SlideShare utilise les cookies pour améliorer les fonctionnalités et les performances, et également pour vous montrer des publicités pertinentes. Si vous continuez à naviguer sur ce site, vous acceptez l’utilisation de cookies. Consultez nos Conditions d’utilisation et notre Politique de confidentialité.
SlideShare utilise les cookies pour améliorer les fonctionnalités et les performances, et également pour vous montrer des publicités pertinentes. Si vous continuez à naviguer sur ce site, vous acceptez l’utilisation de cookies. Consultez notre Politique de confidentialité et nos Conditions d’utilisation pour en savoir plus.
Dr. Furquan Alam
Department of Biochemistry,
SGRRIMHS & SMI Hospital, Dehradun.
The sperm formation involves two steps :
In the first step spermtogenic cells form rounded cells called
In the second step spermatids differentiate into specialized
cells known as sperms.
DR. FURQUAN ALAM 2
Site Of Sperm Formation
Seminiferous tubules of Testis
DR. FURQUAN ALAM 3
Semen is body fluid that is ejaculated at the time of
orgasm, contain sperm & secretion of seminal vesicle,
prostate, Cowper's gland & urethral gland.
DR. FURQUAN ALAM 6
Source Volume Characteristics
Urethral and bulbourethral
0.1-0.2cc Viscous, clear
Testes, epididymides, vas
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
DR. FURQUAN ALAM 7
Semen is viscous, neutral or slightly alkaline & whitish
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
It is milky in appearance & also rich in proteolytic enzymes are responsible
for the liquefaction of semen.
DR. FURQUAN ALAM 8
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.
DR. FURQUAN ALAM 9
Normal Values From WHO Manuals, Editions 2- 4 And Lower
Reference Limits From New 5th WHO Manual (2010)
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
50 30 (15) 4
3/13/2016 8:00:38 PM DR. FURQUAN ALAM
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.
DR. FURQUAN ALAM 12
Steps Of Semen Analysis
Between 30 and 60 minutes:
Assessing sperm vitality (if the percentage of motile cells is
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
DR. FURQUAN ALAM 13
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).
DR. FURQUAN ALAM 14
After 4 hours:
Fixing, staining and assessing smears for sperm
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
DR. FURQUAN ALAM 15
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
DR. FURQUAN ALAM 16
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
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
DR. FURQUAN ALAM 17
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.
DR. FURQUAN ALAM 18
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.
DR. FURQUAN ALAM 19
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:
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
DR. FURQUAN ALAM 20
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
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.
DR. FURQUAN ALAM 21
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
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
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).
DR. FURQUAN ALAM 22
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).
DR. FURQUAN ALAM 23
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.
DR. FURQUAN ALAM 24
The semen specimen should be examined immediately
It should be kept at room temperature.
DR. FURQUAN ALAM 25
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
DR. FURQUAN ALAM 26
History To Be Noted:
Date and time of collection
Length of abstinence
Interval between collection and analysis
History of fever
DR. FURQUAN ALAM 27
Sample Form For
DR. FURQUAN ALAM 28
Prolonged abstinence will lead to increased volume, but
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
DR. FURQUAN ALAM 30
Assessment Of Semen:(according To WHO)
3. Sperm concentration
4. Total sperm count
8. White blood cells
9. Immunobead test
DR. FURQUAN ALAM 31
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
DR. FURQUAN ALAM 32
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
High volume: >10ml: Dilutional Oligozoospermia
Absence of ejaculate
B/l ejaculatory duct obstruction
DR. FURQUAN ALAM 33
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.
Tumor of the genital tract.
Increased turbidity indicates inflammatory process in some part
of the reproductive tract.
DR. FURQUAN ALAM 34
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
Protein fragments are further degraded into free
amino acids and ammonia.
Failure to liquefy indicates inadequate prostatic
secretion. To liquefy, add bromeline, plasmin or
DR. FURQUAN ALAM 35
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
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
DR. FURQUAN ALAM 36
Measured by using a pH meter or pH paper.
Semen is the strongest buffer in the body.
Seminal vesicle & vas deference secretions alkaline
Prostatic secretion acidic(due to citric acid, proteolytic enzymes,
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
pH>8 is associated with acute infection of prostate, seminal vesicles
DR. FURQUAN ALAM 37
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
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.
DR. FURQUAN ALAM 38
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
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
DR. FURQUAN ALAM 39
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.
Cover slip- 22mm x 22mm
Semen- 10µl , depth of 20µm.
Phase contrast microscopy – 400x/600x at 37 °C.
DR. FURQUAN ALAM 41
Rapid progressive motility
Slow/Sluggish progressive motility
Non - progressive motility
DR. FURQUAN ALAM 42
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
Many immotile live sperms direct towards immotile cilia syndrome.
Do electron microscopy.
DR. FURQUAN ALAM 43
Normal Motility: > 32%
If less than this, asthenozoospermia.
Prolonged heat exposures
Progressive loss of motility by 5%/hr after 3hrs.
After liquefaction or
within 60mins after
DR. FURQUAN ALAM 44
Thin smears similar to blood smears are made feathering
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.
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.
Body &tails: red/pink. 3/13/2016 8:00:38
DR. FURQUAN ALAM 45
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 =
DR. FURQUAN ALAM 46
Computer Aided Sperm Analysis:
Automation seeks to establish standard methods of
analysis and set acceptable levels of accuracy in
measuring various parameters to promote
Subjective errors avoided
Motility can be assessed quantitatively
Capable of handling many samples without undergoing
Morphological defects can be made out
DR. FURQUAN ALAM 47
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.
DR. FURQUAN ALAM 48
Can occur in the:
a)Serum of male or female
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.
d)Equatorial part of acrosome
DR. FURQUAN ALAM 50
Antisperm antibodies are found in following
Obstruction of ducts
DR. FURQUAN ALAM 51
Techniques of detecting antibodies:
Mixed antiglobulin reaction (MAR) test
DR. FURQUAN ALAM 52
Round cells :
1) Germinal cells (single or double highly condensed nucleus with
2) Leucocytes < 1 million/ml or 1-2/hpf. Increased no. in infection of
RBCs : normally absent.
1. TB of seminal vesicles
2. rupture of blood vessels
3. Infection of prostate
4.Vit. C deficiency
Epithelial cells from urogenital tract.
DR. FURQUAN ALAM 53
Determination of fructose:
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.
Red colored ppt. in 30 seconds.
In quantitative assays, this is compared with a known fructose standard at
Normal level of fructose: 150-300mg/dl.
Seminal vesicle dysfunction.
High sperm count
DR. FURQUAN ALAM 55
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
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.
DR. FURQUAN ALAM 56
Sperm Function Tests:
Factors responsible for defective sperm function:
Peroxidase damage by excessive generation of reactive oxygen
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
DR. FURQUAN ALAM 57
a. Sperm penetration assay:
Uses zona denuded golden hamster eggs as hosts for
the penetration of human sperms. This measures
Sperm oocytes fusion
Sperm incorporation in oocytes
Decondensation of sperm chromatin
b. Hemizona assay:
Utilizes unfertilized oocytes
DR. FURQUAN ALAM 58
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.
DR. FURQUAN ALAM 59
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
Therefore assessment of membrane function is a useful
indicator of fertilizing ability of spermatozoa.
DR. FURQUAN ALAM 60
E. Cervical mucous penetration test/ Post coital test/ Sims-
To study the quality of cervical mucous.
To know the ability of spermatozoa to penetrate the cervical mucous and
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
DR. FURQUAN ALAM 61
Examination For The Presence Of Sperms In
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.
1)Examination for sperms:
Direct smears from vagina
Smears from aspirate
Washings from fabric after centrifugation Stain with H & E.
DR. FURQUAN ALAM 62
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
6) Determination of sperm specific LDH isoenzyme.
DR. FURQUAN ALAM 64
TYPICAL HEAD DEFECTS
3/13/2016 8:00:38 PM DR. FURQUAN ALAM 65
TYPICAL MID PIECE DEFECTS
implantation Too thin Irregular
3/13/2016 8:00:38 PM DR. FURQUAN ALAM 66
TYPICAL TAIL DEFECTS
drop Double Coiled Stumped
3/13/2016 8:00:38 PM DR. FURQUAN ALAM 67
DR. FURQUAN ALAM 68