Semen Analysis: Verian John Hoo - Sudario, RMT

Semen Analysis
Verian John Hoo – Sudario, RMT

College of Medical Technology
FORMATION & PHYSIOLOGY
FOUR FRACTIONS OF SEMEN

• 5% SPERMATOZOA
1. Semineferous tubules (Testes)
• Site of Spermatogenesis
• Leydig cells – testosterone secretion
• Sertoli cells – serve as nurse cells for
developing sperm cells
2. Epididymis
• Sperm Maturation
•60-70 % SEMINAL VESICLE FLUID
• Provides nutrients for sperm & fluid
• Rich in FRUCTOSE for sperm motility
FOUR FRACTIONS OF SEMEN
•20-30 % PROSTATE FLUID
• Acidic fluid containing ACP, Citric acid, & Zinc
• Provides proteolytic enzymes for coagulatinon
& liquefaction
•5% BULBOURETHRAL GLAND FLUID
• Thick, alkaline mucus to neutralize prostate
secretions & vagina
• If the vaginal acidity is not neutralized, sperm
motility would be diminished
SPECIMEN COLLECTION
• SEXUAL Abstinence of 2-3 days but NOT > 5
or 7 days
• Prolonged abstinence – higher volume,
decreased sperm motility & increased Flavin
giving semen a yellowish color
•Collect the ENTIRE ejaculate
• First portion of the ejaculate contains the
highest concentration of sperm
• If First Portion of ejaculate is missing –
↓ sperm ct, ↑ pH, spx will not liquefy
• If Last Portion of ejaculate is missing –
↑ sperm ct, ↓ pH & volume, spx will not clot
SPECIMEN COLLECTION
• METHODS OF COLLECTION
1. Self-productIon or MASTURBATION –
PREFERRED METHOD
2. Coitus interruptus – withdrawal method
3. Vaginal vault aspiration – aspiration of
semen from the vaginal vault after coitus
4. Condom method – use only nonlubricant
containing rubber or silastic condom
SPECIMEN COLLECTION
•PRESERVATION
• Specimen, if collected at home, should be kept
at Room temp & delivered w/in 1 hr of
collection
• Specimen awaiting analysis must be kept at
37ᵒC
•Take note of the time of specimen collection,
specimen receipt & liquefaction
•Analysis should be done AFTER liquefaction
(usually 30-60 mins)
• Specimen that doesn’t liquefy must be treated
with Amylase or Bromelin
Semen
analysis
MACROSCOPIC EXAM
APPEARANCE
, TRANSLUCENT – NORMAL
• With Musty or Bleach odor
•Increased Turbidity – Infection
(↑WBCs)
Coloration – Presence of Blood
Coloration – Prolonged abstinence
(due to the elevation of Flavin), urine, or
medication
MACROSCOPIC EXAM
VOLUME
•NORMAL: 2-5 mL
•Increased in: Prolonged or Extended
Abstinence
•Decreased in: Infertility, Incomplete
collection
pH
 Normal: 7.2 – 8.0
 Increased pH: Infection
 Decreased pH: Increased prostatic fluid
MACROSCOPIC EXAM
VISCOSITY
•NORMAL: Pour in Droplets
•Incompletely liquefied specimens – clumped
& highly viscous
•Reporting
• O = Watery to 4+ = Gel Like
LIQUEFACTION
 Incomplete liquefaction will impede sperm
motility
 Failure of liquefaction w/in 60 mins may be
caused by prostatic enzyme deficiency
Sperm concentration
•Normal Value = 20-60 million/mL
•METHODS
1. Improved Neubauer Counting Chamber
• Dilution = 1:20
• Diluting fluids (to immobilize sperm)
• Traditional: 5% sodium bicarbonate or 1%
formalin
• Routine: Saline or Cold Tap Water
2. Makler Counting Chamber
• For UNDILUTED specimen
• Uses heat to immobilize sperm
Sperm concentration
BASIC FORMULA FOR CELL COUNT
# 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑺𝒑𝒆𝒓𝒎 𝑪𝒐𝒏𝒄 𝒖𝑳 = 𝐴𝑟𝑒𝑎 ×𝐷𝑒𝑝𝑡ℎ
To Convert Sperms/uL to Sperms/mL, MULTIPLY by 1000
SHORTCUT METHODS
Using 5 RBC squares
𝑺𝒑𝒆𝒓𝒎𝒔 𝒊𝒏 𝒎𝒊𝒍𝒍𝒊𝒐𝒏 𝒑𝒆𝒓 𝒎𝑳
= # 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 1,000,000
Using 2 WBC squares
𝑺𝒑𝒆𝒓𝒎𝒔 𝒊𝒏 𝒎𝒊𝒍𝒍𝒊𝒐𝒏 𝒑𝒆𝒓 𝒎𝑳
= # 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 100,000
Sperm COUNT
•Normal Value = ≥40 million/ejaculate
•FORMULA
𝑺𝒑𝒆𝒓𝒎 𝑪𝒐𝒖𝒏𝒕
= 𝑆𝑝𝑒𝑟𝑚 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 × 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛 𝑉𝑜𝑙𝑢𝑚𝑒
SAMPLE PROBLEM
1. Using a 1:20 dilution, an average of 60 sperm are
counted in the 5 RBC squares on both sides of the
hemocytometer. Calculate the sperm conc/mL &
total sperm count in a spx with a volume of 4 mL
2. In a 1:20 dilution, 600 sperm are counted on 2
WBC squares. Calculate the sperm conc/mL & the
total sperm count in a spx with a volume of 2 mL
Sperm MOTILITY
•Place a drop of semen in a slide & cover it
with cover slip
•GRADING
GRADE WHO WHO CRITERIA
4.0 a Rapid, straight line motility
3.0 b Slower speed, some lateral movement

Slow forward progression, noticeable
2.0 b
lateral movement
1.0 c No Forward progression
0 d No Movement
Sperm MOTILITY
•NORMAL:
• Minimum of 50% w/ a rating of 2.0 after 1 hr
COMPUTER ASSISTED SEMEN ANALYSIS

(CASA)
 Provides objective
determination of both
Sperm Velocity &
Trajectory
 Sperm Concentration &
Morphology also included
in the analysis
Sperm MORPHOLOGY
•At Least 200 sperm cells under OIO should be
evaluated from a thinly smeared stained slide
•STAINS FOR SPERM MORPHOLOGY
1. Wright’s stain
2. Giemsa stain
3. Papanicolau’s stain – STAIN of CHOICE, best
staining method, no heat fixation required
•Evaluated with respect to the head (Ovum
penetration), neckpiece, midpiece, & tail
(Motility)
Sperm MORPHOLOGY
•HEAD
• Oval shaped head – approx 5 um L & 3 um W
• Acrosomal cap – should occupy appox ½ of
head & should cover approx 2/3 of the sperm
nucleus
• Enzyme-containing
• Critical to ovum penetration
•NECK PIECE – Attaches the head to the tail
•MIDPIECE – Appox 7 um long & is the thickest
part due to the mitochondrial sheath that
produces
Sperm MORPHOLOGY
•TAIL – Approx 45 um long
• Only flagella in the human body
Sperm MORPHOLOGY
ROUTINELY IDENTIFIED ABNORMALITIES
•HEAD – assoc w/ Poor Ovum penetration

• Double heads, giant & amorphous heads,
pinheads, tapered heads & constricted heads
•NECK PIECE – interfere w/ Motility
• Abnormally long neckpiece may cause the
sperm head to bend backward
•TAIL – Doubled, coiled, or bent
Sperm MORPHOLOGY
ROUTINELY IDENTIFIED ABNORMALITIES
Sperm MORPHOLOGY
NORMAL VALUES
•ROUTINE CRITERIA:>30% normal forms
•KRUGER’S STRICT CRITERIA: >14% normal
forms
•KRUGER’S STRICT CRITERIA
• Measures head, neck, tail size, acrosome size
& presence of vacuoles
• Requires use of a stage micrometer or
morphometry
• Not routinely performed in the clinical
laboratory but it WHO recommended
Sperm VIABILITY/vitality
•Assessed w/in 1 hr of ejaculation by mixing
the specimen w/ EOSIN-NEGROSIN stain
•Number of dead cells in 100 sperm is
counted using a bright-field or phase-contrast
microscopy
• Living Sperm = Bluish White
• Dead Sperm = Red against Purple background
Sperm VIABILITY/vitality
•NORMAL VALUE
• 50% or more living sperm cells
•Decreased sperm vitality may be suspected
when a specimen has a normal sperm
concentration w/ markedly decreased
motility
• High number of vital but immobile cells =
Defective Flagellum
• High number of immotile & nonviable cells =
Epididymal pathology
SEMINAL FLUID FRUCTOSE
•Low or Absent Fructose levels may cause low
sperm concentration
•Should be tested w/in 2 hrs or FROZEN to
prevent fructolysis
•Screening Test:
• RESORCINOL Test: Orange color production
when fructose is present
•Normal Fructose Level: ≥13 umol/ejaculate
ANTI-SPERM ANTIBODIES
•Detected in Semen, Cervical mucosa, or
Serum
1. Mixed Agglutination Reaction (MAR)
• Screening test that detects the presence of IgG
• Normal: <10% motile sperm attached to
particles
2. Immunobead test
• Specific test that detects IgG, IgM, & IgA
• Demonstrates what area of the sperm (head,
neckpiece, midpiece or tail) the autoantibodies
are affecting
SEMINAL FLUID FRUCTOSE
•Low or Absent Fructose levels may cause low
sperm concentration
•Should be tested w/in 2 hrs or FROZEN to
prevent fructolysis
•Screening Test:
• RESORCINOL Test: Orange color production
when fructose is present
•Normal Fructose Level: ≥13 umol/ejaculate
Microbial testing
•Round cells
• WBCs & Spermatids (immature sperm cells)
• STAGES of Spermatogenesis
1. Spermatogonium (earlies)
2. Primary Spermatocyte
3. Secondary Spermatocyte
4. Spermatid
5. Spermatozoon
• Normal Value = < 1 million/mL
• > 1 million WBCs/mL = Infection
• > 1 million spermatids/mL = Disruption of
Spermatogenesis
Additional tests
•CHEMICAL TESTS
• Epididymis disorder = ↓ neutral α-glucosidase,
glycerophosphocholine, L-carnitine
• Decreased Prostatic fluid – ↓ zinc, citric acid,
glutamyl transpeptidase, & ACP
•FLORENCE TEST
• Test for Choline
• Reagent: Potassium iodide & iodine crystals
• (+) Brown rhombic crystals examined under
the microscope
Additional tests
•BARBIERO’S TEST
• Test for Spermine
• Reagent: Picric acid & TCA
• (+) Yellow leaf-like structures
•SPINBARKEIT TEST
• Test for the tenacity of mucus
•SIMS HUHNER TEST
• Post-coital test
• Test for the ability of sperm cells to penetrate
the cervical mucosa
POST VASECTOMY
SEMENALYSIS
•Vasectomy
• Cutting of vas deferens so that the ejaculate will
not contain any sperm cell
•Specimens are routinely tested at monthly
intervals
•Beginning at 2 mos Post Vasectomy
•Continued until two consecutive monthly
specimen show NO SPERMATOZOA
TERMINOLOGIES
1. ASPERMIA
• No Ejaculate
2. AZOOSPERMIA
• Absence of sperm cells
3. NECROSPERMIA
• Immotile/dead sperm cells
4. OLIGOSPERMIA
• Decreased sperm concentration
Normal values
MACROSCOPIC EXAMINATION
•COLOR: Grayish white to Pearly white
•TRANSPARENCY: Translucent
•VOLUME: 2 to 5 mL/ejaculation
•ODOR: Fishy, Distinct, “Chlorox-like” or Musty
•VISCOSITY: Pours in Droplets
•LIQUEFACTION TIME: 30 mins to 1 hr AFTER
Collection
•pH: 7.2 to 8.0
Normal values
MICROSCOPIC EXAMINATION
•SPERM CONCENTRATION: >20 million/mL
•SPERM COUNT: >40 million/ejaculate
•MOTILITY: >50% w/in 1 hr
•QUALITY: >2.0 or a, b, c
•MORPHOLOGY:
• > 30% normal forms (routine criteria)
• > 14% normal forms (strict criteria)
•ROUND CELLS: < 1.0 million /mL
Additional testing for abnormal
semenalysis
ABNORMAL POSSIBLE
TEST/S
RESULT ABNORMALITY
Decreased Motility Vitality Eosin-Negrosin stain
& Normal Count
Lack of Seminal
Decreased Count Vesicle support Fructose level
medium
Decreased Motility Male Anti-Sperm MAR & Immunobead
W/ Clumping Antibodies Sperm Agg w/ Male serum
Normal Analysis
W/ Continued Female Anti-Sperm Sperm Agg w/ Female
Infertility Antibodies serum
END.

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Semen Analysis

Verian John Hoo – Sudario, RMT

FOUR FRACTIONS OF SEMEN

To Convert Sperms/uL to Sperms/mL, MULTIPLY by 1000

4.0 a Rapid, straight line motility

3.0 b Slower speed, some lateral movement

COMPUTER ASSISTED SEMEN ANALYSIS

•HEAD – assoc w/ Poor Ovum penetration

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