semen analysis.ppt

semen

1. Seminal fluids

2. Introduction
•Asemenanalysismeasurestheamountofsemenaman
producesanddeterminesthenumberandqualityofspermin
thesemensample.
•Asemenanalysisisusuallyoneofthefirsttestsdonetohelp
determinewhetheramanhasaproblemfatheringachild
(infertility).
•Aproblemwiththesemenorspermaffectsmorethanone-third
ofthecoupleswhoareunabletohavechildren(infertile).

3. Purpose of seminal fluid analysis
•There are basically four indications for the
examination of seminal fluid:
1.The investigation of fertility: male infertility is
primarily responsible in 30%-50% of infertile
marriages.
2.To determine the effectiveness of vasectomy.
3.To determine the suitability of semen for
artificial insemination.
4.Medicolegal: testes to detect semen are
frequently requested in alleged rape

4. Seminal Fluids
•Composition of Semen
•Spermatozoa
•Fluids to provide nutritional support and media ormechanism for delivery
% of total Description / Purpose
Spermatozoa 2-5% Formed in testes,stored in epididymisand vasa
differentia
Seminal Fluid60-75% Alkaline fluid,;primarily responsible for
nutritional support through: amino acids,
enzymes, fructose. Also to suppress possible
immune response by female
Prostate Fluid25-30% Acid phosphatase, citric acid, proteolytic
enzymes and zinc
Bulbourethral
glands
1 –5% Galactose, mucous

5. Seminal Fluids
•Anatomy, composition and formation.
•Testes–source of sperm (2-5%)
•Seminal vesicles–provides fructose & nutrients and is
primary provider of fluid (60-75%)
•Prostate gland–Provides enzyme, acid phosphatase,
citric acid, zinc, and proteolytic enzymes (for coagulation
and liquification). 2
nd
source of fluid(25-30%)
•Bulbourethral glands5%. Thick alkaline mucous-like fluid
that neutralizes acids.

6. Seminal Fluids
•The spermwithin the semen are the cells that
actually fertilize the egg and are therefore the most
important to assess.
•The sperm account for only 1-2 % of the seminal fluid
volume.
•Problems with the surrounding fluid may also
interfere with the movementand functionof the
sperm. Therefore, both the sperm and the fluid must
be tested

7. Coagulation and liquefaction
Coagulation and subsequent liquefaction are
believed to be three stage processes:
Coagulation results from the actions of a prostatic
clotting enzyme on a fibrinogen-like precursor formed
by the seminal vesicles.
Liquefaction is initiated by enzymes of prostatic origin.
The protein fragments are degraded further to free
amino acids and ammonia by the action of several
poorly characterized proteolytic enzymes, including an
amino peptidase and pepsin.

8. Specimen Collection
Pre warmed (21
o
C), sterile, non-toxic, wide-mouthglass or plastic
container.
It is important to note that contamination of the semen sample
with soap, water ect…may adversely affect sperm quality.
Specimen should be delivered to the laboratory within 1 hour of
collection.
Shortly after collection, the semen coagulates because of the action of a
clotting enzyme, formed in the prostate, on a fibrinogen like precursor
substance that is produced by the seminal vesicles.
Semenanalysis should not be performed immediately following
sample production.
The sample should be mixed well in the original container by
swirling for several seconds prior to removing the lid. Do not invert
the container.

9. •The sample should be transported upright, at body
temperature if possible, and should be delivered to
laboratory as soon as possible after collection and
certainly within one hour of collection. If the sample
is cold on receipt, this should be noted in laboratory
records.
The sample should be clearly labeled with:
1.The patient's name
2.ID or clinic number (if available)
3.Date and time of sample collection

10. Examination of seminal fluid
•When evaluating semen specimens in cases of
infertility, the following parameters are
routinely measured:
volume
viscosity
pH
sperm count (concentration)
motility
morphology.

11. Seminal Fluids
•Physical characteristics
–Liquefaction –fresh specimen will clot, then
liquefy within 30 –60 minutes
–Persistence of clot is abnormal
–All further evaluation must wait until
liquefaction is complete.

12. Semen: Appearance
Opaque, Gray, white, light
yellow
Normal
Shades of yellow Correlate with flavinconcentration
Could also indicate contamination
with urine.
Deep yellow Associated with certain drugs
Brown or red May contain blood (infection, cancer)
Highly turbid Usually contains leukocytes
indicating infection or inflammation
Macroscopic examination of Seminal Fluids

13. Semen: Appearance, cont.
Volume 2.0 –5.0 mL
Measured in serological pipette
Recorded to 1 decimal place
pH Slightly alkaline7.2 –8.0
Measured with pH paper
Acid may indicate increased prostatic fluids
pH > 8 may indicate infection
Macroscopic examination of Seminal Fluids

14. Macroscopic examination of Seminal Fluids
•Viscosity:
•Slightly viscous and easily drawn into a pipette.
•Incompletely liquefied specimens will be clumped and highly
viscous.
•Increased viscosity and incomplete liquefaction will impede
sperm motility.
•Droplets with threads longer that 2 centimeters are
considered highly viscous.
•Ratings of 0 (watery) to 4 (gel-like) can be assigned to the
viscosity report.
Factor affects semen viscosity:
•Dehydration, infection of prostate and seminal vesicle and
drug

15. Microscopic Analysis

16. Semen: Microscopic Analysis
•Microscopic examination
–Must begin as soon as the specimen is liquidifiedwhich is
generally performed 30 -60 min after collection
–Must be after liquidificationhas occurred
•Motility (sometimes referred to as the "mobility")
•Concentration / cell count
•Morphology
•Agglutination
•Viability

18. •Motility
•This describes the percentage of sperm, which are moving.
60% or more of the sperm should be moving. In order to
achieve fertilization, a sperm must not only be able to move
but be capable of movement that results in forward
progression is often also known as progressive activity.
–Motility is a very necessary quality of sperm and
critical to fertility.
–Must be evaluated within the 1
st
hour following
collection
–As it Will decrease over time
–It is important that the accurate time of collection
has been recorded and the sample is watched
carefully

19. To evaluate sperm motility, place a small drop of
liquefied semen on a pre warmed slide and cover
slipped.
•At least 200 spermatazoa counted (under 40X).
•Normally ≥ 60% sperm should be motile.
•“Asthenospermia"or Asthenozoospermia:
•Is the medical term for reduced sperm motility.
•The movement of sperm is evaluated and may be
subjectively estimated or counted into four
categories

20. sperm motility
A. Rapidly progressive spermatazoa:Moving fast
forward in a straight line.
B. Slowly progressive spermatazoa: Slow linear or
nonlinear, eg. Crooked/curved movement.
C. Non progressive spermatazoa:Movement of tails,
but with no forward progress sperm are also described
as twitching or shaking
D. Immotile spermatazoa: No movement at all.

21. Causes of Asthenospermia
• Infections: like mumps, tuberculosis, brucellosis,
gonorrhea and Chlamydia, typhoid, influenza, smallpox,
and syphilis.
• Epididymitis
• Prostatitis
Nutrition:
• Vitamin deficiencies (vitamin C,Vit B12 & E,
selenium,zinc and folate, specifically) can cause low
motility.
• Anabolic steroids ,cigarettes, alcohol

22. Semen: Microscopic Analysis
•Sperm count(sperm concentration)
•This is a measurement of how many million sperm there are in each
milliliter of fluid.
•Average sperm concentration is (60-150 million/ml).
•Counts of less than 20 million per milliliter (<20 million/ml) are
considered sub fertile.
–NV= 20 –160 million/mL

23. Azoospermia: After centrifuge sperm count is zero/HPF).
Oligozoospermia:Sperm concentration of less than 20
million/ml(sperm count 5-10 sperm/HPF) (Few spermatozoa)
Severeoligospermia: Sperm count 1-2 sperm/HPF.
Polyzoospermia:sperm concentration in excess of 350
million/ml
Aspermia-complete lack of semen
Hypospermia-Smal semen volume
Teratospermia-Sperm with abnormal morphology
Asthenospermia (Asthenozoospermia)-Reduced sperm
motility

24. •Sperm count(sperm concentration)
•Make 1 to 20 dilution with sodium bicarbonate and formalin,
count 5 small squares (within the center large square) of the
Neubauer hemacytometer.

25. Methods of measuring sperm concentration
A.By using hemacytometer
1.The sperm count is performed in the same manner as blood
and CSF counts; that is by diluting the specimen and counting
the spermatozoa in a neubauerchamber.
2.Sperm can be counted by make dilution 1:20 in WBC pipette
or by automatic pipette (which is more accurate) with a
solutioncontaining sodium bicarbonate (5g) and formalin
(1ml)(immobilize & preserve the spermatozoa), tap water
(100 ml) will suffice as a diluent.
3.The sperm should then be counted -do not count headless or
"pin-heads" sperm and do not count tailless heads.
4.Traditionally, the sperm concentration is expressed in millions
per milliliter(x10
6
/ml) of semen and the total sperm/ejaculate
is reported in millions (x10
6
) per ejaculate.

26. Calculations
1.Using a 1:20 dilution and two large WBC’s squares counted the sperm
concentration/ml = No of sperms counted x 100,000
2.Using a 1:20 dilution and five small RBC’s squares counted The sperm
concentration /ml = No of sperms counted x 1,000,000
•Using a 1:20 dilution, an average of 60 sperm are counted in the five RBC
counting squares on both sides of the hemocytometer. Calculate the
sperm concentration per milliliter and the total sperm count in a specimen
with a volume of 4 mL.
–60 sperm counted x1,000,000 = 60,000,000 sperm/mL
–60,000,000 sperm/mL x4 mL = 240,000,000 sperm/ejaculate

27. •Calculations
–standard method to begin calculation of # cells
(mature sperm) per microliter:

28. Calculations
–Example: 52 cells (mature sperm) x 20
• 5 (squares) x 0.004
This provides results as ___ cells / uL;
Normal values are reported as ___ cells / mL
–Must multiply X 1000 to convert uLto mL
•= 52.0 x 10
6
/ mL

29. B.Direct smear
•The application of a drop of well-mixed semen to a clean glass
slide under a lightly applied glass cover slip will allow
visualization of the sperm in a specimen of semen.

30. Semen: Sperm Morphology
•A smear is prepared by spreading a drop of seminal fluid on a
glass slide, stained and percentage of normal & abnormal
spermatozoa are counted.
•200 spermatozoa should be counted under oil immersion.
•Normal spermatozoa consists of : head,neck & tail.

32. Causes of abnormal sperm morphology
•Causes of abnormal sperm morphology
•Infections
•High fever
•Organic solvents causes coiled tails
•Usage of illegal drugs (cannabis, heroin, cocaine)
•Alcohol consumption
•Congenital testicular abnormalities
•Cigarette smoke

33. Sperm Viability
•Eosin –Nigrosinstain
–supravitalstain
–Add to drop of fresh sample
–Smear is made and allowed to dry
–Evaluated on oil immersion (1000x)
–Viable / live sperm do not take up the stain and remain
colorless or blue-white
–Non-viable / dead sperm stain orange-red
•Reported as % viable
•Normal >75%

34. Vitality Assessment
1.Eosin-nigrosin(dead sperm stain pink/red)
2.Eosin (1%) (dead sperm stain pink/red)
3.Trypan(0.4%) blue(dead sperm stain blue)
4.Hypo-osmotic swelling test (HOS) (live sperm shows tail curling
•Test 1, 2 and 3 for diagnostic uses. Usually 1:1 ratio of semen to dye
mixture, mix well and smear onto a slide. Read immediately at x40
objective, count 200 sperms
•Test 4 is use to choose live (immotile) sperm for ICSI .Dead sperms will
not react in HOS while live sperm will take up fluids causing their tails
to curl within 5 min and stabilize at 30 min. Therefore viable sperms
may be selected for ICSI

35. •Eosin stain is used to differentiate live
(unstained) and dead (stained) spermatozoa

36. Other cells in semen
1.leukocytesnormally (1-4/HPF), increase number
(leukocytospermia) indicates reproductive tract infection
2.Epithelial cells normally (1-2/HPF)
3.Spermatocytes(Immature germ cells) 1-2/HPF.
4.Erythrocytes(1-2/HPF). Increased number may indicate a
reproductive tract infection or damage to a small capillary
during sample production.
5.Bacteria and protozoan such as Trichomonasvaginalisare
uncommon in human semen but their presence is
indicative of possible male reproductive tract infection
and should be reported to the referring doctor for further
evaluation.

38. Sperm Agglutination
•Observed while performing motility
evaluation.
•Few clumps are normal.
•Distinctly head-to-head or tail-to-tail clumping
may indicate the presence of ant isperm
antibodies.
–IgG
–IgA

39. Sperm Agglutination
•The presence of agglutination should be recorded as
this may indicate immunological infertility.
•Assess the spermatozoa in 10 random fields -estimate
the average percentage of spermatozoa clumped
together to the nearest 5%.
•Only count motile sperm attached to other motile
sperm -do not assess immotile sperm stuck together
or motile sperm adhering to mucus threads, other cells
or debris , this is non-specific aggregation.

40. Semen: Chemical Analysis
•pH
–Measure within 1 hour of collection
–Normal 7.2 8.0
•Acid Phosphatase
–Used to evaluate the secretory function of the prostate
–Also used in forensic analysis –as prostatic fluid acid phosphatase is
higher than other fluids. (>200 units)
•Fructose
–Provides energy / nutrition to sperm
–Indication of viability
–Presence of fructose –screen using resorcinol test
•Hormones
–Testosterone, LH, FSH

41. Post-Vasectomy Analysis
–Post-vasectomy semen analysis
•Specimens tested at monthly intervals starting 2
months post-vas.
•2 consecutive months of negative microscopic for
sperm
•Wet prep with phase microscopy
•Examination of centrifuged specimen as well

42. Semen: Analysis
–Microbial testing
•Increased WBC (>1 million/ mL) suggestive of infection
•Aerobic and anaerobic cultures

45. Semen: Forensic Analysis
•Examination of fluid as to being semen
(forensic)
–Acid phosphatase –highly sensitive, as no other
body fluid contains as high level (2500 units
compared to 5 units)
–ABO, HLA typing
–DNA analysis
–UV light scan, semen fluoresces green/white