTS :
1. Mixed antiglobulin reaction test (MAR test)
2. Immunobead test (IB test)
• The MAR test is performed on a fresh semen sample while the IB test uses washed
spermatozoa.

88. THE MIXED ANTIGLOBULIN REACTION TEST
(SPERM MAR TEST)
• Inexpensive, quick and sensitive screening test.
• Disadvantage : Provides less information than the Direct immunobead test.
• The MAR test is used primarily to detect the presence of IgG antibodies.

89. • The semen sample containing the motile sperm is incubated with IgG
Antihuman globulin (AHG) and a suspension of IgG coated latex
particles.
• The bivalent AHG will bind simultaneously to both the antibody on
the sperm and the antibody on the latex particles forming
microscopically visible clumps of sperm and particles.
• Less than 50% of the motile sperm attached to the particles is
considered normal.

90. Microphotography of MAR test.Mixed Antiglobulin Reaction Test

91. IMMUNOBEAD TEST
• This Immunobead test (IBT) is a technically simple assay that evaluates
immunologic infertility by identifying the classes of anti sperm
antibodies and their binding sites on the sperm surface.
• The test uses polyacrylamide beads coated with antibodies against
human immunoglobulins to bind to antibody coated sperm.
• This test is one of the most informative and specific of all the assays
currently available to detect anti sperm antibodies.

92. • The Immunobeads are microscopic polyacrylamide spheres which carry
covalently bound rabbit antibodies directed against human Immunoglobulins.
• By using beads coated with Ig class specific second antibodies one can
identify the different antibody classes involved (IgG, IgA, IgM).
• IgAs are considered to be more detrimental than IgGs to sperm function and is
associated with poor prognosis.
• Immunologic infertility secondary to vasectomy shows predominance of IgG.

94. • The Immunobead test also identifies the location on the sperm cell (head,
midpiece, tail) to which the antibody is bound.
• Depending on the region of the sperm to which the antibody is attached,
different sperm functions may be affected.
• Antibodies attached to the sperm tail may hinder sperm motility and transport
and those attached to the head region could interfere with sperm binding to the
zona pellucida or block entry of the sperm into the ooplasm itself.
• Head-directed antibodies, particularly of the IgA class, can interfere with
fertilization likely through interference with sperm–zona interaction.

95. The direct method
• This IBT can be performed directly to assess native antibodies on sperm by
binding immunobeads to the target sperm surface.
• This is the method of choice for assessing men who have adequate numbers of
motile sperm in their semen.
2 METHODS

96. The indirect method
• This is performed after passive transfer of antibody from a body fluid (blood,
serum, seminal plasma, cervical mucus or follicular fluid) to donor sperm.
• Donor sperm that have been found to be negative for anti sperm antibodies by
testing previously by the direct method are used for the passive transfer method.
• ABNORMAL - > 50% or more adherence of Immonobead particles to motile
spermatozoa.
• Suggests strong evidence for an immunological cause of infertility.

98. Biocemical analysis for accessory sex organ function
• Prostate gland function
– Zinc
– Citric acid
– Acid phosphatase
– α-glutamyl transpeptidase
• Seminal vesicles
– Fructose
– Prostaglandins
• Epididymis
– Neutral Alpha glucosidase
– Free L-carnitine
– Glycerophosphocholine (GPC)

99. Estimation of fructose
• Fructose in semen reflects the secretory function of the seminal vesicles.
• Test used – Seliwanoff’s test.
• Seliwanoff’s reagent preparation : Dissolve Resorcinol - 50mg in Conc.
HCl - 33ml and add distilled water- 67ml to make up the volume to 100
ml.

100. Principle :
• Under the influence of heat and low pH, fructose forms a coloured
complex with indole.
• Fructose + Indole – complex which absorbs light of wavelength 470
nm.

101. Procedure:
• 5 ml resorcinol reagent + 1 ml of semen in a test tube
• Mix and place in a boiling water bath for 5 min or heat.
• Positive test is indicated by formation of Red coloured product in 30
seconds.
• In quantitative assays, this is compared with a known fructose standard at
490 nm.
• Normal level of fructose: 150-300mg/dl.
• Reduced levels: Seminal vesicle dysfunction, High sperm count,
obstructed vas deferens.

102. COMPUTER -AIDED SPERM ANALYSIS
• CASA is a semiautomated technique.
• Provides data on Sperm density, Motility and Morphology.
Advantages:
- High precision.
- Quantitative assessment of sperm kinetics.
- Capable of handling many samples without undergoing fatigue.
- Morphological defects can be made out Interlaboratory comparison
possible.

103. Disadvantages:
- Expensive equipment.
- Participation of a technician.
- Hence not used for routine semen analysis.
- Commonly done in high volume andrology labs.

104. Normal values (lower reference limits) of semen analysis
(WHO 2010)

105. Biochemical variables of semen analysis

106. SPERM FUNCTION ASSESSMENT
• Sperm- mucus interaction assay
• Acrosome reaction testing
• Sperm penetration assay
– Cervical mucus penetration assay
– Hamster egg penetration assay

107. SPERM-MUCUS INTERACTION/ POSTCOITAL TEST/
SIMS – HUHNER TEST
• Assess cervical environment as a cause of infertility.
• Cervical mucus - heterogenous fluid - cyclical changes in consistency .
• Postcoital test (PCT)- Conducted when the cervical mucus is thin and clear
just before ovulation.
• Cervical mucus is aspirated and examined 2 to 8 hours after normal
intercourse.
• Progressively motile sperm > 10 to 20 per HPF is designated as normal.
• Abnormal test - advised to proceed with Intra uterine Insemination.
– Inappropriate timing testing / intercourse
– Anatomic abnormalities
– Semen or cervical mucus anti sperm antibodies
– Abnormal sperm.

108. SPERM PENETRATION ASSAYS (SPA)
• SPA analyses the ability of a man’s sperm to attach to the egg
membrane, penetrate the egg and undergo decondensation.
• These are the necessary first steps of actual fertilization.
• Indication - Unexplained infertility / IVF failure
• Principle - A normal spermatozoa can bind and penetrate the oocyte
membrane.

109. HAMSTER EGG PENETRATION ASSAY
• A man’s sperm is mixed with hamster eggs resembling human ovum that
have been pre treated with enzymes to remove the zona pellucida in a
laboratory.
• The oocytes are examined microscopically for sperm penetration.
• Penetrations are indicated by swollen sperm heads within the oocyte
cytoplasm.
• Normally, 10% to 30% of ova are penetrated.
• Oligozoospermic and severely teratospermic men – Negative.

110. Sperm capacitation index (SCI)
• It is a variant of the SPA test assessing the mean number of penetrations per
ovum.
• Normal - > 5
• ICSI ( Intra cytoplasmic sperm injection) has been recommended - SCI less
than 5 instead of standard IVF procedures.

111. Cervical Mucus Penetration Test
• In this test, greatest distance travelled by the sperm in seminal fluid
placed and incubated in a capillary tube containing bovine mucus is
measured.
• Majority of fertile men show score >30 mm, while most infertile men
show scores <20 mm.

112. ACROSOME REACTION
• The Acrosome is a membrane-bound organelle covers the anterior 2/3
of the sperm head.
• Acrosome reaction is an important prerequisite for successful
fertilization.
• During fertilization a sperm must first fuse with the plasma membrane
penetrate the female egg cell in order to fertilize it - Involves fusion of
acrosomal membrane and plasma membrane.

113. • Acrosin and Hyaluronidase – required to digest the oocyte cumulus
cells and zona pellucida.
• Acrosome reaction testing - not widely practiced in laboratories -
research interest.
• Profound abnormalities of head morphology.
• Unexplained infertility.

116. In - vitro
fertilization
The egg and the
sperm (of which
there are multiple)
are left in a petri
dish to fertilise on
their own.

117. Intra cytoplasmic
sperm injection
One sperm is
directly injected
into the egg.