Semen analysis dr kamlesh

Semen analysis as per WHO guideline, Presentation for PG students.

1. www.jaailab.com

2. Fluid Fractions of semen
1.Urethralglands(2-5%volume)-mucouslike.
2.Prostate:(20%to30%volume)
acidicfluid
Containsacidphosphataseandproteolyticenzymes.
Actontheseminalfluidandcausesliquefaction.
3.Seminalvesicles(46-80%volume)
Viscous,alkaline(acidicenvironmentinthevagina).
Richinfructose,vitaminC,prostaglandin.
Nourishandactivatethespermwhilepassingthrough
femaletract.
4.Testis&Epididymis(5%volume)
Epididymisprovidesatemporarystorage
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3. Semen Analysis
It provides information on
1.Sperm production.
2.Patency of the male ducts.
3.The function of the accessory glands.
4.Ejaculative function.
Indications:
1.Male infertility (30% ).
2.Effectiveness of a vasectomy (6 weeks after the vasectomy).
3.Evaluation of rape victim.
4.Suitability for artificial insemination (ART).
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4. Methods of collection
1.Masturbation.
2.Bycondom(containspermicidalagents).
3.Bycoitusinterrupts
4.Assistedejaculation:usedinparaplegics
5.PercutaneousEpididymalSemenAspiration(PESA).
Onlyindicatedfortherapeuticreasonsinobstructiveazoospermic.
6.Testicularspermextraction(TESE/TESA):OpenTesticular
Biopsy:
Highlyinvasive,opensurgicalprocedure.
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5. Instructions for collection
•Abstinence 2-6 days.
•Sample container should be provided from laboratory.
•Container must be properly labelled.
•Collection should be in the vicinity of laboratory.
•Collected after passing urine.
•Preferably by masturbation
•Entire sample must be collected into container (No spillage).
•Time of collection must be noted.
•Keep the sample at body temperature, no sunlight
•Deliver the sample within one hour of ejaculation
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6. Since semen samples may vary from day to day.
2 or 3 samples must be evaluated within a 3-6
month period for accurate testing.
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7. Steps of semen analysis
1.In the first 5 minutes:
Placing the specimen container at 37 °C for liquefaction.
2.Between 30 and 60 minutes:
Assess liquefaction and appearance.
Measure semen volume, pH.
Prepare wet preparation for motility, count, viability.
Assess for round cells.
Perform the mixed antiglobulin reaction (MAR) and
biochemical examination (if required).
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8. Steps of semen analysis
3.Within 3 hours:
if required send samples to the microbiology laboratory.
4.After 4 hours:
Assess for sperm morphology by staining.
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9. Macroscopic Examination
AppearanceNormal: Whitish to grey
Yellow (jaundice); Pink/Reddish/Brown (RBCs)
LiquefactionNormal: 15–30 minutes after collection
Lumpy>60 min : may be sign of prostatic infection, lack of
prostatic protease.
Viscosity Normal Smooth and watery
High viscosity impede sperm movements
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10. Macroscopic Examination
Volume Normal:>1.5 ml per ejaculation
Lowvolume(<1ml)-problem with the seminal vesicles and
prostate, block or retrograde ejaculation.
Low semen volume cannot neutralize vaginal acidity
pH Normal:≥7.2(alkaline)
Acidic pH (<7.0) with low volume indicates problem with
seminal vesicle flow or ejaculatory duct obstruction.
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11. Sperm Motility Assessment
A very important parameter.
Add 10µl semen under the coverslipon a glass slide.
Wait 1-2 minutes before reading.
At least 200 sperms should be counted under 40x magnification.
Motility noted as follow:
PR=Progressive motility (forward movement, large circles)
NP=Non progressive (on the spot movement, twitching)
IM=immotile (no movement)
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12. Fourdifferentgrades:
Grade4:Progressivemotility.Swimfastinastraightline.
(denotedmotilitya).
Grade3:non-linearmotility:Thesealsomoveforwardbuttravel
inacurvedorcrookedmotion.(denotedmotilityb).
Grade2:Non-progressivemotility-donotmoveforward
Grade1:Immotile.
Sperm Motility Assessment
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13. Sperm count
Mix 10µl semen to 190 µl semen diluting fluid* (1:20).
After shaking for 3minute we charge the hemocytometer with
10µl of this mixture.
Wait 2-3 min to settle
Sperms counted in 5 square of central grid (n).
Multiplied with 10
6
to get the sperm count/ ml.
We count only whole sperms, not pinheads
Use the “L” rule
* semen diluting fluid: 5gm sod. Bicarbonate and 1ml formalin in
100ml distilled water, other 0.5% chlorazene.
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14. Sperm count calculation
With chamber depth of 100µm, each large grid (1x1mm) holds 0.1
µl (100nl) volume.
The central grid contains 25 squares = 4nl per square.
Count = Number of sperms / volume x dilution factor
Example :
N (per 5 squares) x 20 (DF) = N x 10
6
sperms per ml
volume of 5 square (20 nl)
1 ml = 1x10
6
nanolitre
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15. Semen count
Makler Chamber
A chamber specially designed
for semen analysis
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16. Vitality Assessment
1 part semen mixed with 5 part sod bicarbonate and
centrifuge for 2to 3 minutes at 2000 to 3000 rpm.
Discarding supernatent and add normal saline and again
centrifuge.
Sediment is used to prepare a smear by using wire loop.
This smear stained by any of the following methods:
Dead sperms take up the stain while live doesn’t.
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17. Vitality Assessment
1.Eosin-nigrosin (dead sperm stain pink/red)
2.Eosin (1%) (dead sperm stain pink/red)
3.Trypan blue (0.4%) (dead sperm stain blue)
4.Hypo-osmotic swelling test (HOS) (live sperm shows tail
curling
Usually 1:1 ratio of semen sediment and dye used for staining.
At least 200 sperms must be counted.
Test 4 is use to choose live (immotile) sperm for ICSI
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18. Vitality Assessment
Dead sperms are stained pink/redLive sperms shows curling tails
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19. Assessment of morphology
Staining methods are:
Hematoxylin-Eosin Staining (The acrosomal area –pink, Post-acrosomal
area -dark purple).
Diff-Quik Staining (Acrosome –red, Post acrosomal area -dark red).
Carbol fuschin-methylene blue (Acrosomal area red)
Head:
oval, smooth with L-3-5 µm and W-2-3µm.
The head should have a well defined acrosome area of 40-70%.
Mid-piece:
Must be straight and slender, 0.5 µm in width and 7-8µm long.
Tail:
Must be straight and 45-50 µm long.
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20. Assessment of morphology
To be classified as normal, the sperm must be normal
in all portions (head, mid-piece, tail).
At least 400 sperms must be scored on randomly
chosen fields.
Normal Forms (%) = normal sperms / the total
number of sperms evaluated x 100.
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21. Morphology Assessment
Morphologically normal sperms
correlated well with the
fertilization rate in-vitro and
pregnancy rate.
Sperms with defective heads are
more likely to be immotile than
sperms without defects.
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22. Abnormal Sperms
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23. Abnormal Sperms
1 2
43
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24. Round Cells Assessment
Round cells include neutrophils, lymphocytes, macrophages,
epithelial cells and spermatogenic cells.
The presence of neutrophils denotes infection/inflammatory
reaction
Normal values in high power (40x)
Leukocytes:1-4/HPF
Epithelial cells:1-2/HPF
Spermatocytes:(Immature germ cells): 1-2/HPF
Erythrocytes:1-2/HPF
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25. Semenbiochemistry
Markers of prostatic function:
Acid phosphatase (>200U/ ejaculate)
Citric acid (>52 µmol/ ejaculate)
Zinc (>2.4 µmol/ ejaculate)
Marker for seminal vesicle function
Fructose (>13µmol/ ejaculate)
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26. Other tests
Post coital test (Sim’s and huhner’s test)
Woman’s cervical aspirate taken 2 hours after intercourse.
1x10
5
/ hpf sperms seen in normal condition.
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27. Studies and guidelines for SA
Guzick’s study (1991): included three parameters of semen
analysis-Concnetration, motility and morphology.
Group Concentration
(Million/ ml)
Motility (%)Morphology
(% of normal
sperms)
Fertile >48.0 >63 >12
Intermediate13.5-48.0 32-63 9-12
Subfertile <13.5 <32 <9
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28. WHO 2010 guideline
Reference range is derived from 4500 semen analysis.
Semen sample were collected from fertile men whose partner had
pregnancy within 12 months.
Sample collected from people of 14 countries over 4 continents.
From Asia only Chinese and Singaporean were included.
No Indians.
Lower reference range was 5
th
percentile.
No high reference range is given.
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29. WHO 2010 guideline
Parameter Lower Reference Limit
Semen volume 1.5 ml
Sperm concentration 15 x 10
6
/ml
Total sperm number 39 x10
6
/ejaculate
Progressive motility (PR)32%
Total motility (PR +NP) 40%
Vitality (live sperms) 58%
Sperm morphology (NF) 4%
pH* ≥7.2
Leucocyte* <1 x10
6
/ml
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30. Terminologies
Hypospermia –semen volume < 1.5 ml
Hyperspermia –semen volume > 6.0 ml
Aspermia –no semen volume
Oligozoospermia –sperm concentration <15 million/ml
Azoospermia–no spermatozoa in semen
Polyzoospermia–++ high sperm concentration, >200M/ml
Asthenozoospermia–<40% grade (PR+NP) or < 32 PR%
Teratozoospermia–<4% spermatozoa
Necrozoospermia–“dead” sperm
Pyospermia–leukocytes present in semen, >1M/ml
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31. Retrograde Ejaculation
Semen is ejaculated into the bladder.
The acidity of the urine will kill sperms quickly.
Alkalination of the urine is very important in order to recover
live motile sperms.
The patient is instructed to take sodium bicarbonate, 3g
dissolved in a glass of water in the night.
In the morning patient must empty his bladder completely.
Again he drinks another glass of sodium bicarbonate before
coming to the laboratory.
Ask the patient to empty his bladder before semen collection.
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32. Retrograde Ejaculation
Provide two containers for collection
A small one for semen
Larger one for urine.
Instruct the patient to collect the semen by masturbation
and to urinate immediately after masturbation.
The urine is divided into tubes and centrifuged for 10
min at 1500 rpm.
The supernatant is removed leaving behind the
sediments which analyzed as semen analysis.
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33. Semen examination for medicolegal
purpose
Look for presence of semen on stained clothing or
materials or linen.
Presence of semen may be indicated by:
1.Presence of spermatozoa.
2.Presence of biochemical of semen by microchemical
test (Florence test)
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34. Advantages of semen analysis
Noninvasive
Simple
Inexpensive
Fast result
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35. Limitations of semen analysis
Patient may be too embarrassed to provide sample.
Sample from outside laboratory may not be reliable.
Wrong sampling.
Improper transportation
Subjective test so inter-observer differences high.
Semen quality vary from day to day hence single test is
not reliable.
Reproducibility is low as it is subjective test.
Hesitation in reporting due medicolegal issues.
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36. Computer Assisted SA (CASA)
Uses video and computer software technology to capture the
typesand speed of sperm.
Additional parameters can be measured such as
Curvilinear velocity (VCL)
Straightline velocity (VSL)
Linearity
Flagellar beat frequency
Amplitude of lateral head (ALH)
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37. Computer Assisted SA (CASA)
Advantages
More objective and reproducible measurement
Superior in measurement of sperm motility
Disadvantages
Not reliable if sperm density is <2x10
6
/ml or >50x10
6
/ml.
lots of debris/immotile sperm.
Parameters not standardized between laboratories –difficult
to interpret results
Can not distinguishing fertilizing capacity of semen.
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38. SQA-V
TheSQA-V(SpermQualityAnalyzer-V)
Verypopularbecauseofit’sspeed,accuracy,andprecision
parametersincludesaremotilityandmorphology.
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39. Integrated Semen Analysis System(ISAS)
ISAScanbeconsideredasthemostcompleteandeasiest-to-
usesysteminmarket.
ItalsoincludesDNAfragmentationanalysis.
Integrated Visual Optical System for sperm analysis (IVOS)
Onlysystemwhichincludes
directvisualizationofsperms.
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40. CASA for Research
This study was conducted in Calcutta, India
Groups were studied for risk factor exposure by using CASA.
The parameters considered among CASA results were: VCL,
VSL, VAP, STR.
1.Tobacco-exposed group-VCL and STR were declined.
2.Heavy metal-exposed group-VCL and ALH were
declined.
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41. Factors affecting SA result
Poor SA can result from factors such as:-
•Incorrect semen collection technique
•Spillage
•Delay in delivering sample
•History of recent illness –flu or high fever may depress
sperm counts
•Long period of abstinence –increased abnormal sperm
morphology and decrease motility
•Short abstinence period –lower sperm count
As it take 10 weeks (64-70 days) for a new batch of sperm to
be generated by the testes, it is best to repeat SA after a
period
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42. Factors affecting SA results
Medicines (cimetidine, male and female hormones,
sulfasalazine, nitrofurantoin)
Caffeine, alcohol, cocaine, marijuana, and smoking tobacco.
Temperature -sperm motility decreased if sample cold.
Exposure to radiation, some chemicals (certain pesticides)
Prolonged heat exposure.
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43. Conditions with Abnormal SA
Low or absent sperm count:
Orchitis
Varicocele
Klinefelter syndrome
Radiation
Mumps
Chronic illness (diabetes) may cause retrograde ejaculation
Hormonal imbalance
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44. Improving SA
Quality Assurance Program
SOP
Documentation
Proper labeling and reporting
External QC
Internal QC
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45. 1.Invitrofertilization(IVF)
2.Zygoteintrafallopiantransfer(ZIFT):
3.Gameteintrafallopiantransfer(GIFT)
4.Intracytoplasmicsperminjection(ICSI)
Assisted reproductive technology (ART)
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46. Sperm preparation
The semen is a mixture of motile and dead spermatozoa with cells,
cellular debris and sometimes micro-organisms present.
A variety of methods have been developed to separate the motile
sperms from the ejaculate.
The most common methods are based on washing and
centrifugation
1.Simple sperm wash
2.Swim up
3.Gradient
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