Joshua David S.

Abejo
MALE REPRODUCTIVE ORGANS AND THEIR
ROLE IN SEMEN PRODUCTION:

• TESTES – paired glands found in the scrotum

• EPIDIDYMIS – for sperm maturation and storage until ejaculation
• DUCTUS DEFERENS – propel sperm to the ejaculatory duct
• SEMINAL VESICLES – provides transport medium for the sperm
• PROSTATE GLAND – provides proteolytic enzymes
• BULBOURETHRAL GLAND – produces thick, alkaline mucus
SEMEN COMPOSITION
• 60-70% seminal vesicle fluid
• 20-30% prostate gland
• 5% bulbourethral gland fluid
• 5% sperm cells
CHEMICAL CONSTITUENTS
• Acid phosphatase – distinguishes semen from other body fluids
• Fructose – major nutrient of spermatozoa
• Spermine and Choline – antibacterial substances from the prostate
glands
• Potassium, Citric Acid, Ascorbic Acid
SPECIMEN COLLECTION
• SEXUAL ABSTINENCE – at least 2 days to not more than 7 days
Prolonged Abstinence = higher volume, decreased motility and
increased flavin

First portion of the ejaculate contains the highest concentration of

sperm

• EMPTY BLADDER BEFOE EJACULATION!!!!!

• TIME OF COLLECTION PREFERABLY MORNING.
METHODS OF COLLECTION:
**MASTURBATION – PREFERRED METHOD
• COITUS INTERRUPTUS
• VAGINAL VAULT ASPIRATION
• CONDOM METHOD – Nonlubricated containing condoms or polyurethane
condoms should be used
PRESERVATION
• Keep specimen at room temperature if it needs to be transported
• Specimen must be kept at body temperature
• For Artificial Insemination, it can be preserved in frozen state and
stored for 1 year at -85C
• Specimens for fructose levels should be tested within 2 hours or
frozen to prevent fructolysis
ANALYSIS OF SEMEN
MACROSCOPIC:
Color – Grayish to pearly white
Transparency – Translucent
Volume – 2-5 mL/ejaculation
Odor – Chlorox-like
Viscosity – pours in droplets
Liquefaction time – 30 minutes to 1 hours
pH – 7.2-8.0
MICROSCOPIC:
Sperm Motility – at least 50% with a grade of 2.0 after 1 hour
Sperm Concentration - >20 million/mL
Sperm Count - >40 million/ejaculate
Morphology –
Routine: >30% normal morphology
Kruger’s Strict Criteria: >14% normal morphology
KRUGER’S STRICT CRITERIA:
- Measures head, neck, tail size, acrosome size and presence of vacuoles
- Requires micrometer or morphometry
- Recommended by WHO
VARIATIONS/ABNORMALITIES
• COLOR: rusty red to brown, yellowish
• TRANSPARENCY: Clear, white turbidity
• VOLUME: Increased – Prolonged abstinence
Decreased – Infertility, Incomplete collection
• VISCOCITY: Incomplete liquified specimen

Failure of liquefaction within 60 minutes may be caused by prostatic

enzymes deficiency and should be reported.
*Dulbecco’s Phosphate buffered saline, alpha-chymotrypsin or
bromelain may be added to induce liquefaction
MICROSCOPIC EXAMINATION
• SPERM MOTILITY – performed on a well-mixed, liquified specimen
Grade WHO SPERM MOTILITY ACTION

4.0 a Rapid, straight-line motility

3.0 b Slower speed, some lateral movement

2.0 b Slow forward progression, noticeable lateral movement

1.0 c No forward progression

0 d No movement

• COMPUTER ASSISTED SEMEN ANALYSIS (CASA)

- Determines both sperm velocity and trajectory (direction of motion)
- Sperm concentration and morphology is also included in the analysis
• SPERM CONCENTRATION AND COUNT
- Sperm cells are counted by diluting the specimen and counting using
the Neubauer counting Chamber
- Makler: undiluted specimens, sperms are immobilized by heating,
sperm motility is assessed in the unheated portion

SPERM COUNT:
- Both sides of the hemocytometer are loaded and counted
- Brightfield/Phase-contrast microscope is used
>1.0 million/mL WBCs = inflammation
>1.0 million/mL spermatids = disruption of spermatogenesis
• Dilution – 1:20
• Diluting fluids: 5% Sodium carbonate or 1% formalin

• Clinical significance:
Azoospermia – total absence of sperm
Necrospermia – presence of sperm cells whether completely dead or
immobile
Oligospermia – deficiency in the number of sperm cells or presence of
few motile cells
• SPERM CONCENTRATION

sperms/uL = #of sperms counted x dilution

#of squares counted x volume of square

To convert uL to mL, multiply by 1000

• SPERM COUNT
Multiply sperms/mL by volume of semen
SPERM MORPHOLOGY
• At least 200 sperm cells under OIO should be evaluated from a thinly
smeared, stained slide
• Stains for Sperm Morphology: Papanicolau, Giemsa, Wright or Shorr

• Evaluated from the head, neckpiece, midpiece and tail

- Head: Oval - shaped approximately 5um long and 3 um wide
- Acrosomal Cap – ½ of the head and 2/3 of the nucleus
- Neckpiece – attaches the head to tail
- Midpiece – approximately 7 um long and the thickest part of the tail
- Tail – 45 um long
SPERM VITALITY/BLOOMS TEST
• Assessed within 1 hour of ejaculation by mixing specimen with
EOSIN-NIGROSIN DYE

Living sperms – bluish-white

Dead sperms – red against purple background

Normal Value: >50% living sperm cells

 SEMINAL FLUID FRUCTOSE
• Low or absent fructose levels may cause low sperm concentration, caused by
abnormalities of the SEMINAL VESICLE

• RESORCINOL TEST
Reagent: 50 mg resorcinol in 33 mL concentrated HCl diluted to 100 mL water
Mix 1 mL of semen and 9 mL of reagent
Boil
Result: ORANGE
Normal Fructose Level: >13umol/ejaculate

TESTED WITHIN 2 HOURS OR FROZEN!!!!!!

ANTI-SPERM ANTIBODIES
MIXED AGGLUTINATION REACTION – screening procedure to detect IgG
antibodies
- Less than 10% of motile sperm attached to particles is considered
normal

IMMUNOBEAD TEST – more specific test and detects IgM, IgG and IgA
antibodies and demonstrates what area of sperm the autoantibodies
are affecting
- Less than 50% of the sperm with attached beads is considered normal
CHEMICAL TESTS
Decreased Neutral alpha-glucosidase, Choline, L-carnitine: Epididymis
Decreased Zinc, Citric acid, Acid phosphatase: prostatic fluid

REFERENCE SEMEN CHEMICAL VALUES

Neutral alpha-glucosidase >20 mU/ejaculate

Zinc >2.4 umol/ejaculate

Citric Acid >52 umol/ ejaculate

Acid phosphatase >200 Units/ejaculate

• POSTVASECTOMY SEMEN ANALYSIS
- Specimens are routinely tested at monthly intervals
- Beginning at 2months post vasectomy
- Continuing until two consecutive monthly specimens show no
spermatozoa

• MICROBIAL TESTS
- Routine aerobic and anaerobic cultures and tests for C. trachomatis,
M. hominis and U. urealyticum
• FLORENCE TEST: test for Choline
- Potassium Iodide and Iodine crystals
- Positive: brown rhombic crystals

• BARBIERO TEST: test for Spermine

- Picric acid and TCA
- Positive: yellow leaf-like structures

• SPINBARKEIT TEST: test for the tenacity of Mucus

• SUMS-HUHNER: test for the ability of sperm cells to penetrate the mucosa