Global Standards for Human Semen Analysis: A Comparative Guide (WHO 6th, ISO 23162, ESHRE, & ASRM)
In 2021, the landscape of human andrology was redefined by the dual release of the WHO 6th Edition Manual and the ISO 23162:2021 standard. These documents have moved the field toward rigorous standardization, emphasizing that accuracy is only possible through strict quality control and the reduction of human subjectivity.
1. Pre-analytical Phase: The Baseline of Quality
Standardization begins before the sample reaches the microscope. Both WHO 6th and ISO 23162 emphasize that pre-analytical variables are the most common cause of "false" sub-fertility results.
- Abstinence Period: Standardized at 2 to 7 days. ASRM notes that while longer abstinence increases volume and count, it significantly reduces motility and increases DNA fragmentation (DFI).
- Collection: Must be performed into a container pre-validated for the Human Sperm Survival Assay (HSSA).
- Macroscopic Assessment: Volume must be measured with high precision. ISO 23162 recommends measuring by weight (assuming a density of 1.0 g/mL) to avoid the "wall-sticking" loss associated with pipettes or graduated cylinders.
- pH: The lower limit remains 7.2. Values below this, combined with low volume, may indicate obstructive issues (e.g., CBAVD).
2. Sperm Concentration and Total Count: The Quantitative Metric
The quantitative assessment of sperm production is a primary predictor of fertility potential.
A. Concentration vs. Total Sperm Number
- Sperm Concentration: The number of sperm per milliliter. WHO 6th lower limit: 16 × 10⁶/mL.
- Total Sperm Number: The total count per entire ejaculate (WHO 6th lower limit: 39 × 10⁶).
- Clinical Value: ASRM and ESHRE emphasize that Total Sperm Number is a more robust indicator of testicular health than concentration alone, as it accounts for the entire reproductive output.
B. The "Gold Standard" Equipment (ISO 23162 vs. Others)
- Improved Neubauer Hemocytometer: Both WHO 6th and ISO 23162 mandate this as the only acceptable manual counting chamber.
- Rejection of Fixed-Depth Chambers: A major point of divergence. ISO 23162 strictly prohibits the use of fixed-depth chambers (e.g., Makler or Leja slides) for definitive concentration assessment because their high Coefficient of Variation (CV) does not meet international precision standards.
- Replicate Counting: ISO 23162 requires two separate replicates (counting at least 200 sperm each). If the results of the two counts do not align within a specific statistical range (Poisson distribution), the test must be repeated.
3. Sperm Motility: Measuring Progressive Competence
Motility is the measure of "vital energy." WHO 6th simplified classification to reduce the high inter-observer variability found in previous editions.
A. The Three-Category Classification
The previous 4-grade system (A, B, C, D) has been replaced by a more objective 3-category system:
- Progressive Motility (PR): Sperm moving actively, either linearly or in a large circle, regardless of speed.
- Non-progressive Motility (NP): Movement with no progression (e.g., swimming in small circles, or only head/tail vibrating).
- Immotility (IM): No movement at all.
- Reference Value: WHO 6th requires ≥30% Progressive (PR) or ≥42% Total Motility (PR + NP).
B. The Thermal Requirement (The "Life-Like" Environment)
- ISO 23162 Mandate: Sperm motility is highly temperature-dependent. ISO 23162 requires analysis to be performed at 37°C.
- Clinical Rationale: Analyzing at room temperature (20-22°C) causes sperm metabolism to slow down, resulting in "falsely" low progressive motility scores. Integrated heating stages (like those found in the SQA-6100VET) are essential for compliance with this standard.
4. Sperm Morphology: The "Strict Criteria"
Morphology provides the deepest insight into the biological "quality control" of the testes.
A. Kruger Strict Criteria (The 4% Rule)
All standards converge on the Kruger (Strict) Criteria. A sperm is only "Normal" if it is essentially perfect. Any "borderline" sperm is categorized as abnormal.
- Normal Threshold: ≥4%.
- Head/Tail Anatomy: Specific dimensions for head length (3.7–4.7 μm) and acrosome size (40–70% of the head area) are required.
B. The Teratozoospermia Index (TZI)
WHO 6th recommends calculating the TZI, which is the average number of defects per abnormal sperm. A high TZI is a marker of significant spermatogenesis dysfunction, even if the total normal percentage is above 4%.
C. Clinical Application (IVF vs. ICSI)
ASRM and ESHRE clarify that morphology's greatest clinical utility is in selecting the mode of fertilization. If morphology is <4%, laboratories often shift from standard IVF to ICSI (Intracytoplasmic Sperm Injection) to ensure the best-looking sperm are manually selected for fertilization.
5. Summary Table: Comparing WHO 5th and WHO 6th Standards
This table outlines the key "Lower Reference Limits" (5th percentile) for human semen.
| Parameter | WHO 5th Ed (2010) | WHO 6th Ed (2021) |
|---|---|---|
| Semen Volume (mL) | 1.5 | 1.4 |
| Total Sperm Number (10⁶/ejaculate) | 39 | 39 |
| Sperm Concentration (10⁶/mL) | 15 | 16 |
| Total Motility (PR + NP, %) | 40 | 42 |
| Progressive Motility (PR, %) | 32 | 30 |
| Vitality (Live Sperm, %) | 58 | 54 |
| Normal Morphology (%) | 4 | 4 |
6. Laboratory Quality Assurance (ISO 23162 Central Focus)
Compliance with ISO 23162 requires more than just following the WHO manual; it requires documented proof of precision.
- Intra-observer CV: Technicians must prove they can count the same sample twice and get the same result within a 10% margin of error.
- External Quality Assessment (EQA): Labs must participate in global proficiency testing (like ESHRE or CAP programs) to compare their results against other standardized labs.
- Standardized Reporting: Reports should clearly state which standards were followed and include the reference ranges for each parameter to aid clinician interpretation.
Conclusion: The Path to Precision
The modernization of human semen analysis is defined by a shift away from "technician's intuition" toward objective, automated, and temperature-controlled methodologies. By adhering to the combined requirements of WHO 6th and ISO 23162, laboratories ensure the highest level of diagnostic accuracy, providing patients with a reliable roadmap for their fertility journey.
References & Technical Resources
- World Health Organization (2021). WHO laboratory manual for the examination and processing of human semen, 6th ed.
- ISO 23162:2021. Basic semen examination — Specification and test methods.
- ESHRE Special Interest Group in Andrology. Semen analysis best practice guidelines.
- Practice Committee of the American Society for Reproductive Medicine (ASRM) (2021). Diagnostic evaluation of the infertile male: a committee opinion.