This document provides information about semen analysis, including: 1. Semen analysis evaluates the quantity and quality of semen and sperm to detect male infertility and check the effectiveness of procedures like vasectomy. 2. Normal parameters include a sperm count of 15 million/ml or more, motility grades of 40% or more, and normal morphology of 4% or more according to WHO standards. 3. Samples should be collected after 2-7 days of abstinence and delivered to the lab within 1 hour to examine volume, pH, viscosity, sperm count, motility, morphology, and presence of other cells. Abnormal results can indicate conditions like infection or abnormalities of the reproductive organs.

1. SEMEN ANALYSIS
Presented by: Dr Shamim Ahmad
Moderator : Dr. Ashish Koshti
ASSO. Prof. (M.D.)
Department of Pathology,
G.M.C. Bhopal

2. INTRODUCTION
 Semen is a fluid that is emitted from male genital
tract and contains sperms that are capable of
fertilising female ova.
 Semen analysis also called ‘SEMINOGRAM’
evaluates following characteristics of semen and the
sperms contained within:
>To judge the quantity and quality of semen
> To detect male infertility.

6. SPERMATOGENESIS

7. INDICATIONS FOR SEMEN ANALYSIS
 INFERTILITY:
>First step in the investigation of infertility
 To check the effectiveness of vasectomy by
confirming the absence of sperm.
 To support or disprove a denial of paternity on the
ground of sterility
 For selection of assisted reproductive technology :
>IVF
>GIFT

8. INDICATIONS
 Medicolegal case:
>To examine vaginal secretions or clothing stains
for presence of semen (in case of rape)
• For artificial insemination (for selection of donors)

9. SAMPLE COLLECTION AND TRANSPORT
 The patient should be given clear written oral
instruction concerning the collection, handling and
transport of the semen.
 Sample s/b collected after min of 48 hrs of
abstinence, but not later than 7 days (ideal 3-5 days)
 Ejaculate should be collected in privacy near the
laboratory.
 It should be delivered to the lab within 1 hr of
collection.

10. SAMPLE COLLECTION……
 The sample is obtained by MASURBATION in a
clean wide mouthed container made up of glass or
plastic.
 Condom must not be used because spermicides
(menfegol etc).
TESA : Testicular sperm extraction.

11. SAMPLE COLLECTION…
The sample should
be clearly labeled
with:
the patient's name
ID or CR number
Date and time of
sample collection

13. EXAMINATION OF SEMINAL FLUID
PHYSICAL EXAMINATION
: COAGULATION- Ejaculated in liquid state, it get coagulated.
Absence of coagulation indicates CAVD.
: LIQUEFATION – Liquified within 30 minutes after ejaculation.
If not liquified then t/t with plasmin or
chymotrypsinogen may be needed.
: ODOUR- Pungent.
Absence of smell=Impaired prostate.
: COLOUR- Cloudy white to grey.
Yelllow coloured= Pyospermia
white= Azoospermia,
Brown/ Red= Haemospermia

14. : Volume- 1.5 to 4.5 ml
Less than 1.5 ml= Abnormal
: PH- Measured in PH paper.
Normal 7.2 to 8.0
< 7 = Dysgenesis of vas deferens or
Seminal vesicle or
Epididymis.
> 8= Bacterial contamination.

15. MICROSCOPIC EXAMINATION
SPERM COUNT
 Count is done after liquefaction in improved neubaur’s chamber.
 Semen is diluted 1:20 with semen diluting fluid (NaHCO3 formalin)
 Sperm count per ml is calculated by:
 Sperm counted X Correction factor for diluent
…..……………………………………………………………............ X 1000 (sperms/ml)
No. of square counted X Volume of One Square
Sperm counted X 20
 …………………………....... X 1000 (sperms/ml)
4 X 0.1
 Sperm counted X 50000 (sperms/ml)

16. SPERM MOTILITY
 All motile and non motile sperms are counted in randomly chosen field in a
wet preparation under 40x.
 A drop of semen is placed in a glass slide, covered with a coverslip and
examined under 40 x objective.
 Minimum 200 sperms are counted.
 Motility is graded as:
> Grade IV- Progressive motility: Actively moving.
> Grade III- Slow or sluggish motility: Zig-zag movement.
> Grade II- Non progressive motility: Twitching or shaking.
> Grade I - Immotility: Non motile.

17. MOTILE SPERMS SEEN IN MICROSCOPE

18. SPERM MORPHOLOGY
60 MICRON

19. NORMAL SHAPED ABNORMAL SHAPED
 Head is oval shaped
and smooth.
 Midportion of sperm is
thinner than head.
 Tail is long and thinner
than head and mid
portion.
 Head is either
abnormally large or
small and shape is
other than oval.
 Double headed,
vacuolations present
 Mid portion is divided.
 Tail defect.
NORMAL VS ABNORMAL MORPHOLOGY

20. ABNORMAL FORMS

21.  If normal morphology of sperms is less than 4 %
then it is considered as ABNORMAL.
 Abnormal morphology play an important role in
investigation of infertility but other factors are also
important:
> Sperm concentration
>Semen volume
>Sperm motility

22. NORMAL vs ABNORMAL

23. ABNORMAL FORMS

24. VITALITY ASSESSMENT
 Eosin-nigrosin stain
 Eosin (1%) stain
Usually 1:1 ratio of semen to dye mixture, mix well
and made smear into a slide.
Read immediately at 40 x objective.
Dead sperm stains pink/red and viable sperm stains
white colour.

25. VITALITY ASSESMENT

26. OTHER CELLS IN SEMEN
Leukocytes :- Normal Range…….1-4/HPF
Leukocytospermia:- >increased in number
>in reproductive tract infection
Epithelial cells:- normal Range …1-2/HPF
Spermatocytes :- >Immature germ cells
>Normal Range …….1-2/HPF.

27. OTHER CELLS IN SEMEN
 Erythrocytes :- Normal Range…..1-2/HPF.
Increased number may indicate
A reproductive tract infection or
Damage to a small capillary during
sample production.
 Bacteria and protozoan such as
Trichomonas vaginalis are uncommon in human semen.
but their presence is indicative of possible male
reproductive tract infection.

28. BIOCHEMICAL ANALYSIS
 Fructose : Seminal vesicle marker
Low or absent indicates CAVD.
(Congenital absence of vas deferens)
 Acid phosphatase, Zinc and citric acid:
>Low level indicate prostatic dysfunction
 Neutral glucosidase and Carnitine:
>Epididymis marker

29. WHO REFERENCE VALUES(2010)

30. WHO NOMENCLATURE FOR SEMEN
VARIABLES
 Normozoospermia > Normal semen parameters.
 Oligospermia > Sperm count less than 15 million/ml.
 Azoospermia > Absence of sperms in Seminal fluid.
 Aspermia > Absence of ejaculate.
 Asthenzooospermia > Reduced sperm motility
 Teratozoospermia > Incresed abnormal form
 Lekocytospermia > More than 1 million WBCs/ ml
 Necrozoospermia > All non motile and non viable
sperms.

31. IMMUNOLOGICAL ANALYSIS
 Detects antisperm antibodies.
 IMMUNOBEAD TEST.
 MIXED ANTIGLOBULIN REATION: Detect Ig G
and Ig A antibodies against sperm surface.

32. SPERM FUNCTION TEST
 Post Coital test (Sim’s Huhnar’s test)
 Cervical mucous penetration test.
 Hamster egg penetration assay.
 Computer assisted semen analysis.

33. EXAMINATION OF SEMEN IN MEDICOLEGAL
CASES
 Sample obtained from vagina, stain from clothing,
skin, hair or other body parts for semen.
 TESTS : >Microscopic examination for sperm.
 >Acid Phosphatase detection.
 >Florence test- For presence of choline.

34. TO CHECK EFFECTIVENESS OF VASECTOMY
 Aim is to detect presence or absence of spermatozoa.
 Successful Vasectomy: Count should be zero (within
12 weaks).
 Semen is considered free of sperm: If 2 successsive
samples are negative for sperm.

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