NARMADA PRASAD TIWARI MGM MEDICAL COLLEGE INDORE

1. Moderator :- Dr. Shailendra Singh Thakur

2. The sperm formation involves two steps : in
the first step spermtogenic cells form
rounded cells called spermatids which in
the second step differentiate into
specialized cells known as sperms. These
processes are labeled respectively as
Spermatocytogenesis
Spermiogenesis

3. Seminiferous tubules of Testis

5. The primitive sex cells
appear earliest in 4th week
of intra uterine life in the
wall of yolk sac as
primordial germ cells
They migrate to the
developing testes and lie
dormant among the cells
lining the seminiferous
tubules

6. At puberty the germ cells awaken
and start the actual process of
spermato genesis
These cells increase in number by
simple mitosis to form cells known
as spermatogonia ; type- A and
type- B.
Type-B spermatogonia, replicate
DNA to have 46 double structured
chromosomes to begin meiosis-1
and are called primary
spermatocytes.

7. In prophase1 pairing and crossing over
of chromosomal segments takes place
and genetic recombination occurs
In metaphase1 homologous pairs
arrange on equator
In anaphase 1 homologous pairs
separate to go to opposite poles
After telophase 1 meiosis 1 ends and 2
secondary spermatocytes form each
with 23 double structured
chromosomes with X or Y sex
chromosome complement
It takes about 22 days to complete
meiosis-1

8. Meiosis 2 follows immediately
without DNA replication. Only 23
double structured chromosomes
are involved
2 secondary spermatocytes
quickly undergo meiosis-2
(Centromeres split in
metaphase 2) and end with the
formation of 4 sperrmatids each
with 23 single structured
chromosomes and1N DNA
Two spermatids bear X
chromosome complement and
other two bear Y chromosome
complement

9. As steps of spermatogenesis
continue the
spermatocytes
progressively move from
basement membrane to
the luminal side of
seminiferous tubule
The cells of Sertoli provide
nutrition and pockets of
support to developing
spermatocytes

10. The spermatocytes in
different stages of
development remain
attached by
cytoplasmic bridges
All the spermatocytes
are not in the same
stage of development
in the seminiferous
tubules

11. Spermatids are rounded cells.
They modify to assume
specific shape of the sperm.
This process is called
Spermiogenesis. In it they
elongate and reorganize
internal structure to acquire
the particular shape.

12. The changes include ;
Golgi apparatus forms
acrosomal cap-proteolitic
enzymes
Nucleus is condensed
Centriols: make collar
around neck
Microtubules, forrm
flagellum,
Mitochondria arrange as
spiral around neck
Excess cytoplasm cast off as
residual body
Cyto plasmic bridges break
and sperms release from
Sertoli cells to lie free in
lumen of seminiferous
tubules.
About 64 days are required to
go from a spermatogonium
to a sperm

13. A mature sperm has head, neck
and tail
From lumen of seminiferous
tubules sperms enter duct of
epididymis
They take 20 days to travel this
4-6 meter long tortuous duct
If ejaculation does not occur
they die and degenerate

16. Semen is body fluid that is
ejaculated at the time of orgasm,
contain sperm & secretion of
seminal vesicle, prostate,
cowper’s gland & urethral gland.

17. SEMEN
Source Volume Characteristics
Urethral and 0.1-0.2cc Viscous, clear
bulbourethral glands
Testes, 0.1-0.2cc Sperm present
epididymides,vasa
deferentia
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

18. 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.

19. 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.

20. 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.

21. The process of ejaculation result in the
mixing of these distinct fraction of
semen.
These enter the urethra individually in the
rapid succession.

22. The function of first clear fluid fraction may
be to cleanse & lubricate the urethra in
preparation for the bulk of following
ejaculate - It originate from urethral &
cowper’s gland.

23. The second fraction consist of small
amount of secretion from epididymis &
vasdeferns & large proportion of
prostatic secretion which contain
spermatozoa.

24. 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.

26. WHO edition and year
Semen parameter
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
Cooper, 2007 (ESHRE campus meeting)

27. Sample should be collected in wide mouth
clean & dry bottle.
1. Semen collected following 3 days of
abstinence.
2.The specimen should be collected by
MASTURBATION in the clinical pathology
laboratory.
This allow a complete examination of
semen particularly liquefication time.

28. 3. Alternate method for collection is in
pateint’s house by coitous interuruptus or
masturbation.
The specimen should be deliverd within 30
min to the laboratory.( This specimen
may be not satisfactory).

29. 1. 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.

30. The semen specimen should be examined
immediately after collection.
It should be kept at room temperature.

31. To determine the fertility of the man.
After a male has undergone vasectomy to
check the completeness of the
procedure.
In medicolegal situations such as disputes
about the paternity of a child.
After reversal of vasectomy to confirm the
success of the procedure.

32. 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

33. 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.

34. 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

35. Optional tests:
• Alpha galactosidase(neutral):
20mU or more
• Zinc (total): 2.4micromol or more
• Citric acid(total):52micromol or
more
• Acid phosphatase:200U or more
• Fructose:13micromol or more

36. 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

37. COLOUR:
Homogenous grey opalescent appearance.
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.
•Tumour of the genital tract.
Increased turbidity indicates inflammatory process in some part
of the reproductive tract.

38. 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.

39. 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.

40. 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.

41. 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
4
=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.

42. 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.

43. Normal count – 15 to 150 million/ml.
Oligozoospermia - < 15million/ml.
Causes:
• Mumps orchitis
• Prostatitis
•Hypopituitarism
•Hypogonadotropic hypogonadism
•Estrogen secreting tumours
•Hypo/Hyperthyroidism
•Drugs – Sulfasalazine, Cimetidine, Estrogen,
Nitrofurantoin, Caffeine, Alcohol, Cocaine, Smoking
tobacco, Herbal medications, Chemotherapeutic
agents

44. MOTILITY:
Routinely used technique:
•Place a drop of liquefied semen on a glass slide.
•Cover with coverslip 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,
Coverslip- 22mm x 22mm
Semen- 10ul , depth of 20um.
Phase contrast microscopy – 400x/600x at 37deg C.

45. Grading:
Rapid progressive motility
Slow/Sluggish progressive motility
Non - progressive motility
Immotility

46. 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.

47. Normal Motility:
After liquefaction or
If less than this, asthenozoospermia. within 60mins after
ejaculation.
Causes:
Cold
Radiation
Spermicides, Pesticides
Prolonged heat exposures
Prolonged abstinence
Autoimmunity
Progressive loss of motility by 5%/hr after 3hrs.

48. 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.
Body &tails: red/pink.

49. 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

50. 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

51. 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.

52. 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

53. 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

54. Techniques of detecting antibodies:
•Agglutination
•Immobilization
•Precipitation
•Complement fixation
•Passive hemagglutination
•Cytotoxicity
For screening,
•Mixed antiglobulin reaction test
•Immunobead method

55. 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.

56. BIOCHEMICAL ASSAYS:
Prostate gland function:
- zinc
-citric acid
-acid phosphatase
Seminal vesicles:
- fructose
- prostaglandins
Epididymis:
- alpha glucosidase
- L-carnitine
- glycerophosphocholine

57. 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 coloured ppt. in 30 secs.
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

58. 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 & anerobic
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.

59. 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-M+B)  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

60. 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

61. 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.

62. 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 assesment of membrane
function is a useful indicator of fertilizing
ability of spermatozoa.

63. f. 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.

64. 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.

65. 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.

66. TYPICAL HEAD DEFECTS

67. Bent Flat Too
implantation Too thin Irregular thick

68. Cytoplasmic Double
drop
Hairpin Stumped
Coiled