Semen Reviewer

Here's your comprehensive exam reviewer for Seminal Fluid & Semen Analysis:

SEMINAL FLUID & SEMEN ANALYSIS — Exam Reviewer

MLS 419 | AUBF | Ray Gomez, RMT, MPH

I. OVERVIEW & APPLICATIONS OF SEMEN ANALYSIS

Semen — the secretion men produce to fertilize the female ovum.

Why is semen analyzed?

• Andrology — evaluates male fertility; guides need for in-vitro fertilization (IVF)

• Assisted Reproductive Technology (ART)

• Fertility studies

• Post-vasectomy — even ONE motile sperm = vasectomy failure

• Forensic analysis — evidence in rape/sexual assault cases

II. PHYSIOLOGY & COMPOSITION

Semen = combination of 4 secretions from 4 structures. ALL FOUR must be present for normal function.

III. STRUCTURES CONTRIBUTING TO SEMEN

A. Testes

• Paired glands in the scrotum → produce sperm via spermatogenesis

• Contain seminiferous tubules

• External location → lower temperature → optimal for sperm development

Spermatogenesis pathway:

1. Mitosis of germ cells (epithelial cells)

2. Germ cells undergo meiosis — Sertoli (nurse) cells provide nutrients

3. Development of immature, non-motile spermatids

4. Spermatids mature in the epididymis for 90 days or until ejaculated

5. Ejaculated via vas deferens → ejaculatory duct

Spermatids:

• Immature, no flagellum (tail) → cannot propel toward ovum

• Resemble WBCs microscopically → called "round cells"

• ↑ Round cells = infection (if WBC) OR disrupted spermatogenesis (if spermatid)

B. Ejaculatory Ducts

• Receives mature sperm + seminal vesicle secretions

C. Seminal Vesicle

• Produces 60–70% of semen fluid

D. Prostate Gland

• Located below the bladder

• Functions: secretes prostatic fluid + helps propel semen during ejaculation (contracts)

• Prostatic fluid = 20–30% of semen

• Milky and acidic; contains acid phosphatase, citric acid, zinc, proteolytic enzymes

• Responsible for liquefaction of semen — without it, sperm cannot swim

E. Bulbourethral Gland

• Located below the prostate

• Secretes alkaline mucus (5% of semen)

• Neutralizes acidity of prostatic fluid and vaginal environment

• Sperm cannot thrive in acidic conditions → alkaline buffer is essential

IV. SEMENALYSIS — 7 ROUTINE PARAMETERS

a. Appearance | b. Volume | c. Viscosity | d. pH | e. Sperm concentration & count | f. Motility | g. Morphology

A. SPECIMEN COLLECTION

Abstinence requirement: 2–7 days

Fertility testing: WHO recommends 2–3 collections, 1–3 weeks apart; 2 out of 3 abnormal = significant

Preferred method: Masturbation at hospital/lab premises

If collected at home: Must be delivered within 1 hour at room temperature

Specimen awaiting analysis: Store at 37°C

Acceptable containers: Non-spermicidal, non-lubricant rubber or polyurethane condoms

NOT recommended:

• Ordinary condoms → contain spermicides

• Coitus interruptus → first portion may be lost; vaginal pH affects motility

Consequences of improper collection:

After collection, note:

1. Patient info

2. Period of abstinence

3. Time of collection (basis for liquefaction time)

4. Any abnormalities in specimen quality

B. APPEARANCE

Normal: Gray-white, translucent, musty odor (odor NOT reported)

C. LIQUEFACTION

• Normal: Liquefies within 30–60 minutes

• Failure to liquefy = prostatic enzyme deficiency or incomplete collection

• If not liquefied within 2 hours, induce with:

  • Dulbecco's phosphate-buffered saline (DPBS)

  • Proteolytic enzymes (alpha-chymotrypsin or bromelain)

  Note: Liquefaction agents can affect biochemical tests, motility, and morphology — must be documented.

D. VOLUME

• Normal: 2–5 mL

• Measured by graduated cylinder

• ↑ Volume = prolonged abstinence

• ↓ Volume = impaired seminal vesicle function, infertility, incomplete collection

E. VISCOSITY

• Normal: Easily drawn into pipette; forms droplets, not clumped or stringy

• Abnormal: Incompletely liquefied; threads longer than 2 cm (WHO)

• Scale: 0 (watery) to 4 (gel-like) OR reported as low/normal/high

• ↑ Viscosity affects: motility, sperm concentration, anti-sperm antibody detection, biochemical markers

F. pH

• Normal: 7.2–8.0 (alkaline)

Testing methods: Reagent pad (routine), pH paper, pH meter (specialized labs)

G. SPERM CONCENTRATION & SPERM COUNT

Sperm count formula: Concentration × Volume

Counting method — Neubauer Counting Chamber:

1. Dilute semen 1:20 with diluting fluid (sodium bicarbonate + formalin) — immobilizes sperm for easy counting

2. Load on both sides of chamber

3. Stand 3–5 minutes to settle

4. Use RBC counting area (5 boxes only)

5. Count only mature sperm (with tail); round cells are NOT counted

6. Both sides must agree within 10% margin; if not → repeat

Shortcut: With 1:20 dilution, multiply average count × 1,000,000

Diluting fluid alternatives: Ice-cold water (most accessible)

Round cells:

• Use peroxidase stain to differentiate peroxidase-positive granulocytes from spermatids and lymphocytes

H. SPERM MOTILITY

• Assessed as progressive, forward motility

• 10 µL on glass slide → evaluate ≥20 HPF OR examine 200 spermatozoa with cell counter

Grading System 1 — Speed & Direction:

Normal: >50% of sperm at grade 2.0 or higher

WHO 2010 Alternative Grading:

High % of immotile sperm → further testing for vitality or sperm agglutinins

I. SPERM MORPHOLOGY

Evaluated structures: Head, midpiece, tail

Method:

• 10 µL smear → stain with Wright's, Giemsa, Shorr, or Papanicolaou

• Evaluate at least 200 spermatozoa

Reporting:

V. ADDITIONAL TESTING (done when routine parameters are abnormal)

A. Sperm Vitality

• Indicated when: normal concentration + decreased motility

• Stain: Eosin-negrosin

  • Living cells → bluish white (can't take up stain)

  • Dead cells → red against purple background

• Count dead cells per 100 spermatozoa

• Normal: ≥50% living sperm with grade 2.0 motility

• Vital but immotile cells → defective flagellum

• Large numbers of dead cells → epididymal pathology

B. Seminal Fructose

• Low/absent fructose → low sperm concentration (immotile sperm)

• Screening test: Resorcinol test → (+) = orange-red color

• Quantitative test: Spectrophotometry → Normal: >13 micromoles/ejaculate

• Must be performed within 2 hours or specimen frozen (prevents fructolysis)

C. Anti-Sperm Antibodies

In males:

• Disruption of blood-testes barrier → sperm antigens trigger antibody production

• Causes: surgery, vasectomy reversal, trauma, infection

• Sign: clumps of sperm during semenalysis

In females:

• Damaged sperm → antibody production

• Infertility despite normal semenalysis

Detection methods:

Immunobead clinical significance:

• Head-directed antibodies → block cervical mucosa or ovum penetration → cannot do anything

• Tail-directed antibodies → impede movement through cervical mucosa → IVF still possible

• Reported as: e.g., "IgG head antibodies," "IgM tail antibodies"

D. Microbial & Chemistry Analysis

Microbial cultures for:

• Chlamydia trachomatis

• Mycoplasma hominis

• Ureaplasma urealyticum

Chemistry (by spectrophotometry):

Forensic markers:

• Acid phosphatase → enzyme in semen; confirms presence of semen

• PSA (Prostate-Specific Antigen) → confirms semen in absence of sperm

Forensic timelines:

VI. POST-VASECTOMY ANALYSIS

• Evaluate presence/absence of sperm to confirm vasectomy success

• Test at monthly intervals starting at 2 months post-vasectomy

• Continue until two consecutive specimens show no spermatozoa

• Method: Wet preparation

  • If negative → centrifuge for 10 minutes → examine sediment

• Single motile sperm on wet prep = unsuccessful vasectomy

• Nonmotile sperm = considered successful (cannot fertilize)

VII. SPERM FUNCTION TESTS

VIII. AUTOMATION — CASA SYSTEMS

CASA = Computer Assisted Semen Analysis

• Determines: velocity, trajectory, concentration, morphology

• MT manually records only: volume and pH — system handles the rest

Three CASA instruments:

1. Sperm Class Analyzer (SCA) — microscope with digital camera, motorized heating stage, image analysis software; filters debris; measures concentration, motility, morphology, velocity, sperm motility index

2. CEROS CASA systems

3. Automated Sperm Quality Analyzers (ASQA)

QUICK REFERENCE — NORMAL VALUES

HIGH-YIELD MNEMONICS & TIPS

• "Every last drop counts" — incomplete collection gives false results

• Spermatids = round cells → look like WBCs → use peroxidase stain to differentiate

• Fructose absent = immotile sperm (no energy source)

• Flavin = gray color + UV fluorescence → key in forensics

• Prostatic fluid = liquefaction → no prostate = sperm can't swim

• Bulbourethral = buffer → without it, sperm die in acidic vaginal environment

• Vasectomy: 1 motile sperm = failed; nonmotile = acceptable (can't fertilize)

• IVF clue: If immunobead antibody is on the tail → IVF still possible; if on head → cannot penetrate ovum, IVF not useful