Semen Examination , Speical and routine tests by suchit kumar

1. SEMEN EXAMINATION
SPECIAL & ROUTINE TEST
Moderated by:- Presented
by:-
Dr. S.P. Khanna Suchit Kumar
(Assoc. Prof. ) Msc(mlt) 1st
year
Department of pathology (MMIMSR)
M.M. Deemed to be university

2. INTRODUCTION
 Semen (seminal fluid ) consists of
spermatozoa(sperms) and fluid part.
 About 40% cases of infertility are due to
abnormalities in semen.
 Semen analysis is the first test to be performed
while investigating for infertility.
 Defect of sperms may be quantitative (absence f
sperms , lacks of enough sperms) and qualitative.

3. INDICATIONS OF SEMEN ANALYSIS
 Assessment of fertility/ infertility.
 Success of vasectomy.
 Success of varicocele surgery ( after 3-4 months of
surgery)
 Medicolegal purpose: In alleged rape cases,
vaginal pool smear are examined to detect sperms.
 For selection of assisted reproductive technology
(e.g. in vitro fertilization, gamete intra-fallopian
transfer technique).

4. DIFFERENT PROCEDURES OF SEMEN ANALYSIS
1. Sample collection
2. Physical examination
3. Microscopic examination
4. Chemical examination
5. Immunological assays
6. Microbiological assays
7. Sperm function tests
8. Semen cryopreservation
9. Sperm donation

5. COLLECTION OF THE SEMEN SAMPLE
 Patient is asked to collect the semen by
masturbation after a minimum of 2 days and a
maximum of 7 days of sexual abstinence.
 Specimen should be collected in a clean,dry,wide
mouthed plastic/glass container.
 Collection of condom sample is not advisable.
 The sample submitted in the laboratory immediately
within one hour of collection for examination.

6. PHYSICAL EXAMINATION
Semen is examined grossly for the following features:
colour ,volume,viscosity,pH reaction and
liquefaction.
 Colour normally , volume of semen is b/w 2.5 to 5
ml.
 Viscosity when ejaculated , semen is fairly viscid
and it falls drop by drop.
 PH reaction normally, it is slightly alkaline with pH
b/w 7-8.
 Liquefaction occurs because of presence of
fibrinolysis. Normally liquefaction occurs at room
temperature within 10-30 minutes.

7. MICROSCOPIC EXAMINATION
Semen is examined microscopically
for motility, count and morphology.

8. MOTILITY
 Place a drop of liquefied semen on clean glass
slide.
 Put a coverslip over it and examined it under the
microscope .
 Normally , within 2 hours of ejaculation , more than
60% of spermatozoa are vigorously motile and in 6-
8 hours 25-40% are still motile is.
 If motility is less than 50%, a stain for viability such
as methylene blue or eosin Y with nigrosin as
countersatin can be done.
 Heads of non-motile sperms are stained positive for
the dye.
 Sterile moles have <25% motility of sperms.

9. COUNT
 This is done in Neubauer’s (hemacytometer) chamber
using a WBC pipette.
 Draw liquefied semen in WBC pipette up to marks 1
and then the diluting fluid up to mark 11.
 The composition of diluting fluid is as under:
Sodium bicarbonate 5 gm
Formalin (neutral) 1 ml
Distilled water 100 ml
continued….

10. COUNT
 After mixing it properly, charge the chamber.
 Allow the spermatozoa to settle down in 2 minutes.
 Examine under microscope and count the number of
spermatozoa in one large peripheral square.
Calculations:
number of sperms= n
In 1 µl , no. of sperms = n X 10
no. of sperms per µl = n X 10 X 10
no. of sperms per ml = n X 10 X10 X1000
n X 100,000 ( 1 lakh)
Average no. of sperms counted in 1 mm²
Normal value = > 60 million/ml
Abnormal value = < 20 million/ml

12. MORPHOLOGY
 Prepare a thin smear from liquefied semen on a
glass slide and stain with a Romanowsky stains ,
Pap or H&E stain.
 Observe at least 200 sperms for any abnormality in
their morphology
 Normally 80% of sperm are normal
 The abnormal forms of sperms are with double
head , swollen and pointed head , double tail and
rudimentary forms.

13. MORPHOLOGY OF SPERM

14. CHEMICAL EXAMINATION
 Chemical analysis of semen consist of two
main tests :
Fructose test
Acid phosphatase test

15. FRUCTOSE TEST
 This test determines androgen deficiency or
ejaculatory obstruction of semen ; the level of
seminal fructose is low in both these
conditions.
 Normal seminal fructose level is 150-
600mg/dl
 Fructose is measured by qualitatively by
resorcinol test.

16. ACID PHOSPHATASE TEST
The test is used for seminal stain and on
veginal aspirate in medicolegal cases
Normally , semen has 2500 KA units /ml of
acid phosphatase

17. IMMUNOLOGICAL ASSAYS
 The presence of sperm antibody binding to
head or tail antigen considered specific for
immunologic infertility.
 The antibodies are usually of immunoglobulin
A (IgA) or IgG, and rarely of IgM class.
 These are detected by direct or indirect
mixed agglutination test .

18. MICROBIOLOGICAL ASSAYS
 Genital track infections by bacteria , yeast
and sexually transmitted diseases may have
significant adverse effect on male infertility.
 If the concentration of bacteria exceed 1000
CFUs per ml ,
The colonies should be identified and test
for antibiotic sensitivity .

19. SPERM FUNCTION TEST
 Defective sperm function may affect various
fertilizing functions .
 Most importantly , it includes transport of sperms in
the male and female reproductive tracts ,
which is responsible for fertilization activity such as
specific zona binding , penetration and formation of
male pronucleus .
continued…

20. SPERM FUNCTION TEST
 A list of common sperms function test is as
under :
1. Sperm penetration assay to test the success
of penetration of egg by the spermatozoa.
2. Hypoosmatic swelling test to test the
membrane integrity of the sperms.
3. Cervical mucus penetration test to test the
relative ability of motile sperms to pass
through the cervical mucus of the partner
collected at mid-cycle.

21. SEMEN CRYOPRESERVATION
 Cryopreservation or semen banking is
indicated in the following conditions :
1. For assisted reproduction
2. For donor insemination
3. For men undergoing vasectomy
4. In men before starting cancer therapy
5. In life-threatening jobs (e.g. military service )

22. SPERMS DONATION
 Sperm donation is a procedure in which a man
donates semen — the fluid containing sperm that is
released during ejaculation — to help an individual
or a couple conceive a baby.
 Donated sperm can be injected into a woman's
reproductive organs (intrauterine insemination) or
used to fertilize mature eggs in a lab (in vitro
fertilization). The use of donated sperm is known as
third-party reproduction.