What are the World Health Organization (WHO) standardized methods for semen analysis?

WHO Standardized Semen Analysis Methods

The WHO standardized semen analysis evaluates seven core parameters with specific reference limits: volume ≥1.4 mL, sperm concentration ≥16 million/mL, total sperm number ≥39 million per ejaculate, progressive motility ≥30%, total motility ≥42%, vitality ≥54% live, morphologically normal forms ≥4.0%, and pH >7.2. 1, 2

Core Semen Parameters and Reference Values

The WHO reference limits are derived from analysis of 3,589 males from 12 countries across 5 continents, specifically selecting fertile men whose partners achieved pregnancy within 12 months. 1 These represent the 5th percentile lower reference limits with 95% confidence intervals:

• Volume: 1.4 mL (1.3-1.5 mL) 1, 2
• Total sperm number: 39 million per ejaculate (35-40 million) 1, 2
• Sperm concentration: 16 million/mL (15-18 million/mL) 1, 2
• Progressive motility: 30% (29-31%) 1, 2
• Total motility (progressive + non-progressive): 42% (40-43%) 1, 2
• Vitality: 54% live spermatozoa (50-56%) 1, 2
• Morphologically normal forms: 4.0% (3.9-4.0%) 1, 2
• pH: >7.2 2

Collection and Handling Requirements

Proper specimen collection is critical, as improper technique invalidates all results. 3, 2

Pre-Collection Requirements

• Abstinence period: 2-3 days of sexual abstinence is mandatory, as inadequate abstinence significantly affects volume and concentration. 3, 2

Collection Methods

• Primary method: Masturbation 2
• Alternative method: Intercourse using specialized semen collection condoms (not regular condoms, which contain spermicides) 2

Transport and Timing

• Temperature maintenance: Keep specimen at room or body temperature during transport 2
• Analysis timing: Examine within one hour of collection, as delayed analysis significantly affects motility assessment 3, 2

Laboratory Procedures and Quality Control

A critical pitfall is that many laboratories do not adhere to WHO standardized methods, leading to high variability and unreliable results. 3, 2 The WHO manual emphasizes internal and external quality control to identify and correct both incidental and systematic errors. 4

Standard Evaluation Components

• Macroscopic evaluation: Volume, pH, liquefaction time, appearance 5, 6
• Microscopic evaluation: Sperm concentration, motility (progressive and non-progressive), morphology, vitality 5, 6
• Cellular elements: White blood cell count (Endtz test for leukocytes) 6

Specialized Testing When Indicated

• Fructose testing: Identifies seminal vesicle dysfunction or ejaculatory duct obstruction, though considered relatively unreliable 1, 2
• Post-ejaculatory urinalysis: Mandatory if ejaculate volume <1 mL (except in bilateral vasal agenesis or hypogonadism) to rule out retrograde ejaculation 3, 2
• Sperm antibody testing: For suspected immunological infertility 2

Frequency of Testing

At minimum, order two semen analyses at least one month apart for initial screening. 3 A single properly performed analysis may be sufficient for initial evaluation, but if abnormalities are detected, a second confirmatory analysis at least one month after the first is mandatory. 2 This accounts for biological variability in semen parameters. 3

Clinical Interpretation Patterns

Obstructive Patterns

Low volume (<1.4 mL) with acidic pH (<7.0) indicates distal genital tract obstruction, typically from congenital bilateral absence of vas deferens or ejaculatory duct obstruction. 1, 2 These males will have absent or minimal seminal vesicle contribution. 1

Azoospermia Confirmation

When azoospermia is suspected, the laboratory must centrifuge the ejaculate and examine the pellet under microscopy for rare sperm, as this identifies motile or non-motile sperm in approximately 18-23% of men initially diagnosed with azoospermia. 7

Critical Pitfalls to Avoid

• Single parameter focus: Evaluation of the entire profile is necessary, as focusing on one parameter is insufficient for fertility assessment 3, 2
• Assuming normal equals fertility: 25% of infertility cases remain unexplained despite normal conventional parameters 3
• Laboratory quality variability: Many clinical laboratories fail to adhere to WHO standardized methods, lacking comprehensive quality control programs 3
• Improper collection invalidates results: Temperature, timing, and abstinence period must be strictly followed 3, 2

Recent Updates in the 6th Edition (2021)

The 6th Edition of the WHO manual abandoned the notion of fixed reference thresholds, suggesting instead to replace them with "decision limits" that can be adjusted based on clinical context. 8 The manual now includes well-described tests for sperm DNA fragmentation and seminal oxidative stress, and has integrated a chapter on cryopreservation of spermatozoa. 4, 8