2. SEMEN
■ Concentrated suspension of spermatozoa in seminal plasma
■ Ejaculate four fractions:
■ PRE – EJACULATORY FRACTION :
– COWPER’S or LITTER’S GLANDS
■ PRELIMNARY FRACTION :
– PROSTRATE GLAND.
– ODOUR
– Enzymes which liquefies the spermatozoa coagulum.
■ MAIN FRACTION :
– SEMINALVESICLES,TESTES, EPIDIDYMIS
– Prostate gland partially
■ TERMINAL FRACTION :
– Seminal vesicles
– Gelatinous
– Immotile spermatozoa.
Majority of spermatozoa
3. FRACTION OF SEMEN CONTRIBUTED BY
VARIOUS GLANDS
■ Urethral glands 2 - 4%
■ Prostate 20-30%
– Acidic fluid
– citrate, zinc, acid phosphatase and proteolytic enzymes
■ Seminal vesicles 46-80 %
– alkaline viscous
– yellowish secretion rich in fructose, vitaminC, prostaglandin, protein kinase
■ Testis & Epididymis: 5-10%
– Epididymis α-glucosidase isoenzyme
4. ■ Human sperm cell is about 65 µm long.
■ The head size: 4-5µm
– Nucleus - contains the 23 chromosomes
– Acrosome
■ Mid-piece: 4-5µm
– The energy for motility is generated
■ Tail: 55µm
– Motility -Propagated along the tail.
5. 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 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, e.g. in vitro fertilization, gamete
intrafallopian transfer technique.
7. After reversal of vasectomy to confirm the success of procedure.
6. Semen analysis involves the following steps :
■ First 5 minutes:
Placing the specimen container on the bench or in an incubator (37°C) for liquefaction.
■ Between 30 and 60 minutes:
– Liquefaction and appearance
– Semen volume.
– Semen pH
– Wet preparation
■ microscopic appearance
■ sperm motility
■ dilution required
– Sperm vitality
– Sperm morphology.
– Sperm concentration.
7. – Sperm number.
– Mixed antiglobulin reaction (MAR) test (if required).
– Assessing peroxidase-positive cells (if round cells are present).
– Immunobead test (if required).
– Centrifuging semen (if biochemical markers are to be assayed).
■ Within 3 hours:
– Sending samples to the microbiology laboratory (if required).
■ After 4 hours:
– Sperm morphology.
■ Later on the same day (or on a subsequent day if samples are frozen):
– Accessory gland markers (if required).
– Indirect immunobead test (if required).
8. Sample collection - Preparation
■ Sexual abstinence – 2- 7 days
■ Two separate samples at least 7 days apart should be analyzed
■ The duration of abstinence should be constant
■ Sample collection - private room in the same laboratory
■ Recommended procedure – Masturbation
■ Pre warmed (21oC), sterile, non-toxic, wide-mouth container.
9. Sample collection - Preparation
■ Pass urine
■ Wash hands and penis with soap
■ Rinse away the soap
■ Dry hands and penis with a fresh disposable towel
■ Ejaculate into sterile container
■ Precautions :
– Lubricants should be avoided - interfere with motility
– Collect the entire sample -70% of sperms is in the first part of the ejaculate.
10. ■ If retrograde ejaculation is suspected, a post-ejaculate urine sample is collected and
examined for presence of sperms.
■ Other methods of collection:
– Coitus interruptus
– Condom collection
■ Longer period of abstinence increase volume & reduced motility
■ Shorter period of abstinence lower sperm count
11. LABELLING OF SAMPLE
■ Patient name
■ Age
■ Clinic or Doctor name
■ Laboratory analysis form:
■ The period of abstinence (in days)
■ Date &Time of collection
■ Place of collection
■ Method of collection
■ Complete or incomplete
■ The time interval from collection to analysis.
12. Collection of semen at home / condom
collection
■ Only in exceptional circumstances :
■ At home:
– demonstrated inability to produce a sample by masturbation in the clinic
– the lack of adequate facilities near the laboratory.
■ Condom collection during sexual intercourse
– demonstrated inability to produce a sample by masturbation.
– Special non-toxic condoms given
■ Instructions about collection and transport of the semen sample.
■ Semen sample needs to be complete
– Should be noted in report if the sample is incomplete.
■ The man should record the time of semen production
■ Should deliver the sample to the laboratory within 1 hour of collection.
13. ■ During transport sample kept between 20 °C and 37 °C.
■ The report should note that the sample was collected at home or another location
outside the laboratory.
■ Coitus interruptus not recommended because the
– first portion of the ejaculate may be lost.
– Cellular and bacteriological contamination of the sample
– Low pH of the vaginal fluid could adversely affect sperm motility.
14. Safe handling of specimen
■ Semen samples may contain dangerous infectious agents and should be handles as bio - hazard
– human immunodeficiency virus (HIV),
– hepatitis viruses
– herpes simplex virus
■ What are the safety precautions????
15. TIMING OF ANALYSIS
■ Semen is placed at 37° C in a incubator for 30 minutes.
■ Continuous gentle mixing or rotation of the sample on a two-dimensional shaker is
mandatory to produce a homogenous sample after liquefaction
■ The semen sample should be examined, Ideally within 30 mins ; Absolutely within 1
hour of collection.
■ Motility decreases significantly after 2 hours
18. 1. Liquefaction
■ Immediately after ejaculation semen is typically a semisolid coagulated mass
due to enzyme PROTEIN KINASE from seminal vesicles.
■ Liquefaction is initiated by enzymes of prostatic origin.
■ Protein fragments degraded into free amino acids and ammonia
■ Failure to liquefy indicates inadequate prostatic secretion
■ Within a few minutes the semen begins to liquefy (become thinner), at which time a
heterogeneous mixture of lumps will be seen in the fluid.
■ As liquefaction continues, the semen becomes more homogeneous and quite watery,
■ In the final stages only small areas of coagulation remain.
19. ■ Normal – 15- 30 minutes after collection
■ The complete sample usually liquefies within 15 minutes at room temperature
■ If the semen does not liquefy within 30 minutes, do not proceed with semen analysis
but wait for another 30 minutes.
■ Rarely it may take up to 60 minutes or more.
■ If complete liquefaction does not occur within 60 minutes, this should be recorded.
■ Semen samples collected at home or by condom will normally have liquefied by the
time they arrive in the laboratory.
20. ■ Normal liquefied semen samples may contain jelly-like granules (gelatinous bodies)
which do not liquefy – no clinical signifi cance.
■ The presence of mucus strands indicate incomplete liquefaction
■ Liquefaction recognized both macroscopically and microscopically.
■ Immobilized spermatozoa gain the ability to move as the semen liquefies.
■ If immobilized spermatozoa seen in 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 set at 37 °C
help to produce a homogeneous sample.
21. ■ Delayed liquefaction
■ Additional treatment, mechanical mixing or enzymatic digestion may be necessary.
■ Treatment with PLASMIN 0.35 – 0.50 units/ ML or CHYMOTRYPSIN 150 units / ml or
BROMELAIN ( 1ml semen +10µL reagent x 30 mins)
■ Addition of an equal volume of physiological medium (e.g. Dulbecco’s phosphate-
buffered saline), followed by repeated pipetting.
■ Inhomogeneity can be reduced by repeated (6–10 times) gentle passage through a
blunt gauge 18 (internal diameter 0.84 mm) or gauge 19 (internal diameter 0.69 mm)
needle attached to a syringe.
22. ■ ABSENCE OF INITIALCOAGULATION :
■ 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.
23. 2. 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.
■ Viscosity of the sample estimated After liquefaction
■ Procedure : gently aspirate sample into a wide-bore 5ml 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 viscosity is abnormal
the drop will form a thread more than 2 cm long.
■ Alternative method introduce a glass rod into the sample and observe the length of the
thread that forms upon withdrawal of the rod.
24. ■ Viscous semen specimen
– homogeneous stickiness
– consistency will not change with time
■ The methods to reduce viscosity are the same as those for delayed liquefaction.
■ High viscosity interfere with determination of sperm motility, sperm
concentration, detection of antibody-coated spermatozoa and measurement of
biochemical markers.
■ Absence of viscosity points to reduced cell content.
■ The semen from males with b/l congenital absence of vas deferens & seminal vesicles
fails to coagulate due to absence of substrate.
25. 3. Appearance of the ejaculate
■ Assessed after liquefaction
■ Normal homogeneous, grey-opalescent appearance.
■ Less opaque if sperm concentration is very low
■ 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 (haemospermia)
■ Trauma to the genital tract
■ Inflammation
■ Tumor of the genital tract.
– Rust colour - Small bleedings in seminal vesicles
– Increased turbidity - inflammatory process in some part of the reproductive tract.
26. 4.Semen volume
■ Precise measurement of volume essential
■ Normal : ≥ 1.5ml
■ Needed for calculation of total number of spermatozoa and non-sperm cells
■ The volume is best measured by weighing the sample in the vessel in which it is collected.
■ Collect the sample in a pre-weighed, clean, disposable container.
■ Weigh the vessel with semen in it.
■ Subtract the weight of the container.
■ Calculate the volume from the sample weight, assuming the density of semen to be 1 g/ml (Auger et
al., 1995) (volume = mass / density).
■ Semen density varies between 1.043 and 1.102 g/ml (Huggins et al., 1942; Brazil et al., 2004a; Cooper
et al., 2007).
27. ■ Alternative method : Measured by aspirating into a pipette or by using a syringe(non-toxic
1,2 or 5ml)
■ Low semen volume causes:
– Obstruction of the ejaculatory duct
– Congenital bilateral absence of the vas deferens (CBAVD), a condition in which the seminal
vesicles are also poorly developed.
– Partial retrograde ejaculation
– Androgen deficiency
– Inadequate erection & improper mood at collection
– Incomplete collection
■ High semen volume
– may reflect active exudation in cases of active inflammation of the accessory organs.
28. 5. Semen pH
■ Semen is the strongest buffer in the body.
■ Reference value - ≥ 7.2
■ 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.
■ Procedure:
■ For normal samples: pH paper in the range 6.0 to 10.0 should be used.
■ For viscous samples, the pH of a small aliquot of the semen can be measured using a pH meter
designed for measurement of viscous solutions
29. ■ pH < 7.0
– With absence of sperm ejaculatory duct obstruction or congenital bilateral absence of the vas
deferens, a condition in which seminal vesicles are also poorly developed.
– contaminated with urine.
■ Semen pH increases with time, as natural buffering decreases, so high pH values may provide
little clinically useful information.
31. ■ Scan the wet smear at x100 magnification
■ Look for:
– mucus strand formation
– sperm aggregation or agglutination
– cells other than spermatozoa, e.g. epithelial cells, “round cells” (leukocytes and
immature germ cells) and isolated sperm heads or tails.
■ The preparation should then be observed at ×200 or ×400 total magnification
■ For:
– assessment of sperm motility
– determination of the dilution required for accurate assessment of sperm number
32. Making a wet preparation
■ Mix the semen sample well
■ Remove an aliquot of semen immediately after mixing
■ Place a standard volume of semen, e.g. 10 µl, onto a clean glass slide.
■ Cover it with a coverslip, e.g. 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.
33. ■ NOTES:
■ Depth < 20 µm constrains the rotational movement of spermatozoa
■ If chamber too deep difficult to assess spermatozoa as they move in and out of
focus.
■ If the number of spermatozoa per visual field varies considerably, the sample is not
homogeneous.
■ In such cases, the semen sample should be mixed again thoroughly and a new slide
prepared as above.
■ Lack of homogeneity – other causes :
– abnormal consistency
– abnormal liquefaction
– aggregation of spermatozoa
– sperm agglutination.
34. 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
■ NON- SPECIFICAGGREGATION / ? Accessory gland infection
35. 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
further testing is required
■ Severe agglutination can affect the assessment of sperm motility and concentration.
38. Sperm Motility
■ Examine the slide with phase-contrast optics at ×200 or ×400 magnification
■ Assess minimum 200 spermatozoa in a total of at least five fields per replicate for the
percentage of different motile categories.
■ Assesed after liquefaction , at 30 mins
■ Compare the replicate values to check if they are acceptably close.
■ If so, proceed with calculations; if not, prepare new samples.
■ Reference range:
– Total motility (PR + NP) ≥40%
– Progressive motility (PR) ≥ 32%
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, the flagellar force hardly displacing the head, or when only a
flagellar beat can be observed
■ Immotility (IM): no movement.
■ Previous edition: PR divided into rapidly progressive
and slowly progressive
■ Slowly progressive – crooked, curved, slow forward
movement
40. How to assess motility?
■ Where to look for?
– 5 mm away from the edges of the coverslip (to prevent observation of effects of drying
on motility)
■ Systematically scan the slide to avoid repeatedly viewing the same area.
■ Change fields often and randomly
■ Start scoring a given field at a random instant.
■ Do not wait for spermatozoa to swim into the field to begin scoring.
■ 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
41. ■ Aids to assessing sperm motility
(a) An eyepiece reticle makes it easier to count motile and immotile spermatozoa.
(b) Systematic selection of fields for assessment of sperm motility, at least 5 mm from
the edges of the coverslip.
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.
44. Sperm vitality
■ Mandatory - if the percentage of motile cells is low
■ Estimated by assessing the membrane integrity of the cells
■ Assesed after liquefaction , at 30 mins
■ Can be done routinely on all samples
■ 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 )
45. ■ 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
46. Vitality test using eosin–nigrosin
■ one-step staining technique
■ Nigrosin used to increase the contrast between the background and the sperm heads
■ Can be assessed with eosin alone also.
■ Method :
– Mix the semen sample well
– Remove a 50µl aliquot of semen and mix with an equal volume of eosin– nigrosin
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
47. ■ Examine each slide with brightfield optics at ×1000 magnification and oil immersion.
■ Count the number of stained (dead) or unstained (vital) cells
■ Evaluate 200 spermatozoa in each replicate for low sampling error
■ Leaky neck membrane :
■ Stain is limited to only a part of the neck region, and the rest of the head area is unstained
■ NOT a sign of cell death and total membrane disintegration
■ These cells counted as alive
48.
49.
50. 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 37deg 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.
51. ■ Scoring :
■ Live spermatozoa are identified by changes in the shape of the cell, as indicated by
coiling of the tail Swollen spermatoxoa
■ Dead spermatozoa no change
55. Sperm number
■ “TOTAL SPERM NUMBER” 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 total number of spermatozoa in the entire ejaculate and is
obtained by multiplying the sperm concentration by the semen volume.
■ Normal - ≥ 39 x 106
56. 8 Steps for estimation of Sperm Number
1. Examine a wet mount to determine the appropriate dilution and appropriate
chambers to use
2. Mixing semen and preparing dilutions with fixative
3. Loading the haemocytometer chamber and allowing spermatozoa to settle in a
humid chamber.
4. Assessing the samples within 10–15 minutes (after which evaporation has noticeable
effects on sperm position within the chamber).
5. Counting at least 200 spermatozoa per replicate.
6. Comparing replicate counts to see if they are acceptably close. If so, proceeding with
calculations; if not, preparing new dilutions.
7. Calculating the concentration in spermatozoa per ml.
8. Calculating the total number of spermatozoa per ejaculate.
57. Counting chamber
■ 100 µm deep haemocytometer chamber recommended
■ Most commonly used – Improved Neubauer chamber
■ Disposable chambers also available
■ Shallow chambers not recommended
■ Improved Neubauer chamber – consists 9 grids
■ Each grid hold 100nL
■ Cover slip to be used – thickness no 4 , 0.44mm
■ After cleaning , soak reusable chambers and coverslips overnight in disinfectant to
avoid contamination with potentially infectious agents in semen.
60. 2 & 3. Preparing dilutions &Loading the
haemocytometer chamber
■ Fixative for diluting semen – SODIUM BICARBONATE FORMALIN DILUTING FLUID
– Dissolve 50 g of sodium bicarbonate (NaHCO3) and 10 ml of 35% (v/v) formalin in 1000 ml
of purifi ed water.
– Store at 4°C
Appropriate dilutions made in 2 test tubes
Neubauer chamber loaded with both dilutions
Kept for 3-4 minutes
61. 4&5. Assessing sperm numbers in the counting chambers
and counting atleast 200 spermatozoa
■ Count at least 200 spermatozoa in each replicate, for low sampling error
■ First assess the central grid (number 5) of one side of the improved Neubauer chamber, row
by row
■ Continue counting until at least 200 spermatozoa have been observed and a complete row
(of five large squares) has been examined.
■ Counting must be done by complete rows; do not stop in the middle of a row.
■ If 200 spermatozoa are not observed in the five rows of the central grid, continue counting
in the rows (of 4 large squares) of the two adjacent grids
62. ■ Make a note of the number of rows assessed to reach at least 200 spermatozoa
■ Switch to the second chamber of the haemocytometer
■ The same number of rows will be counted from the other chamber of the
haemocytometer (the same volume) as the first replicate, even if this yields fewer than
200 spermatozoa.
■ If fewer than 200 spermatozoa are found in grids 4, 5 and 6, do not continue to count
in grids 1, 2, 3, 7, 8 or 9, since the volume of each row in these grids differs from that of
the rows in grids 4, 5 and 6
■ Calculate the sum and difference of the two numbers.
■ Determine the acceptability of the difference
■ If the difference is acceptable, calculate the concentration
64. 7 &8 . Calculation of the concentration of spermatozoa in
semen & total number of spermatozoa per ejaculate
■ Spermatozoa concentration =
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑝𝑒𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑖𝑛 𝑏𝑜𝑡ℎ 𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠
𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑤ℎ𝑖𝑐ℎ 𝑡ℎ𝑒𝑦 𝑎𝑟𝑒 𝑓𝑜𝑢𝑛𝑑
x dilution factor
■ Total number of spermatozoa per ejaculate = sperm concentration x volume of whole ejaculate
■ Each grid = 100 nL
■ Grid 4,5 & 6 contain 5 rows. So , each row contains = 100 /5 = 20 nL
■ Grid 1,2,3,7,8 & 9 contain 4 rows each , so each row = 25 nL
■ In 1: 20 and 1:5 dilution , only grids 4,5,& 6 counted, that too row wis because each row have same
volume
■ In 1:2 dilution, all grids are assessed.
65. ■ Calculation of sperm concentration for grid 4,5,6:
■ Spermatozoa concentration(per nL) =
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑝𝑒𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑖𝑛 𝑏𝑜𝑡ℎ 𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠
𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑤ℎ𝑖𝑐ℎ 𝑡ℎ𝑒𝑦 𝑎𝑟𝑒 𝑓𝑜𝑢𝑛𝑑
x dilution factor
■ i.e, SC =
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑝𝑒𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑖𝑛 𝑏𝑜𝑡ℎ 𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠 (𝑁)
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑜𝑤𝑠 (𝑛) 𝑥 20
x dilution factor
■ i.e, C = (N/n) × (1/20) × dilution factor
■ Example 1. : With a 1:20dilution, replicate 1 is found to contain 201 spermatozoa in seven rows, while
replicate 2 contains 245 spermatozoa in seven rows.
– SUM = (201 + 245) is 446 in 14 rows
– difference (245–201) is 44.
– See the acceptable difference in table = 41
– so new replicate dilutions are made
66. ■ Example 2 :With a 1:20 dilution, replicate 1 is found to contain 220 spermatozoa in four
rows, while replicate 2 contains 218 spermatozoa in four rows.The volume of whole
ejaculate is 3ml.
– Sum = (220 + 218) = 438 in eight rows
– Difference (220–218) is 2.
– See table
– Values are accepted.
– C (per nL) = (N/n) × (1/20) × dilution factor
– C = (438/8) × (1/20) x 20
= 54.75 spermatozoa/nl
= 55 × 106 spermatozoa per ml of semen
– Total sperm count = 55 x 106 x 3
= 165 x 106 spermatozoa/ ejaculate
67. ■ Example 3 :
■ With (1:20) dilution, replicate 1 is found to contain 98 spermatozoa in 15 rows (grids 5,
4 and 6), while replicate 2 contains 114 spermatozoa in 15 rows (grids 5, 4 and 6).
Volume of ejaculate 2.5ml
■ Sum (98 + 114) = 212 in 30 rows
■ Difference (114–98) is 16
■ See table
■ The values are accepted.
■ C = (212/30) × 1/20 x 20 = 7.07 spermatozoa/nl
= 7.1 × 106 spermatozoa per ml of semen
■ Total sperm number = 7.1 x 2.5 x 106 spermatozoa per ejaculatate
■ =18 X 106 spermatozoa per ejaculatate
■ Note: In this example, the sample has been overdiluted, since fewer than 200
spermatozoa were found in grids 5, 4 and 6; a 1 + 4 (1:5) dilution would have been
more appropriate.
68. ■ Example 4 :With a 1:5 dilution, replicate 1 is found to contain 224 spermatozoa in four
rows, while replicate 2 contains 268 spermatozoa in four rows.
■ The sum =(224 + 268) = 492 in eight rows
■ Difference (268–224) is 44.
■ FromTable, this is seen to exceed the difference expected by chance alone (43),
■ so new replicate dilutions are made.
69. ■ Example 5 :With a 1:5 dilution, replicate 1 is found to contain 224 spermatozoa in eight
rows, while replicate 2 contains 213 spermatozoa in eight rows.Volume of ejaculate
3ml
■ Sum (224 + 213) = 437 in 16 rows
■ Difference (224–213) is 11.
■ FromTable this is seen to be less than that found by chance alone (41), so the values
are accepted.
■ C = (437/16) x 1/20 x 5 = 6.825 spermatozoa/nl, or 6.8 × 106 spermatozoa per ml of
semen
■ Total sperm count = 6.8 x3 x 106 spermatozoa per ejaculate
■ = 20.4 x 106 spermatozoa per ejaculate
71. ■ When no spermatozoa are observed in either wet preparation, the sample can be
centrifuged to determine if any spermatozoa are present in a larger sample
■ Remove a 1ml aliquot of semen and centrifuge at 3000g for 15 minutes
■ Decant most of the supernatant and resuspend the sperm pellet in the remaining
approximately 50 µl of seminal plasma.
■ Place one 10 µl aliquot of the pellet on each of two slides and create two wet
preparations
■ The presence of spermatozoa in either replicate indicates cryptozoospermia
■ The absence of spermatozoa from both replicates suggests azoospermia.
73. Counting of cells other than spermatozoa
■ Non-sperm cells in semen:
– Epithelial cells
– Round cells (germ cells and leukocytes)
– Isolated sperm heads or tails
■ The presence of non-sperm cells in semen indicate:
■ Testicular damage (immature germ cells)
■ Pathology of the efferent ducts (ciliary tufts)
■ Inflammation of the accessory glands (leukocytes).
74. Non sperm cells
■ Leukocytes: normally (1-4/HPF)
– Leukocytospermia as levels above 1 × 106 WBC/mL – infection
■ Epithelial cells: normally (1-2/HPF)
■ Spermatocytes: (Immature germ cells) 1-2/HPF
■ Erythrocytes: (1-2/HPF). Increased number may indicate a reproductive tract infection or
damage to a small capillary during sample production.
■ Bacteria and protozoan such asTrichomonas vaginalis are uncommon in human semen but
their presence is indicative of possible male reproductive tract infection
75. ■ Estimated in fixed and stained semen smears made from undiluted semen
■ N number of round cells counted in the same number of fields as 400 spermatozoa
■ S concentration of spermatozoa
■ Then, concentration (C) of round cells can be calculated by
■ C = S × (N/400).
■ Calculations same like that of sperm count
■ If fewer than 25 round cells are counted, report the number of round cells observed with
the comment “Too few for accurate determination of concentration”.
■ If the estimate of round cell concentration exceeds 1 × 106 per ml, their nature should be
assessed by peroxidase activity or leukocyte markers
76. ■ peroxidase-positive granulocytes are the predominant form of leukocytes in semen,
■ Staining cellular peroxidase using ortho-toluidine
■ This test is quick and inexpensive, and is a useful initial screening for granulocytes.
78. 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% 0f head
– 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.
79. Preparation of semen smears
Normal semen sample
■ Undiluted semen
■ Feathering method 10µL + 450
angle + 1 second
■ Low sperm concentration :
Centrifuge – 600rpm x 10 mins
Viscous semen sample
■ Washed semen sample ( 0.2-0.5ml
semen + 10 ml saline centrifuge at
800rpm x10 mins)
■ Pipette method by Pasteur pipette
■ Sperm morphology affected
80. Staining
■ Air dried – 4 hours in pap and shorr stain
■ Fixation – ethanol / methanol
■ Recommended stains : Papanicolaou, Shorr , Diff-Quik stain (rapid method)
■ Mounted
■ Examine the morphology of at least 200 sperms
■ Normal > 4 % of sperm should have normal morphology.
81. Interpretation
■ Head - pale blue in acrosomal region and dark blue in the post-acrosomal region.
■ Midpiece - red
■ Tail - blue or reddish
■ Excess residual cytoplasm, usually located behind the head and around the midpiece
– stained pink or red (Papanicolaou stain)
– reddish-orange (Shorr stain)
82. 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
– small or large acrosomal areas (<40% or >70% of the head area)
– double heads, or any combination of these
■ Neck and midpiece defects:
– asymmetrical insertion of the midpiece into the head
– thick or irregular
– sharply bent
– abnormally thin, or any combination of these.
83. ■ Principal piece defects:
– Short
– Multiple
– Broken
– smooth hairpin bends
– sharply angulated bends
– irregular width
– coiled, or any combination of these.
■ Excess residual cytoplasm (ERC):
– associated with abnormal spermatozoa produced from a defective spermatogenic
process.
– 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
90. Antisperm antibodies
■ Immune response can be generated against sperm antigens
■ Since spermatogenesis begins during puberty
■ 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.
■ This unique barrier can be violated,
■ InTesticular torsion,Vasectomy,Testicular trauma, testicular surgeries
■ Sperms are exposed to immune system l/t formation of antisperm antibodies
91. Anti- sperm antibodies
■ 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 % WHO 2010
92. ■ Direct test :Tests for antibodies on spermatozoa
■ IndirectTest :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
■ The MAR test is performed on a fresh semen sample while the IB test uses washed
spermatozoa
Direct and Indirect
93. The mixed antiglobulin reaction test (Sperm
MARTMTest)
■ Inexpensive, quick and sensitive screening test
■ Disadvantage : provides less information than the direct immunobead test
■ Principle :
■ A “bridging” antibody (anti-IgG or anti-IgA) is used to bring the antibody-coated latex
particles into contact with unwashed spermatozoa bearing surface IgG or IgA.
■ Procedure :
■ The direct IgG and IgA MAR tests are performed by mixing fresh, untreated semen
separately with latex particles or treated red blood cells coated with human IgG or IgA.
■ To the suspensions is added a monospecific anti-human-IgG or anti-human-IgA.
94. ■ Atleast 200 motile spermatozoa are examined
■ If the spermatozoa have antibodies on their surface, antihuman immunoglobulin will
bind IgG or Ig A-coated latex particles to IgA or IgGon the surface of the spermatozoa
■ This will cause attachment of latex particles to spermatozoa, and motile, swimming
sperms with attached particles will be seen.
■ If the spermatozoa do not have antibodies on their surface, they will be seen
swimming without attached particles
■ The latex particles will show clumping due to binding of their IgG to antihuman
immunoglobulin.
95. ■ In indirect SpermMARTM tests, fluid without spermatozoa (e.g. serum etc) is tested
for the presence of antisperm antibodies.
■ Procedure : the diluted, heat-inactivated fluid suspected of containingASAs is
incubated with antibody-free donor spermatozoa that have been washed free of
seminal fluid.
■ Any ASAs in the suspect fluid will bind specifically to the donor spermatozoa,
which are then assessed in a direct test, as above.
■ For reliable results, it is important to allow sufficient time for the sperm–antibody
interaction, since it may take up to 10 minutes for the mixed agglutination to become
visible.
96. Immunobead test
■ The direct immunobead test
■ More time-consuming than the MAR test
■ Provides information about antibodies on spermatozoa that have been removed from
possible masking components in seminal plasma.
■ Beads coated with covalently-bound rabbit anti-human immunoglobulins against IgG
or IgA are mixed directly with washed spermatozoa.
■ The binding of beads with anti-human IgG or IgA to motile spermatozoa indicates the
presence of IgG or IgA antibodies on the surface of the spermatozoa.
97. ■ The indirect immunobead test
■ Used to detect anti-sperm antibodies in heat inactivated, sperm-free fluids (serum,
testicular fluid, seminal plasma or bromelain-solubilized cervical mucus).
■ Antibody-free donor’s spermatozoa take up anti-sperm antibodies present in the
tested fluid and are then assessed as in the direct immunobead test.
■ If >50% of spermatozoa show attached latex particles or beads in any tests,
immunological problem is likely.
99. Clinically used markers to determine secretory capacity
of accessory glands
■ Prostate gland function
– Zinc
– Citric acid
– Acid phosphatase
– α-glutamyl transpeptidase
■ Seminal vesicles
– Fructose
– Prostaglandins
■ Epididymis
– Neutral Alpha glucosidase
– Free L-carnitine
– glycerophosphocholine (GPC)
Infection cause decrease in the secretion of these markers
100. Estimation of fructose
■ Resorcinol method used
■ Resorcinol reagent: resorcinol-50mg, Conc.HCl-33ml, distilled water-67ml
■ 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.
■ Procedure:
■ 5 ml resorcinol reagent + 0.5 ml of semen in a test tube
■ Mix and place in a boiling waterbath for 5min or heat.
■ Observations: Red colored ppt. in 30 seconds.
■ In quantitative assays, this is compared with a known fructose standard at 490nm.
■ Normal level of fructose: 150-300mg/dl.
■ Reduced levels: Seminal vesicle dysfunction, High sperm count, obstructed vas deferens
102. ■ Indications:
■ 1) Accessory gland infection.
■ 2) High number of leucocytes in semen(>1million/ml)
■ Precautions should be taken to avoid contamination.
■ Culture should be done to detect both aerobic & anaerobic organisms.
■ If >1000CFUs/ml, then do antibiotic sensitivity tests.
■ E. Coli can cause sperm agglutination and immobilization.This is mediated by mannose and
mannose binding cell surface structures present on both cell types.
104. COMPUTER -AIDED SPERMANALYSIS
■ 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
■ Disadvantages:
– Expensive equipment and still requires the subjective
participation of a technician.
■ Hence not used for routine semen analysis
■ Commonly done in high volume andrology labs
105. Different CASA types:
■ ISAS (Integrated Semen Analysis System)
■ SCA (Sperm ClassAnalyzer)
■ IVOS (IntegratedVisual Optical System )
■ SQA-V (Sperm QualityAnalyzer)
■ The use of fluorescent DNA stains with CASA allows the concentration of motile
sperm and percentage motility to be determined accurately
106. ISAS (Integrated Semen Analysis System)
■ ISAS is a CASA system based on image analysis.
■ ISAS analyzes motility and concentration in more than 17 sperm parameters
■ ISAS also do DNA fragmentation analysis
107. SCA (Sperm Class Analyzer)
■ SCA provides fast, accurate and repeatable results.
■ SCA Motility & Concentration
■ SCA DNA Fragmentation
■ Morphology
■ SCAVitality
108. IVOS (IntegratedVisual Optical System )
■ The IVOS is unique in that it is the only CASA system that integrates the optical system
within the unit, so that an external microscope is not needed.
■ Able to analyze sperm of multiple species (rat)
■ Research institutes, IVF clinics, pharmaceutical companies, reproductive toxicology labs,
veterinary and animal breeding centres
■ A single field - analyzed in just 0.5 second.
109. SQA-V (Sperm QualityAnalyzer)
■ Fully automated
■ SQA-V semen analysis eliminates inter-operator variation.
■ Electro-optics, computer algorithms and video microscopy
■ Provide a precise and accurate
■ Less than 2 minutes
■ The SQA-V ( 16 clinical parameters )
117. DNA Fragmentation test in CASA
■ DNA damage in human sperm is associated with impaired conception, disrupted
embryonic development, increased rate of miscarriage and morbidity in offspring
■ Indications:
1. Couples planning for IVF or ICSI
2. Unexplained infertility
3. Recurrent pregnancy loss
4. IVF failure
5. Selection of best donor
6. Auto-preservation of sperms
118. ■ Interpretation:
■ 200-500 spermatozoa are examined under 400 magnification
■ Identify sperms with tail and observe the head as follow:
A. SPERMWITH HALO
■ Identify and mark centre of head
■ Minor core :
– Mark periphery of central dark area
– This area is known as minor core
– Note down diameter of minor core (XY)
■ Major core
– Mark periphery of chromatin dispersion
– Area from inner line of minor core to outer periphery
is known as HALO
– Note down the thickness of halo (YZ)
■ Ratio
– Calculate ratio of thickness of halo with diameter of core
– R=YZ/XY
119. B. Sperm without HALO
C. Degraded sperm
■ CATEGORISATIONOF SPERM
1. NON - FRAGMENTED DNA
– Ratio ® > 0.33 ( sperm with halo)
2. FRAGMENTED DNA
– Ratio ® < 0.33 ( sperm with halo)
– Ratio ® = 1( sperm without halo)
3. DEGRADED SPERM
– Sperm with shrunken size
120.
121.
122.
123. Nuclear protein assay by CASA
■ Sperm head with blue stain sperm with immature nuclear protein
■ Sperm head with red stain sperm with mature nuclear protein
124. Normal values (lower reference limits) of
semen analysis (WHO 2010)
Test Result WHO 1999
1.Volume ≥ 1.5ml ≥ 2ml
2. pH ≥ 7.2 7.2 - 8
3. Sperm concentration ≥ 15 x 106/ml ≥ 20 x 106/ml
4.Total sperm count per ejaculate ≥ 39 x 106 ≥ 40 x 106
5. Sperm morphology ≥ 4.0% normal forms ≥ 15 % normal forms
6.Vitality ≥ 58% live forms ≥ 75% live forms
7.White blood cells (peroxidase +ve) ≤ 1.0 x 106/ml ≤ 1.0 x 106/ml
8. Motility
–Total motility (PR + non – progressive)
- Progressive motility (PR)
≥ 40%
≥ 32%
≥ 50% RAPID + SLOW
≥ 25% RAPID
9. Mixed antiglobulin reaction (MAR test) < 50% motile sperms with adherent
particles
< 50% motile sperms with
adherent particles
10. Immunobead test < 50 % motile sperms with bound beads < 50 % motile sperms with
bound beads
129. SPERM-MUCUS INTERACTION/ POSTCOITALTEST/
SIMS – HUHNERTEST
■ 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 IUI.
– Inappropriate timing testing / intercourse
– Anatomic abnormalities
– Semen or cervical mucus antisperm antibodies
– Abnormal sperm.
130. SPERM PENETRATIONASSAYS
■ Unexplained infertility / IVF failure
■ Principle - a normal spermatozoa can bind and penetrate the oocyte membrane.
■ HAMSTER EGG PENETRATION ASSAY
■ Incubating zona-free hamster oocytes in sperm droplets for 1 to 2 hours.
■ 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 testing
■ Sperm capacitation index (SCI) is a variant of the SPA test, assessing the mean number of
penetrations per ovum.
■ NORMAL - > 5
■ ICSI has been recommended - SCI less than 5 instead of standard IVF
proceduresADVANCED SPERMTESTING
131. Cervical Mucus PenetrationTest
■ In this test, greatest distance traveled 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.
132. 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.
■ Involves fusion of acrosomal membrane and plasma membrane
■ Acrosin and Hyaluronidase – required to digest the oocyte cumulus cells and ZP
■ Acrosome reaction testing - not widely practiced in laboratories - research interest.
■ Profound abnormalities of head morphology
■ Unexplained infertility
133. Limitation of semen analysis
■ Clinical research has shown,
■ Normal semen analysis may not reflect the true fertility status of an individual
■ Men with poor sperm parameters can cause spontaneous pregnancies
■ Men with good sperm parameters are still subfertile
■ Only 50% of subfertile men have recognizable causes detectable by semen analysis.
■ Semen analysis is only a surrogate test to measure the man’s fertility potential
