SPERMATOGENESIS

SEMEN
1. Spermatogonia
REASONS FOR SEMINAL FLUID 2. 1° Spermatocyte
ANALYSIS 3. 2° Spermatocyte
● FERTILITY TESTING 4. Spermatid (no tail)
● POST VASECTOMY SEMEN 5. Spermatozoa (w/ tail)
ANALYSIS
● FORENSIC ANALYSIS (SUSPECTED 
RAPE CASE)

SPECIMEN COLLECTION
PHYSIOLOGY &
➔ Abstinence of 2-3 days,but not >7 days
COMPOSITION ➔ Collect the entire ejaculate
SPERMATOZOA (5%) ➔ Specimen should be delivered to the
1. SEMINIFEROUS TUBULES laboratory within 1 hour of collection (at
(TESTES) room temp)
➔ Site of spermatogenesis Method:
➔ Sertoli cells: nurse cells for developing 1. Masturbation
sperms 2. Coitus interruptus / withdrawal
2. EPIDIDYMIS method
➔ Site of sperm maturation (they becomes 3. Condom method
motile) ➔ use non-lubricant-containing rubber.or.
polyurethane condom
SEMINAL FLUID (60-70%)
1. SEMINAL VESICLE
➔ Provide nutrients for sperm and fluid
➔ Secretions rich in fructose and flavin
MACROSCOPIC
PROSTATE FLUID (20-30%)
PROSTATE GLAND
EXAMINATION
➔ Contributes acidic fluid that contains ACP,
Appearance Significance
zinc, citric acid, and other enzymes.
➔ For coagulation and liquefaction Gray-White, Normal
Translucent with
Musty or Bleach
BULBOURETHRAL ALKALINE MUCUS odor
(5%)
Increased White Infection (↑ WBC)
BULBOURETHRAL GLAND Turbidity
➔ Secretes thick alkaline mucus
➔ Neutralizes acidity from the prostatic Red or Brown RBC
secretions and vagina Coloration

Yellow Coloration Urine Contaminant,

Medication, or
↑ Abstinence
 2. Makler-counting chamber
Viscosity Significance
➔ For undiluted specimen
Pour in droplets Normal ➔ Uses heat to immobilizer sperms

Thread >2cm long Abnormal SPERM COUNT

NV: 240 million per ejaculate
↓ viscosity, ↑ sperm motility Formula: sperm concentration x specimen
volume
Reporting
Watery 0

Gel-like 4
SPERM MOTILITY
➔ May also be reported as low, normal, high ➔ Place a drop of semen in a slide and cover
it with coverslip. Allow to settle for 1
minute. Observe in 20 HPF
Normal
pH Significance ➔ >50% motile (within 1 hour)
7.2 to 8.0 Normal ➔ Quality = ≥ 2.0

Increased Infection SPERM MOTILITY GRADING

Decreased ↑ Prostatic fluid Grade WHO CRITERIA

4.0 a Rapid, straight-line motility


3.0 b Slower speed, some lateral movement

2.0 b Slow forward progression, noticeable

SPERM CONCENTRATION lateral movement

Methods: 1.0 c No forward progression

1. Improved Neubauer Counting
0 d No movement
Chamber
➔ Dilution = 1:20
➔ Diluents: CASA (COMPUTER-ASSISTED SEMEN
● Formalin Sodium bicarbonate ANALYSIS)
● Saline ➔ Determines sperm concentration,
● Distilled water morphology, velocity, and trajectory
● Cold tap water (direction of motion)

NV: >20 million sperms/mL SPERM MORPHOLOGY

SHORTCUT METHOD
2 WBC squares = # sperms counted x 100,000 Routine criteria: >30% normal
5 RBC squares = # sperms counted x forms
1,000,000 Krugers' strict criteria: >14% normal
LONG METHOD forms
Standard Neubauer calculation formula ➔ Measures the head, neck, and tail using
micrometer

SPERM MORPHOLOGY
➔ Use 45° degrees when preparing smears SPERM VIABILITY
➔ Stains for Sperm Morphology
● Papanicolau's stain (VITALITY)
● Wright's stain Normal value: 50% living sperms
● Giemsa stain Modified Blom's Test:
➔ Reagents: Eosin and Nigrosin (Blue-black
SPERM MORPHOLOGY BG)
HEAD ➔ Living sperms: unstained, bluish white
Normal: oval shaped ➔ Dead sperms: red
Abnormal: indicates poor ovum
penetration

MIDPIECE
Contains mitochondria

TAIL
Abnormal: poor motility

SEMINAL FRUCTOSE
➔ Tested within 2 hours or frozen to prevent
fructolysis
➔ Resorcinol test: screening test; (+)
orange-red color

VARICOCELE
➔ Hardening of the veins that drain the testes
Most common cause of male infertility
➔ Sperm head tapered

”Seliwanoff's test” - ???

CHEMICAL TEST
Analyte Normal Value Decreased
Value
Indication

Fructose ≥ 13 Lack of
umol/ejaculate seminal fluid

Neutral ≥ 20 Epididymis
A-Glucosidase umol/ejaculate disorder
 Zinc ≥ 2.4 Lack of < 1 million round Normal
umol/ejaculate prostatic cells/mL
fluid
< 1 million Infection
Citric Acid ≥ 52 Lack of WBCs/mL
umol/ejaculate prostatic
fluid
< 1 million Disruption of
Acid ≥ 200 Lack of spermatids/mL Spermatogenesis
Phosphatase umol/ejaculate prostatic
fluid
➔ Decreased glycerophosphocholine and
L-carnitine also indicate an epididymis
disorder
➔ Decreased glutamyl transpeptidase level
also indicate lack of prostatic fluid

IMMUNOLOGIC TESTS
MEDICOLEGAL TESTS
➔ Antisperm antibodies: cause sperm
agglutination; detected in semen, 1. Microscopic exam
cervical mucosa or serum 2. Fluorescence under UV light
MIXED AGGLUTINATION REACTION 3. Acid phosphatase determination
(MAR) 4. Glycoprotein p30 (aka PSA) = more
➔ Detects the presence of IgG antibodies specific method to detect semen
➔ Semen sample AHG+ Latex particles 5. Florence test (not specific)
or treated RBCS coated with IgG Test for choline
Normal: <10% motile-sperm attached to Reagents: iodine crystals potassium iodide
the particles dark brown rhombic crystals
6. Barbiero's test (very specific)
IMMUNOBEAD TEST Test for spermine
➔ Detects the presence of IgG, IgM, and IgA Reagents: saturated picric acid +
antibodies Demonstrates what area of trichloroacetic acid (+) yellow leaf-like
sperm (head, neck, tail) the autoantibodies crystals
are affecting 7. ABO blood grouping
Normal: presence of beads on <50% of the 8. DNA analysis
sperm

POST-VASECTOMY
MICROBIAL TESTS TESTING
➔ Routine aerobic and anaerobic cultures and VASECTOMY
tests for C. trachomatis, M. hominis, and U. ➔ Surgical cutting of vas deferens so that the
urealyticum ejaculate will not contain any sperm cell
Round cells: WBCs or spermatids (immature ➔ Following a vasectomy sperm count ideally
sperm cells) use peroxidase to differentiate should be zero within 12 weeks after the
them.. procedure

POST VASECTOMY ANALYSIS

 ➔ The only concern is presence or absence of
penetration is observed
sperm
microscopically
➔ Done 2 months after vasectomy and
continues until 2 consecutive monthly CERVICAL Observing perm penetration
specimens show no sperm. MUCUS ability of partner's midcycle
➔ If wet prep is negative, centrifuge specimen PENETRATION mucus
for 10 minutes and examine the sediment
➔ The presence of even a single “motile" HYPO-OSMOTIC Sperms exposed to
spermatozoon is evidence of unsuccessful SWELLING low-sodium concentrations
vasectomy are evaluated for membrane
integrity and sperm
viability

ABNORMAL IN VITRO Evaluation of the acrosome

SEMENALYSIS ACROSOME to produce enzymes
REACTION essential for ovum
Abnormal Possible Test penetration
Results Abnormality

Decreased Viability Eosin-nigrosin
motility with stain
normal count

Decreased Lack of Fructose level TERMINOLOGIES

count seminal
Aspermia: Absence of semen during
vesicle
ejaculation.
Decreased Male MAR and Azoospermia: Lack of sperm in semen,
motility with antisperm immunobead indicating infertility.
clumping antibodies tests Sperm Hematospermia: Presence of blood in semen.
agglutination Leukospermia: Elevated levels of white blood
with male cells in semen.
serum Necrozoospermia: Presence of non-motile or
dead sperm in semen.
Normal Female Sperm Oligozoospermia: Low sperm count in semen,
analysis with antisperm agglutination potentially affecting fertility.
continued antibodies wit female
infertility serum
 

SPERM FUNCTION MISCELLANEOUS INFO

➔ Leydig cells secrete testosterone, Sertoli
Test Description cells secrete inhibin .
➔ Shortly after ejaculation, semen coagulates
HAMSTER EGG Sperms are incubated with
because of the action of a clotting enzyme,
PENETRATION species- nonspecific
formed in the prostate, on a fibrinogen-like
hardster eggs and
 precursor substance that is produced in the
seminal vesicle.
➔ When performing fertility testing. WHO
recommends that 2 or 3 samples be
collected, not <7 days or >3 weeks apart,
with 2 abnormal samples considered
significant.
➔ Motile sperm can be detected for upto 24
hours after intercourse, whereas nonmotile
sperm can persist for 3 days.
➔ As the sperm die off, only the heads remain
and may be present for 7 days after
intercourse.