Semen Analysis Procedure: How Labs Test Sperm Count, Motility & Morphology
A semen analysis is a fundamental test in evaluating male fertility. It assesses various parameters of sperm health, including count, motility, and morphology. Understanding the laboratory procedures behind this analysis can provide clarity for those undergoing fertility evaluations.
1. Sample Collection
The process begins with the collection of a semen sample. Typically, the sample is obtained through masturbation into a sterile container, either at the clinic or at home. If collected at home, it's crucial to deliver the sample to the laboratory within one hour, keeping it at body temperature to preserve sperm viability. Abstinence from ejaculation for 2–7 days prior to collection is usually recommended to ensure optimal sample quality.
Patient preparation:
2–5 days of abstinence (per WHO guidelines).
Collected via masturbation into a sterile, pre-weighed container.
Kept at body temperature (37°C) and delivered to the lab within 30–60 minutes.
Lab reception:
Container is weighed to calculate ejaculate volume (1g ≈ 1mL).
Sample placed in a 37°C incubator to liquefy (takes 15–30 minutes).
2. Macroscopic Examination
Once the sample reaches the lab, technicians perform a macroscopic examination through visual analysis. The lab is assessing:
Volume: Measured using a graduated cylinder; normal volume ranges from 1.5 to 6.8 mL.
Appearance: Evaluated for color and clarity; normal semen is whitish-gray and opalescent.
pH Level: Determined using pH strips; normal pH ranges from 7.2 to 8.0.
Liquefaction Time: Time taken for semen to liquefy; typically occurs within 15 to 60 minutes post-ejaculation.
Viscosity: Assessed by observing the thread length when the semen is allowed to drop from a pipette; high viscosity can impede sperm motility.
3. Microscopic Examination
After liquefaction, a microscopic analysis is conducted by using a phase-contrast microscope at 200-400x magnification. This can take time depending on the experience of the lab team. They are literally counting each sperm, so you can imagine how long it might take if those guys are swimming around all over the place! The lab will evaluate:
a. Sperm Concentration (Count)
A diluted semen sample is placed on a counting chamber, such as a Neubauer hemocytometer. Technicians count the number of sperm in specific grid areas under a microscope to calculate the concentration, expressed as millions of sperm per milliliter. A normal concentration is at least 15 million sperm/mL.
Preparation:
10μL of mixed sample placed on a pre-warmed glass slide.
Covered with a 22x22mm coverslip (depth ~20μm).
Manual counting:
Dilution: Mix 1:20 with sperm immobilizing solution (e.g., sodium bicarbonate/formalin).
Loading: Transfer to a hemocytometer (Makler or Neubauer chamber).
Counting: Under 400x magnification, count sperm in predefined grid squares.
Calculation:
Concentration = (Count × Dilution Factor) / Chamber depth × grid area.
b. Sperm Motility
Motility assessment determines the percentage of sperm that are moving and their movement quality. A drop of semen is examined under a microscope, and sperm are categorized
At least 40% of sperm should be motile, with 32% showing progressive motility.
Evaluation:
Viewed under phase-contrast microscopy at 200–400x magnification.
200 sperm are counted and categorized:
Progressive motility: Forward-moving sperm (≥32% normal).
Non-progressive motility: Swimming in circles or sluggish.
Immotile: No movement.
At least 40% of sperm should be motile, with 32% showing progressive motility.
c. Sperm Morphology
Morphology evaluates the size and shape of sperm. A stained semen smear is prepared, and technicians examine at least 200 sperm under high magnification. Sperm are classified based on the appearance of their head, midpiece, and tail. According to strict criteria, a normal sample has at least 4% of sperm with normal morphology.
Slide preparation:
Thin smear of semen on glass slide.
Air-dried and fixed with methanol.
Stained with Diff-Quik or Papanicolaou stain.
Analysis:
Kruger Strict Criteria (WHO 6th ed.):
Head: Smooth oval (3–5μm long, 2–3μm wide).
Midpiece: Slender, 1.5x head length.
Tail: Single, unbroken, 45μm long.
400 sperm evaluated under 1000x oil-immersion microscopy.
Normal ≥4%; borderline 1–3%; abnormal <1%.
**This process is much more time-consuming and requires more specialized steps than checking sperm count and motility. The reason is that sperm are so fast and motile! We need to stain the sperm and then “freeze” them in place so that we can accurately assess the body. Sadly, this process does make the sperm unviable for use, but fortunately, there’s plenty more we can use so it’s for the greater good!
4. Additional Assessments
Depending on initial findings, further tests may include:
Vitality Test: Determines the percentage of live sperm using dye exclusion methods.
White Blood Cell Count: Identifies potential infections or inflammation.
Fructose Level: Assesses seminal vesicle function.
Vitality testing (Eosin-Nigrosin stain):
Dead sperm absorb pink stain; live sperm remain white.
DNA fragmentation: TUNEL assay or SCSA (requires specialized equipment).
Leukocyte detection: Immunochemical staining (peroxidase test).
These tests are rarely done at an IVF center, so it’s something that can be assessed with a specialist, like a urologist.
5. Quality Control and Reporting
Laboratories adhere to strict quality control measures, following guidelines from organizations like the World Health Organization (WHO). Results are compared against established reference values to determine normalcy or identify potential issues.
Internal controls:
Duplicate analysis (≤10% variation allowed).
Special QC Beads (quality control of counting sperm)
External proficiency testing:
Labs participate in CAP/ESHRE certification programs that regularly test labs through proficiency tests.
Blind samples are tested quarterly to maintain accuracy.
6. Lab Equipment Used
Phase-contrast microscope - check motility & count
Hemocytometer - Manual concentration
Staining racks - Morphology preparation
7. Automated Semen Analyzers
There is high-tech equipment that can be purchased to automate the semen analysis report. There are many computer-assisted sperm analysis systems out there, like the LensHooke X12 PRO Semen Analysis System. Would you rather have a human or a computer analyze your sperm?
Check out this new AI method that can predict male infertility based on hormone levels! Is semen analysis going to be a thing of the past?
8. Key Technical Challenges
Semen analysis is helpful, but not perfect. Below are some common issues that many labs face when analyzing semen.
Subjectivity in motility grading: Addressed via CASA and dual-tech review.
Artifacts in morphology: Distinguishing true defects from staining artifacts.
Time sensitivity: Motility degrades quickly – analysis begins within 60 minutes.
Conclusion
Understanding the meticulous procedures involved in semen analysis can demystify the process and highlight its importance in fertility assessments. Accurate testing and interpretation are crucial steps toward identifying and addressing male fertility concerns. Semen analysis is a crucial part of the IVF journey, and men should be more proactive in getting tested. Unlike women, men are fortunate to be able to reverse problematic sperm since it is constantly being produced! Don’t take this for granted, men!
“Funny how the worst samples are the easiest to analyze!”
