Standard Operating Procedure (SOP) for Seminal Fluid Analysis

Introduction

This SOP outlines the procedures for the collection, handling, and analysis of semen samples, focusing on assessing male fertility according to the guidelines provided by Monica Cheesbrough. The analysis evaluates semen volume, sperm count, motility, morphology, and additional parameters.

Equipment and Materials

• Sterile, wide-mouthed containers for semen collection
• Graduated pipettes for volume measurement
• Hemocytometer or Neubauer counting chamber
• Light microscope with phase contrast (for motility and morphology assessment)
• Centrifuge
• Glass slides and cover slips
• Stains (e.g., Eosin-nigrosin for vitality)
• Incubator (37°C)
• pH strips or meter
• Laboratory timer
• Reagents for sperm vitality and morphology staining

Semen Collection

• Patient Preparation:
  • Advise the patient to abstain from sexual activity for 2–7 days before the test.
  • Instruct the patient to avoid using lubricants or spermicidal agents during collection.

 

• Sample Collection:
  • The specimen should be collected via masturbation directly into a sterile, wide-mouthed container.
  • Ensure that the entire ejaculate is collected, as the initial portion contains the highest sperm concentration.
  • If collected outside the lab, the sample should be kept at body temperature (37°C) and delivered to the lab within 1 hour of collection.
  • Record the time of collection.

 

Macroscopic Examination

• Volume:
  • Measure the semen volume using a graduated pipette. Normal volume ranges from 1.5 to 5.0 mL.
• Liquefaction:
  • Allow the semen to liquefy at room temperature. Normal semen liquefies within 30 minutes. If liquefaction is incomplete after 60 minutes, record it as abnormal.
• Appearance:
  • Note the color and consistency of the semen. Normal semen is whitish-gray and opaque.
• Viscosity:
  • Assess the viscosity by allowing the semen to drop from a pipette. Normal semen forms discrete drops. Highly viscous semen is abnormal and should be recorded.
• pH:
  • Use pH paper or a meter to determine the pH. Normal seminal fluid pH ranges from 7.2 to 8.0. Low or high pH can indicate infections or abnormalities in seminal vesicles or the prostate.

 

Microscopic Examination

• Sperm Count:
  • Mix the semen sample thoroughly after liquefaction.
  • Using semen diluting fluid, make 1:20 dilution for the semen as in WBC COUNT.
  • With a Pasteur pipette, fill the Neubauer ruled chamber with well-mixed diluted semen.
  • Wait 3–5 minutes for the spermatozoa to settle and observe under a light microscope with 10x objective.
  • Using the 10x objective with the condenser iris closed sufficiently to give good contrast, count the number of spermatozoa in the 2 large squares.
  • Calculate the number of spermatozoa in 1 ml of fluid by multiplying the number counted by 100,000.

A normal sperm concentration is ≥15 million/mL.

 

• Sperm Motility:
  • Place a fresh drop of well-mixed liquefied semen on a slide and cover with a cover slip.
  • Focus with 10x objective and examine the semen with 40x objective.
  • Classify sperm motility into three categories:
    1. Actively motile/ progressive: Sperm moving forward in a straight line.
    2. Sluggishly motile/ non- progressive: Sperm moving but not in a straight direction.
    3. Non-motile: No movement.
  • Evaluate at least 100 sperm and record the percentage of each motility category.

Normal motility: Over 50% of spermatozoa are motile within 60 minutes of ejaculation.

 

• Sperm Morphology:
  • Examine the sperm cells at 40x magnification to evaluate the sperm’s shape and structure.
  • Record the percentage of sperm with normal morphology. Using strict criteria, normal morphology is ≥4%.

Image source

• Sperm Viability (if indicated):
  • Mix one fresh drop of semen with one drop of 0.5% eosin solution on a slide.
  • After 2 minutes examine the preparation microscopically. Use the 10x objective to focus the specimen and the 40x objective to count the percentage of viable and non-viable spermatozoa.
  • This stain distinguishes live sperm (which remain unstained) from dead sperm (which absorb the stain).

Normal viability: 75% or more of spermatozoa should be viable (unstained).

 

• White Blood Cells (WBC):
  • Check for WBCs, as a high WBC may indicate infection.

• Agglutination:
  • Look for sperm agglutination (clumping), which could suggest the presence of antisperm antibodies.

 

Reporting Results

• Macroscopic Findings:
  • Volume, liquefaction time, pH, color, and viscosity should be reported.

• Microscopic Findings:
  • Report sperm concentration, motility, morphology, and vitality.
  • Any abnormalities such as high WBC count, agglutination, or poor motility should be noted.

• Reference Values (WHO):
  • Volume: ≥1.5 mL
  • Sperm count: ≥15 million/mL
  • Motility: ≥40% motile (progressive and non-progressive)
  • Morphology: ≥4% normal forms (strict criteria)
  • Vitality: ≥58% live sperm

 

References

• Cheesbrough, M. District Laboratory Practice in Tropical Countries, Part 2, 2nd edition. Cambridge University Press, 2006.