a laboratory method for semen analysis.

1. SEMEN
ANALYSIS
Sherin K
MSc MLT Pathology
JIPMER
1

2. HISTORY
Antonie van Leeuwenhoek
 Sperm first observed in 1677 by Antonie van Leeuwenhoek.
 Sperm is the male reproductive cell .
 “sperm “is derived from the Greek word sperma.
2

3. SPERM
 A sperm cell is 70 µm long, acrosomal cap include 40-70%
 Tail is 45 µm, Head is 5×3 µm (length and width).
 The middle section (body) is approximately the same length as
the head.
3

4. 4

5. Male reproductive system includes
 The testicles
 Epididymis
 Vas deferens (Duct system)
 Seminal vesicles and prostate gland
 Penis
5

6. Composition of semen
 Sperm
 Fructose and minerals
 Prostaglandins
 Fibrinogen, Spermidine
 Citrate ,PSA, Zinc
 Proteolytic enzyme etc
6

7.  Spermatogenesis is the process of forming motile
spermatozoa from spermatogonium.
 Duration : 64 days (2 months)
 Spermiogenesis is the production of mature, motile
spermatozoa from spermatids.
7

8. INDICATIONS
Infertility Vasectomy Medicolegal cases
8

9. SEMEN ANALYSIS
 Sample collection
 Macroscopic examination
 Microscopic examination
 Chemical examination
 Immunological assays
 Sperm function tests
 Microbiological assays
9

10. SAMPLE COLLECTION
Methods :
 Masturbation
 Coitus interruptus : withdrawing the penis just before
ejaculation
 Coitus with silicon condom
 Ejaculation stimulated by electricity.
10

11. Patient preparation :
 Patient should be in a period of sexual abstinence for 3 days
but not more than 7 days.
 Should not use any lubricants.
 Avoid any hormonal medications.
 Collect a complete semen specimen in a clean, sterile , wide
mouthed container .
11

12.  Specimen should collected in room temperature.
 Avoid exposing the specimen to extreme hot or cold
climate.
 Deliver it to the laboratory within 1 hr of collection.
 The lab person should record the time of specimen
collection and specimen receipt.
 Should not process the sample immediately. Wait for 30
minutes(liquefaction time).
12

13. Safe handling of specimen :
 Semen sample may contain dangerous infectious agents like
HIV, Hepatitis viruses or Herpes simplex virus etc.
 Therefore handle the specimen as a biohazard.
 Standard precautions must be taken during the analysis.
13

14. MACROSCOPIC ANALYSIS
It has a homogenous, grey-opalescent appearance.
14

15. COLOUR & CONSISTENCY :
 Red-brown - Red blood cells are present.
 Yellow - jaundice , taking certain vitamins or drugs.
 Thick , lumpy or jelly like – male hormone deficiency.
 Less opaque – sperm concentration is low
VOLUME :
 Normal volume : 2-5 ml
 Less than 1.5 ml : abnormal
 Measured in a small graduated glass cylinder.
15

16. LIQUEFACTION :
 Definition : process when the gel formed by proteins from
the seminal vesicles is & the semen becomes more liquid
 Normal Liquefaction time : 15-30 minutes after collection.
 >60 minutes : abnormal, sperms may be trapped in
unliquefied jelly; maybe sign of prostatic infection, lack of
prostatic protease .
 The semen has not liquefied after 2 hr, add alpha-
chymotrypsin (protease enzyme)
16

17. VISCOSITY :
 It is the consistency of the fluid.
 Normal – smooth and watery.
 Incompletely liquefied- highly viscous.
 Normal semen droplets form a thin thread when released
from the pipette.
 Threads >2 cm are considered highly viscous.
 High viscosity impede sperm movements.
17

18. pH :
 The normal pH is alkaline (varies from 7.2 to 8)
 Secretions from the prostate and seminal vesicles
contribute to seminal pH
 Measured using litmus paper.
 Increased pH- infection of reproductive tract.
 Decreased pH-increased prostatic fluid.
 Sperm die at < 6 pH
18

19. MICROSCOPIC ANALYSIS
SPERM
MORPHOLOGY
SPERM
MOTILITY
19

20. 1. Sperm count
a) Improved Neubauer haemocytometer.
20

21.  Liquefied semen – up to 0.5 mark
 Diluting fluid – up to 11 mark
o Examine under microscope and count the number of
spermatozoa in 4 corner squares.
o Number of spermatozoa/μl =N × 50
 Normal count = 60-150 million/ml
 Abnormal count = < 20 million/ml
21
Diluting fluid :
Sodium bicarbonate - 5 g
(counteract with mucus)
Formalin - 1 ml
Diluting fluid
Sodium bicarbonate – 5g
Formalin – 1 ml
Distilled water – 100ml

22. 22
b) Makler counting chamber

23.  Place a small, un-caliberated drop of liquefied well mixed
semen in the center of the chamber.
 The number of spermatozoa counted in any strip of 10
squares indicates their concentration in millions per ml.
23

24. c) Sperm counting chamber
 10×10 Squares
 Depth 0.01mm
 Area 0.01mm2
 No. of sperm counted in any strip of 10 Squares indicates
their concentration in millions/ml
24

25. 2. Sperm motility
 Should be performed on an Undiluted, well mixed, liquefied
semen within 1 hour of collection.
 Place a drop of liquefied semen on a clean glass slide.
 Put a cover slip over it and first examine under low power and
then under high power.
 https://youtu.be/SMe_FvQifwU
25

26. The microscopic slide screened systematically and the motility
of each spermatozoa encountered is graded as A,B,Cand D in %
according to whether it shows:
 A- Rapid progressive motility
 B- Slow progressive motility
 C- Non-progressive motility
 D- Immotility
26

27.  Normally within 2 hours of ejaculation :- > 60% of
spermatozoa are vigorously motile
 After 6-8 hours :- 25-40% motile
 If motility is less than 50%, a stain for viability such as
Eosin Y with Nigrosine as counter stain can be done.
 Red dye accumulates in the heads of non-motile sperms
27

28. 28

29. 3. Sperm morphology
 Make a thin smear using liquefied undiluted semen.
 Air dried and stain with Leishman , Giemsa or H&E stain.
 Examine under oil immersion objective.
 Normally 80% of the spermatozoa are normal.
29

30. A normal sperm has :
 A smooth, oval-shaped head, 5-6 μm long and 2-3 μm wide
 A well-defined cap (acrosome) that covers 40% to 60% of the
sperm head.
 The mid-piece must be straight and slender, 0.5 µm in width
and 7-8µm long, straightly aligned to the head.
 The tail must be straight and 45-50 µm long.
30

31. 31
Total of 400 sperms should be evaluated. Out of which
% of normal form is calculated as;
Normal Forms (%) = normal sperms x 100
the total number of sperms evaluated
Also seen for,
• Agglutination
• Pus cells/HPF
• Debris

32. Categories of defects :
 Head defects, namely large, small, tapered, pyriform , globe,
amorphous heads, vacuolated heads, double heads and small
acrosome.
 Neck and mid-piece defects namely bent neck, abnormal
mid-piece (thin or thick).
 Tail defects namely short, multiple, hairpin, broken, bent tails,
coiled tail
32

33. 33

34. 34

35. TERMS DISCRIPTIONS
Oligospermia Sperm concentration<15 millions/ml
Azoospermia No sperm in semen
Aspermia No semen volume
Pyospermia Leukocyte present in semen >
Necrozoospermia “Dead” sperm
Hematospermia Red cell present in the semen
Asthenozoospermia Reduced sperm motility
Teratozoospermia Sperms with abnormal morphology
35

36. CHEMICAL EXAMINATION
FRUCTOSE TEST:
 Fructose is produced by seminal vesicles and is released in
to the semen during ejaculation
 Testing should be considered for patients with azoospermia
and low volume ejaculation
 Normal seminal fructose level is 150-600 mg/dl
 Fructose is measured qualitatively by resorcinol test/
Seliwanoff’s test
36

37. Procedure
 Take 5 ml of dilute HCl in a test tube.
 Add 1 ml of semen.
 Add 5 mg of resorcinol. Boil and cool it
 Interpretation :
Cherry red colour –
presence of fructose
37

38. IMMUNOLOGICAL ASSAY
 The sperm antibody binding to the antigen present in the
head or tail is considered specific for immunologic infertility.
 The antibodies are usually of IgA or IgG, and rarely of IgM
 These are detected by direct or indirect mixed agglutination
reaction tests
38

39. SPERM FUNCTION TEST
 Sperm functional assay ,which indirectly measures the ability
of one spermatozoa to deliver the correct complement of
chromosomes to an ovum
 This includes sperm penetration assay , sperm zona pellucida
binding test , acrosomal reaction test
 Defective sperm function may affect various fertilizing
activities
39

40. MICROBIOLOGICAL ASSAY
 It includes the culture of semen in order to exclude
microbiological causes of infertility.
 Culture aimed at the isolation of Neisseria gonorrhoeae ,
chlamydia trachomatis and ureaplasma species
 Culture is done if there are pus cells in the semen.
40

41. AUTOMATION
41

42. SQA-V Gold
 It is a high performance analytical medical device that
combines technology in electro –optics, computer
algorithms and video microscopy.
 Analysis time -75 seconds
 Requires 0.5 ml of sample for testing
 It archive up to 500 patient record
 If the sample is viscous treat it with quick check
liquefaction kit
42

43. 43

44. SQUA-V Testing capillary tube
 Disposable, capillary tube
 Motility measured in the “motility section” which requires 20
micro liters of semen
 Concentration is measured in the “cuvette section” which
requires 450 micro liters of semen
44

45. SQUA-V Slide adaptor
 It is a slide adaptor used for the visualization
compartment.
 Use with a standard laboratory slide 76 x 25.6 mm and 22
x 22 mm cover-slip
 A 10 micro liters sample placed 12 mm from the end of
the slide
45

46. 46

47. Step 1
Capillary is inserted in to the measurement compartment
Step 2
• Concentration- An optical density detector measures the
amount of light absorbed by the cells and convert it to
optical density
• The “OD” reading is translated in to sperm concentration
by a microprocessor
47

48. Step 3
• Motility-The movement of motile sperm causes light
disturbances
• The light disturbances are converted in to electrical signals
with “peaks and valleys”
• It is analysed by a microprocessor and translated in to
motility parameters
48

49. SEMEN PARAMETERS OF SQA-V Gold 49

50. THANKYOU
50