As per WHO guideline 2010

1. Dr. Furquan Alam
PG Resident,
Department of Biochemistry,
SGRRIMHS & SMI Hospital, Dehradun.
SEMEN ANALYSIS

2. SPERMATOGENESIS
The sperm formation involves two steps :
 1) Spermatocytogenesis
In the first step spermtogenic cells form rounded cells called
spermatids.
 2) Spermiogenesis
In the second step spermatids differentiate into specialized
cells known as sperms.
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3. Site Of Sperm Formation
Seminiferous tubules of Testis
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6. Seminal Fluid
Semen is body fluid that is ejaculated at the time of
orgasm, contain sperm & secretion of seminal vesicle,
prostate, Cowper's gland & urethral gland.
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7. Source Volume Characteristics
Urethral and bulbourethral
glands
0.1-0.2cc Viscous, clear
Testes, epididymides, vas
deferentia
0.1-0.2cc Sperm present
Prostate 0.5-1.0cc Acidic,watery
Seminal vesicles 1.0-3.0cc Gelatinous, fructose positive
Complete ejaculate 1.5-5.0cc Liquefies in 20-25min
Semen
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8.  Semen is viscous, neutral or slightly alkaline & whitish
opaque.
 60 % semen volume is derived from seminal vesicle which is
also a major source of high FRUCTOSE content of semen.
 Seminal vesicle secretion also provide the substrate for the
coagulation of the semen following ejaculation.
 About 20 % of the volume of semen is contributed by the
prostate gland.
 It is milky in appearance & also rich in proteolytic enzymes are responsible
for the liquefaction of semen.
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9.  About 10 -15 % of semen volume is also contributed by
epididymis, vasdeferens, cowper’s gland & uretheral gland.
 Less than 5 % of semen volume is contributed by Spermatozoa.
 The process of ejaculation result in the mixing of these distinct
fraction of semen.
 These enter the urethra individually in the rapid succession.
 The third & final fraction consist of mucoid secretion resulting from
emptying of seminal vesicle.
 The semen specimen collected for routine examination should
contain all above mention fraction.
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11. Normal Values From WHO Manuals, Editions 2- 4 And Lower
Reference Limits From New 5th WHO Manual (2010)
Semen parameter
WHO edition and year
2nd - 1987 3rd - 1992 4th - 1999 5th - 2010
Volume (ml) 2.0 2.0 2.0 1.5
Sperm concentration (106/ml) 20 20 20 15
Total sperm count (106) 40 40 40 39
Motility (% progressive) 50 50 50 32
Vitality (% live) 50 75 75 58
Morphology (%
normal)
50 30 (15) 4
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12. Steps: Semen Analysis
In the first 5 minutes:
 Placing the specimen container on the bench or in an
incubator (37 °C) for liquefaction.
Between 30 and 60 minutes:
 Assessing liquefaction and appearance of the semen.
 Measuring semen volume.
 Measuring semen pH (if required).
 Preparing a wet preparation for assessing microscopic
appearance, sperm motility and the dilution required for
assessing sperm number.
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13. Steps Of Semen Analysis
Between 30 and 60 minutes:
Assessing sperm vitality (if the percentage of motile cells is
low).
 Making semen smears for assessing sperm morphology.
 Making semen dilutions for assessing sperm concentration.
 Assessing sperm number.
 Performing the mixed antiglobulin reaction (MAR) test (if
required).
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14. Steps
Between 30 and 60 minutes:
Assessing peroxidase-positive cells (if round cells are present).
 Preparing spermatozoa for the immunobead test (if required).
 Centrifuging semen (if biochemical markers are to be assayed).
Within 3 hours:
 Sending samples to the microbiology laboratory (if required).
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15. Steps
After 4 hours:
 Fixing, staining and assessing smears for sperm
morphology.
Later on the same day (or on a subsequent day if
samples are frozen):
 Assaying accessory gland markers (if required).
 Performing the indirect immunobead test (if
required).
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16. Sample Collection:
Preparation:
 The sample should be collected in a private room near
the laboratory, in order to limit the exposure of the
semen to fluctuations in temperature and to control the
time between collection and analysis.
 The sample should be collected after a minimum of 2
days and a maximum of 7 days of sexual abstinence. If
additional samples are required, the number of days of
sexual abstinence should be as constant as possible at
each visit.
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17. Sample Collection:
Preparation:
 The man should be given clear written and spoken instructions
concerning the collection of the semen sample. These should
emphasize that the semen sample needs to be complete and
that the man should report any loss of any fraction of the
sample.
 The following information should be recorded on the report
form: the man’s name, birth date and personal code number,
the period of abstinence, the date and time of collection, the
completeness of the sample, any difficulties in producing the
sample, and the interval between collection and the start of the
semen analysis.
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18. Sample Collection for Diagnostic/ Research purpose:
 The sample should be obtained by masturbation and ejaculated into
a clean, wide-mouthed container made of glass or plastic, from a
batch that has been confirmed to be non-toxic for spermatozoa.
 The specimen container should be kept at ambient temperature,
between 20 °C and 37 °C, to avoid large changes in temperature
that may affect the spermatozoa after they are ejaculated into it. It
must be labelled with the man’s name and identification number, and
the date and time of collection.
 The specimen container is placed on the bench or in an incubator
(37 °C) while the semen liquefies.
 Note in the report if the sample is incomplete, especially if the first,
sperm-rich fraction may be missing. If the sample is incomplete, a
second sample should be collected, again after an abstinence period
of 2–7 days.
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19. Sterile collection of semen for assisted reproduction:
 This is performed as for diagnostic collection but the
specimen containers, pipette tips and pipettes for
mixing must be sterile.
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20. Sterile collection of semen for microbiological analysis:
 In this situation, microbiological contamination from non-semen sources
(e.g. commensal organisms from the skin) must be avoided. The
specimen containers, pipette tips and pipettes for mixing must be sterile.
 The man should:
 Pass urine.
 Wash hands and penis with soap, to reduce the risk of contamination of
the specimen with commensal organisms from the skin.
 Rinse away the soap.
 Dry hands and penis with a fresh disposable towel.
 Ejaculate into a sterile container.
 Note: The time between collection of the semen sample and the start of
the investigation by the microbiological laboratory should not exceed 3
hours.
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21. Collection of semen at home:
 A sample may be collected at home in exceptional circumstances, such as a
demonstrated inability to produce a sample by masturbation in the clinic or the
lack of adequate facilities near the laboratory.
 The man should be given clear written and spoken instructions concerning the
collection and transport of the semen sample. These should emphasize that the
semen sample needs to be complete, i.e. all the ejaculate is collected, including
the first, sperm-rich portion, and that the man should report any loss of any
fraction of the sample. It should be noted in the report if the sample is incomplete.
 The man should be given a pre-weighed container, labelled with his name and
identification number.
 The man should record the time of semen production and deliver the sample to
the laboratory within 1 hour of collection.
 During transport to the lab., the sample should be kept between 20 °C - 37 °C.
 The report should note that the sample was collected at home or another
location outside the laboratory.
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22. Collection of semen by condom:
 A sample may be collected in a condom during sexual intercourse only in
exceptional circumstances, such as a demonstrated inability to produce a sample
by masturbation.
 Only special non-toxic condoms designed for semen collection should be used;
such condoms are available commercially.
 The man should be given information from the manufacturer on how to use the
condom, close it, and send or transport it to the laboratory.
 The man should record the time of semen production and deliver the sample to
the laboratory within 1 hour of collection.
 During transport to the lab., the sample should be kept between 20 °C and 37 °C.
 The report should note that the sample was collected by means of a special
condom during sexual intercourse at home or another location outside the
laboratory.
Note: Ordinary latex condoms must not be used for semen collection because they
contain agents that interfere with the motility of spermatozoa (Jones et al., 1986).
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23. Safe handling of specimens:
 Semen samples may contain dangerous infectious agents
(e.g. human immunodeficiency virus (HIV), hepatitis
viruses or herpes simplex virus) and should therefore be
handled as a biohazard. If the sample is to be processed
for bioassay, intra-uterine insemination (IUI), in-vitro
fertilization (IVF) or intracytoplasmic sperm injection (ICSI),
or if semen culture is to be performed, sterile materials and
techniques must be used. Safety guidelines as outlined in
Appendix 2 should be strictly followed; good laboratory
practice is fundamental to laboratory safety (WHO, 2004).
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24. Precautions
 Specimen should not be collected in ordinary condoms
since the powder or lubricant applied to the condom
may be spermicidal.
 The container in which the semen sample is collected
should be free from detergent.
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25. Storage
 The semen specimen should be examined immediately
after collection.
 It should be kept at room temperature.
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26. Indications For Analysis:
 To determine the fertility of the man.
 After a male has undergone vasectomy to check the
completeness of the procedure (retrograde ejaculation).
 In medico-legal situations such as disputes about the
paternity of a child.
 After reversal of vasectomy to confirm the success of the
procedure.
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27.  History To Be Noted:
 Name
 Date and time of collection
 Length of abstinence
 Interval between collection and analysis
 History of fever
 Drug intake
 Alcohol abuse
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28. Sample Form For
Semen Analysis
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30. NOTE:
Prolonged abstinence will lead to increased volume, but
reduced motility.
The patient should evacuate his bladder before specimen
collection. If retrograde ejaculation is suspected, a post-
ejaculate urine sample is collected and examined for presence
of sperms.
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31. Assessment Of Semen:(according To WHO)
 Standard tests:
1. Volume
2. pH
3. Sperm concentration
4. Total sperm count
5. Motility
6. Morphology
7. Vitality
8. White blood cells
9. Immunobead test
10.MAR test
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32.  Optional tests:
 Alpha galactosidase (neutral): 20mU or more.
 Zinc (total): 2.4 micromol or more
 Citric acid(total): 52 micromol or more
 Acid phosphatase: 200U or more
 Fructose: 13 micromol or more
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33. Volume:
Measured by aspirating into a pipette or by using a syringe(non-toxic 1,2 or
5ml): Normal: 1.5ml or more.
Low volume: <1.5ml
B/l ejaculatory duct obstruction
B/l congenital vasal aplasia
Inadequate erection & improper mood at collection
Incomplete collection
High volume: >10ml: Dilutional Oligozoospermia
Aspermia:
Absence of ejaculate
Retrograde ejaculation
Anejaculation
B/l ejaculatory duct obstruction
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34. Colour:
Homogenous grey opalescent appearance (Normal).
After prolonged abstinence, slightly yellow.
Deep yellow- Pyospermia.
Rust colour - Small bleedings in seminal vesicles.
Red or brown indicates presence of blood.
Trauma to the genital tract.
Inflammation.
Tumor of the genital tract.
Increased turbidity indicates inflammatory process in some part
of the reproductive tract.
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35. Viscosity:
 Freshly ejaculated semen is highly viscous due to
substrate produced by seminal vesicles. The
coagulum liquefies spontaneously to form a
translucent, viscous fluid in a three stage process.
 Action of a prostatic clotting enzyme.
 Liquefaction is initiated by enzymes of prostatic
origin.
 Protein fragments are further degraded into free
amino acids and ammonia.
 Failure to liquefy indicates inadequate prostatic
secretion. To liquefy, add bromeline, plasmin or
chymotrypsin.
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36. Normal liquefaction time: 20-60min
Viscosity of liquefied semen can be
estimated by: Gentle aspiration into a 5ml pipette, then
allowing the semen to drop by gravity. Observe the length of the
thread.
Normal semen leaves as small discrete drops.
Increased viscosity is associated with poor
invasion of cervical mucus in post-coital studies as well as
decreased ability to fertilize ovum.
Absence of viscosity points to reduced cell content.
The semen from males with b/l congenital absence of vas
deferentia & seminal vesicles fails to coagulate due to absence of
substrate.
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37. pH:
Measured by using a pH meter or pH paper.
Normal: 7.2-8
Semen is the strongest buffer in the body.
Seminal vesicle & vas deference secretions alkaline
Prostatic secretion acidic(due to citric acid, proteolytic enzymes,
acid phosphatase)
Motility is reduced in acidic medium.
pH<7 is associated with largely prostatic secretions due to
congenital aplasia of vas & seminal vesicles and when contaminated
with urine.
pH>8 is associated with acute infection of prostate, seminal vesicles
or epididymis.
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38. Sperm Concentration:
WBC Micropipette Semen-0.5mark
Diluting Fluid- 11 mark
Charge in counting chamber.
Count the number in four corner squares.
Sperm/ml = Nx10x20x1000 = Nx50,000
4
If it is very viscid, add mucolytic agent in 1:1 dilution
and multiply by 2.
If count is high(>100m), then use higher dilution.
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39. Composition of diluting fluid:
1. Sodium bicarbonate- 5g. Counteracts mucus and allows even
dilution of this viscous fluid.
2. Phenol-1ml. Kills sperms and stops their movement. Also acts
as preservative.
3. Distilled water-100ml.
A man should not be termed oligozoospermic until atleast 3
samples are evaluated at an interval of 3wks and 3 months.
Azoospermia is a condition where the semen sample has no
spermatozoa in a fresh sample or in a centrifuged resuspended
sample.
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40. Normal count : 15 to 150 million/ml.
Oligozoospermia : < 15million/ml.
Causes:
 Mumps: orchitis
 Prostatitis
 Hypopituitarism
 Hypogonadotropic hypogonadism
 Estrogen secreting tumors
 Hypo/Hyperthyroidism
 Drugs: Sulfasalazine, Cimetidine, Estrogen,
Nitrofurantoin, Caffeine, Alcohol, Cocaine, Smoking
tobacco, Herbal medications, Chemotherapeutic agents.
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41. Motility:
Routinely used technique:
 Place a drop of liquefied semen on a glass slide.
 Cover with cover slip and rim its edges with Vaseline.
 Examine under 40X with reduced illumination.
 Count the number actively motile sperms out of 200.
 Calculate the percentage.
For accuracy:
Cover slip- 22mm x 22mm
Semen- 10µl , depth of 20µm.
Phase contrast microscopy – 400x/600x at 37 °C.
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42. Grading:
Rapid progressive motility
Slow/Sluggish progressive motility
Non - progressive motility
Immotility
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43. Viability:
 If motility is < 40% a viability should be performed.
 Supravital staining by eosin Y with nigrosin. Dead cells take up the
stain. 100 sperms are counted. Live/Dead sperm ratio calculated.
 Hypo osmotic saline test (HOS):
Sperms are added to hypo osmotic saline and incubated at 37deg C.
Swelling of the sperm tail is examined under phase contrast
microscope. Sperms which have active membrane swell after
30mins.
 Many immotile live sperms direct towards immotile cilia syndrome.
 Do electron microscopy.
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44. Normal Motility: > 32%
If less than this, asthenozoospermia.
Causes:
 Cold
 Radiation
 Spermicides, Pesticides
 Prolonged heat exposures
 Prolonged abstinence
 Autoimmunity
Progressive loss of motility by 5%/hr after 3hrs.
After liquefaction or
within 60mins after
ejaculation.
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45. Sperm Morphology:
 Thin smears similar to blood smears are made feathering
technique.
 Before staining, mucoid material is removed by gentle washing with
semen dilution fluid. Then wash gently with buffered distilled water.
 Several staining techniques are used
1. Pap is the best.
2. Haematoxylene technique.
3. Giemsa.
4. Leishman/basic fuchsin (0.25% acqeous).
5. Crystal violet.
6. Diff-Quick stain
 On basic fuchsin: Sperm head caps: light blue.
 Nuclear post: dark blue.
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46. Sperm morphology: Ideal spermatozoon
Menkveld et al. 1991, WHO 1999, ESHRE 2002
Head oval shaped regular contour
Length: 4-5.5 micron
Width 2.5-3.5 micron
Darker posterior region
Base of head should be broad
Single tail symmetrically attached
BORDERLINE FORMS =
ABNORMAL
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47. Computer Aided Sperm Analysis:
Automation seeks to establish standard methods of
analysis and set acceptable levels of accuracy in
measuring various parameters to promote
interlaboratory comparisons.
Advantages:
Subjective errors avoided
Motility can be assessed quantitatively
Capable of handling many samples without undergoing
fatigue
Morphological defects can be made out
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48. Agglutination Of Sperms:
Motile sperms stick to each other in various
orientations like- head to head, midpiece to midpiece,
tail to tail or in combinations depending on the
specificity of antisperm antibodies. Agglutination
points to immunological cause of infertility.
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50. Antisperm Antibodies:
Can occur in the:
a)Serum of male or female
b)Seminal plasma
c)Spermatozoa
Effects:
a)Lowered progressive motility.
b)Decreased ability to penetrate cervical mucus.
c)Decreased ability to penetrate egg.
Antibodies are found to react with:
a)The front part of acrosome.
b)Post-nuclear cap
c)Tail piece
d)Equatorial part of acrosome
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51. Antisperm antibodies are found in following
conditions:
Testicular disease
Autoimmune azoospermatogenesis
Following vasectomy
Repeated infections
Obstruction of ducts
Cryptorchidism
Varicocele
Testicular biopsy
Trauma
Torsion
Genetic predisposition
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52. Techniques of detecting antibodies:
Agglutination
Immobilization
Precipitation
Complement fixation
Passive hemagglutination
Cytotoxicity
For screening,
Mixed antiglobulin reaction (MAR) test
Immunobead method
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53. Other Cells:
Round cells :
1) Germinal cells (single or double highly condensed nucleus with
abundant cytoplasm).
2) Leucocytes < 1 million/ml or 1-2/hpf. Increased no. in infection of
reproductive tract.
RBCs : normally absent.
Present in
1. TB of seminal vesicles
2. rupture of blood vessels
 3. Infection of prostate
 4.Vit. C deficiency
Epithelial cells from urogenital tract.
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54. Biochemical Assays:
 Prostate gland function
 Zinc
 Citric acid
 Acid phosphatase
 Seminal vesicles
 Fructose
 Prostaglandins
 Epididymis
 Alpha glucosidase
 L-carnitine
 Plycerophosphocholine
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55. Determination of fructose:
Procedure:
Pipette 5 ml of resorcinol reagent in a test tube. [Resorcinol reagent:
resorcinol-50mg, Conc.HCl-33ml, distilled water-67ml]
Add 0.5 ml of semen.
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
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56. Microbiological Assays:
 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.
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57. Sperm Function Tests:
Factors responsible for defective sperm function:
 Peroxidase damage by excessive generation of reactive oxygen
species.
 High activity levels of creatine phosphokinase and a low ratio of
muscle( CK-M) to the combined activities of muscle and brain isoforms
( CK-MB )  abnormal activities of mid piece.
The tests include:
a. Sperm penetration assay (SPA)
b. Hemizona assay
c. Acrosin assay
d. Hypo osmotic saline test
e. Cervical mucous penetration test
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58. a. Sperm penetration assay:
Uses zona denuded golden hamster eggs as hosts for
the penetration of human sperms. This measures
Sperm capacitation
Sperm oocytes fusion
Sperm incorporation in oocytes
Decondensation of sperm chromatin
b. Hemizona assay:
Utilizes unfertilized oocytes
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59. c. Acrosin assay:
Measures acrosin, a trypsin like serine protienase
specific to sperm acrosome. It is responsible for sperm
penetration of the zona pellucida, after its release is
triggered by the binding of sperm to zona.
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60. D. Hypo-osmotic Swelling Test:
 For successful union test of the spermatozoa with female
gametes, integrity of sperm membrane is very essential.
 Sperm capacitation, acrosomal reaction & penetration of
egg is also dependent upon membrane integrity of
spermatozoa.
 Therefore assessment of membrane function is a useful
indicator of fertilizing ability of spermatozoa.
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61. E. Cervical mucous penetration test/ Post coital test/ Sims-
Huhner test:
Aims :
To study the quality of cervical mucous.
To know the ability of spermatozoa to penetrate the cervical mucous and
maintain activity.
After 8-10 hrs of coitus during the ovulatory phase, the endocervical
mucous is collected in a Luer syringe. The volume, colour and viscosity
(Spinbarkeit ) of the mucous noted. A drop of mucous is placed on a slide
and examined for the presence of sperms.
Atleast 10 motile sperms should be present per hpf.
The material should be examined for leucocytes, erythrocytes and
trichomanads.
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62. Examination For The Presence Of Sperms In
Medicolegal Cases:
Obtaining the sample:
1. From vagina: direct aspiration or saline lavage.
2. From clothing or other fabrics: preliminary scan with UV light
green fluorescence 1sq cm of stained fabric soak in 1-2ml of
physiological saline for 1hr. Then fluid is subjected to tests.
Tests:
1)Examination for sperms:
 Direct smears from vagina
 Smears from aspirate
 Washings from fabric after centrifugation Stain with H & E.
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63. 3/13/2016 9:16:24 PM DR. FURQUAN ALAM 63

64. 2) Determination of acid phosphatase:
More sensitive 2500 king armstrong units
3) Blood group substances
4) Florence test: screening method
Depends on presence of cholines
5) Precipitin test: Using specific antiserum by capillary tube
reaction.
6) Determination of sperm specific LDH isoenzyme.
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65. TYPICAL HEAD DEFECTS
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66. TYPICAL MID PIECE DEFECTS
Bent
Flat
implantation Too thin Irregular
Too
thick
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67. TYPICAL TAIL DEFECTS
Hairpin
Cytoplasmic
drop Double Coiled Stumped
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68. THANK YOU
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