Viscosity Normal = pour in droplets

SEMENALYSIS Abnormal = Threads >2cm long (highly viscous)

60-70% SEMINAL FLUID SEMINAL VESICLES ↑Viscosity = ↓sperm motility; impede testing for sperm
• Provide nutrients for sperm & fluid; transport medium for concentration, antisperm antibody detection, and measurement of
sperm biochemical markers
• Rich in fructose = sperm motility Reporting
• Flavin – gives semen its gray appearance • 0 = WATERY
20-30% PROSTATE FLUID Milky, acidic fluid that contains ACP, zinc, citric acid & other • 4 = GEL-LIKE
enzymes • May also be reported as low, normal, or high
For coagulation and liquefaction
5% BULBOURETHRAL GLAND Secretes thick, alkaline mucus that neutralizes acidity from the pH Normal = 7.2-8.0
prostatic secretions & vagina • ↑pH = Infection
5% SPERMATOZOA SEMINIFEROUS TUBULES (TESTES) • ↓pH= Prostatic fluid, ejaculatory duct obstruction or poorly
• Spermatogenesis developed seminal vesicles
• Sertoli cells – serve as nurse cells for developing sperms Specific Gravity Normal = 1.010 – 1.030
EPIDIDYMIS Gives presumptive result of sperm concentration
Sperm maturation (sperm becomes motile); entire process takes SPERM CONCENTRATION
approximately 90 days Normal value 20-250 million/mL
Leydig cells Secrete testosterone • 10-20 million/mL – borderline
Sertoli cells Secrete inhibin which inhibits FSH synthesis Dilution 1:20
Specimen collection Sexual abstinence of 2-3 days but not >7 days (Prolonged Diluents • Formalin-Sodium bicarbonate (NaHC03) – traditional
abstinence: ↑ VOLUME, ↓MOTILITY) diluting fluid
Specimen collection for fertility testing Two or three samples be collected not <7 days or >3 weeks apart • Saline
Methods of collection 1. Masturbation (most preferred) • Distilled water
2. Coitus interruptus
• Tap water (cold/chilled)
3. Condom method – use non-lubricant containing rubber or
silastic condom Shortcut method for Sperm Concentration Computation 2 WBC Squares = # sperms counted x 100,000
Specimen should be delivered to the laboratory within ____ 1 hour of collection (at room temperature) (1:20 DF) 5 RBC Squares = # sperms counted x 1,000,000
Specimen awaiting analysis should be kept at _____ 37℃ Long method (Standard Neubauer Formula) Sperm concentration (/mL) =
# 𝒔𝒑𝒆𝒓𝒎𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏
𝒙𝟏𝟎𝟎𝟎
𝒂𝒓𝒆𝒂 𝒙 𝒅𝒆𝒑𝒕𝒉 𝒇𝒂𝒄𝒕𝒐𝒓 (𝟎.𝟏)
Fresh semen is clotted and should liquefy within _____ 30-60 minutes •
Makler Counting Chamber Counting INDILUTED specimens
Component that causes clotting Semogelin • Sperms are immobilized by heating part of the specimen
No liquefaction after 2 hours Add equal volume of physiologic Dulbecco’s phosphate-buffered prior to charging the chamber
saline or proteolytic enzymes such as amylase, bromelain, or • Sperm motility using the unheated portion of the specimen
alpha-chymotrypsin can also be evaluated in the chamber
If part of the first portion is missing Decreased sperm count
Neubauer Counting Chamber • Sperms are usually counted in the four corner and center
Increased pH
squares of the large center square, similar to a manual
Specimen will NOT LIQUEFY
RBC count
If part of the last portion is missing Decreased semen volume
• Both sides of the hemocytometer are loaded and allowed
Increased sperm count
to settle for 3-5 minutes; then they are counted and counts
Decreased pH
should agree within 10%
Specimen will NOT CLOT
• Counts are performed using either Phase or Brightfield
MACROSCOPIC EXAMINATION
microscopy
Appearance Normal = Gray-white, translucent (with musty or bleach odor)
• Only fully develop sperm should be counted; immature
Increased white turbidity = Infection (↑ WBCs)
sperm and WBCs, often referred to as “round cells”, must
Red to brown appearance = ↑RBCs, blood
not be counted
Yellow coloration = ↑Abstinence, Urine contamination, Medications
• Stain included in the diluting fluid aids in differentiating
Volume Normal = 2-5 mL
between immature sperm cells (spermatids) and
Increased in = Abstinence
leukocytes, and they can be counted in the same manner
Decreased in = Infertility, incomplete collection, may indicate
as mature sperm
improper functioning of one of the semen-producing organs,
primarily the seminal vesicles SPERM COUNT
Normal Value ≥40 million/mL
Formula Sperm count = Sperm concentration x Specimen volume
SPERM MOTILITY
Normal Value >50% motile (within 1 hour)
>2.0 or a/b
MUKARAM, SHAIRA R. | BSMT 2023
 How to Evaluate Evaluate approximately 20 HPF or examine 200 sperm per slide Detected in ____ Semen, cervical mucosa, or serum
and count the percentages of the different motile categories using a Mixed Agglutination Reaction • Detects the presence of IgG antibodies
manual cell counter • Semen sample + AHG + latex particles or treated RBC
Sperm Motility Grading coated with IgG
Grade WHO Criteria Sperm Motility Action • Normal = <10% motile sperm attached to the particles
4.0 a Rapid, straight-line motility Immunobead Test • Detects the presence of IgG, IgM, and IgA antibodies
3.0 b Slower speed, some lateral movement • Sperm are mixed with polyacrylamide beads known to be
2.0 b Slow forward progression, noticeable coated with either anti-IgG, anti-IgM, or anti-IgA
lateral movement • Demonstrates what area of sperm (head, neck, tail) the
1.0 c No forward progression autoantibodies is affecting
0 d No movement • Normal = presence of beads on <50% of the sperm
WHO: Interpretation states that within 1 hour, 50% or more of the sperm should be motile in categories a, b, and c, or 25% or more Kibrick Method • Involves incubating fresh, liquefied semen with serum from
should show progressive motility (a and b) the male or serum from his female partner. Agglutination is
observed macroscopically
Alternative Sperm Motility Grading Criteria •
Isojima Method Tests for sperm-immobilizing antibody
Progressive motility (PM) Sperm moving linearly or in a large circle • Comparison is made between sperm motility of fresh,
Nonprogressive motility (NP) Sperm moving with an absence of progression liquefied semen and that of semen incubated with either
Immotility (IM) No movement rabbit or guinea pig complement.
• A sperm immobilization value is calculated by dividing the
SPERM MORPHOLOGY percent of motile sperm in the fresh specimen by the percent
Normal Value for Routine Criteria >30% normal forms of motile sperm in the incubated sample
Kruger’s Strict Criteria >14% normal forms • A value of 2 indicates the presence of antibodies
• Measure the head, neck, & tail using a micrometer
• Measuring acrosome size CHEMICAL TESTING
• Evaluating for presence of vacuoles Analyte Normal Value Increased Value Indicate
• Not routinely performed but recommended but WHO Fructose ≥13 umol/ejaculate Decreased seminal fluid volume
Stains used Papanicolaou’s stain – stain of choice Neutral a-glucosidase ≥20 mU/ejaculate Epididymis disorder
Wright’s stain Zinc ≥2.4 umol/ejaculate Decreased prostatic fluid
Giemsa stain Citric acid ≥52 umol/ejaculate
Shorr stain Acid phosphatase ≥200 Units/ejaculate
Head Oval/flattened oval, 5 um long, 1 um wide
Acrosomal cap Encompasses ½ of the head and cover 2/3 of the nucleus MICROBIAL TESTING
Contains enzymes critical to ovum penetration Round cells WBCs and spermatids (immature sperm cells)
Midpiece ≈7 um long; thickest part of the tail Normal Value <1 million/mL
Surrounded by a mitochondrial sheath that produces the energy • >1 million WBCs/mL = infection
required by the tail for motility • >1 million spermatids/mL = disruption of spermatogenesis
Tail ≈45 um long 1. Spermatogonium
Sperm maturation stages
Formula Normal sperm cells =
𝑛𝑜𝑟𝑚𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
x 100 2. Primary spermatocyte
𝑡𝑜𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
3. Secondary spermatocyte
Most common morphology abnormality in Varicocele Tapered heads 4. Spermatid
SPERM VIABILITY 5. Spermatozoon
Modified Bloom’s Test Reagents = Eosin and Nigrosin C. albicans Impair sperm motility by agglutinate with sperm head
Living sperms = unstained; bluish white Formula
Dead sperms = red
𝑁𝑥𝑆
Normal value = 50% living sperms C= 100
↑ vital but immobile cells Defective flagellum
↑ immotile and nonviable cells Epididymal pathology

SEMINAL FLUID FRUCTOSE

2 hours
MICRO-LEGAL TESTS
Tested within ____ Best results obtained by adding xylene and examining under
Or frozen to prevent fructolysis Microscopic exam
phase microscopy
Screening test Resorcinol/Seliwanoff’s tests = (+) orange-red color
• Motile sperms = can be detected for up to 24 hrs after
Normal quantitative level of fructose ≥13 umol/ejaculate intercourse
ANTISPERM ANTIBODIES • Nonmotile sperm = can persist for 3 days

MUKARAM, SHAIRA R. | BSMT 2023

 • As sperm die off = heads remain and may be present
for 7 days after intercourse
Florence Test (not specific) Use Reagents Positive Result
Test for CHOLINE Iodine crystals + Dark brow rhombic
Potassium iodide crystals
Barberio’s Test (very specific) Use Reagents Positive Result
For mnemonics: Barbers trim the leaves Test for SPERMINE Saturated picric acid Yellow leaf-like
+ Trichloroacetic acid crystals
Glycoprotein 30 (PSA) MORE SPECIFIC METHOD TO DETECT SEMEN; present even in
the absence of sperm
Other tests Fluorescence under UV light
Acid phosphatase detection
ABO blood grouping
DNA analysis
POSTVASECTOMY SEMEN ANALYSIS
Vasectomy • Cutting of vas deferens so that the ejaculate will not
contain any sperm cell
• The only concern is the presence or absence of sperm
• Done 2 months after vasectomy and continued until 2
consecutive monthly specimens show no sperm
Aspermia No ejaculate
Azoospermia Absence or sperm cells
Necrospermia Immotile or dead sperm cells
Oligospermia Decreased sperm concentration
NORMAL VALUES FOR SEMEN ANALYSIS
Volume 2-5 mL
Viscosity Pour in droplets
pH 7.2-8.0
Sperm Concentration ≥20 million/mL
Sperm count ≥40 million/ejaculate
Viability ≥50% within 1 hour
Motility >2.0 or a/b
Morphology >30% normal forms (routine criteria)
>14% normal forms (strict criteria)
Round cells <1 million/mL
ASSESSMENT OF SPERM CELLS
Sperm Morphology Malaria smear in slide and stain it with Papanicolaou’s, Wright’s or
Giemsa
Sperm Viability Stain with Eosin-Nigrosin
Sperm Count Dilute with chilled tap water or formalin-bicarbonate solution
Charge in a Neubauer counting chamber
Sperm Motility Place a drop of semen in a slide and cover it with cover slip
ADDITIONAL TESTING FOR ABNORMAL SEMEN ANALYSIS
Abnormal Result Possible Abnormality Test
Decreased motility with normal count Viability Eosin-Nigrosin stain
Decreased count Lack of seminal vesicle support medium Fructose level
Decreased motility with clumping Male anti-sperm antibodies Mixed agglutination reaction &
immunobead tests
Normal analysis with continues infertility Female anti-sperm antibodies Sperm agglutination with female serum

MUKARAM, SHAIRA R. | BSMT 2023