EAU Guidelines on Semen Analysis Collection and Interpretation
Core Collection Protocol
The 2025 EAU guidelines mandate strict adherence to WHO reference values for semen collection and analysis, requiring 2-7 days of sexual abstinence before collection, with specimens examined within one hour when collected at the laboratory facility 1.
Specimen Collection Requirements
- Abstinence period: 2-3 days of sexual abstinence before collection 1
- Collection method: Masturbation or intercourse using specialized semen collection condoms 1
- Home collection: If collected at home, maintain specimen at room or body temperature during transport and examine within one hour of collection 1
- Laboratory collection: Preferred method to ensure optimal timing for motility assessment 1
Number of Analyses Required
- Single normal analysis: If semen analysis meets WHO criteria on first test, one analysis is sufficient 1
- Abnormal results: Require at least two abnormal tests before proceeding with further andrological investigation 1
- Timing between tests: Perform repeat analysis at least one month apart to account for the 74-day spermatogenic cycle 1, 2
WHO Reference Values for Interpretation
The EAU guidelines reference WHO 2010 standards for normal semen parameters 1:
- Volume: 1.5-5.0 mL (lower reference limit ≥1.5 mL) 1
- pH: >7.2 1
- Sperm concentration: >20 million/mL (WHO 2010: ≥15 million/mL) 1
- Total sperm number: >40 million per ejaculate 1
- Motility: >50% progressive motility 1
- Morphology: ≥4% normal forms using strict criteria 1, 2
Critical Caveat on Interpretation
Semen analysis alone cannot distinguish fertile from infertile men and must be interpreted within the complete clinical context 1, 2. The guidelines emphasize that normal semen parameters do not guarantee fertility, and abnormal parameters do not definitively exclude fertility potential 1.
Laboratory Processing Standards
Motility Assessment
- Fresh uncentrifuged specimen: Examine within two hours of ejaculation for motility evaluation 1
- No centrifugation for routine analysis: Centrifugation may interfere with sperm motility and lead to identification of clinically insignificant sperm numbers 1
- Well-mixed specimen: Ensure thorough mixing before microscopic examination 1
Specialized Testing Considerations
The EAU guidelines do not recommend routine measurement of reactive oxygen species (ROS) or oxidative stress markers, as standardized testing methods are not available and require validation in well-designed RCTs 1.
Clinical Algorithm for Abnormal Results
When to Proceed with Further Investigation
- Severe oligozoospermia (<10 million/mL): Mandatory endocrine evaluation including serum testosterone and FSH 1, 2
- Azoospermia: Requires hormonal evaluation, genetic testing, and potentially scrotal/transrectal ultrasonography 1
- Low volume (<1 mL): Perform post-ejaculatory urinalysis to exclude retrograde ejaculation, except in bilateral vasal agenesis or hypogonadism 1
Genetic Testing Indications
- Karyotype analysis: Indicated for sperm concentration <5 million/mL 2
- Y-chromosome microdeletion: Consider with severe oligozoospermia 2
Emerging Evidence on Abstinence Period
While the EAU guidelines maintain the WHO recommendation of 2-7 days abstinence 1, recent research suggests shorter abstinence periods may improve sperm quality parameters. Studies show that 1-day abstinence produces better motility, lower oxidative stress, and improved DNA integrity compared to 4-day abstinence 3, 4, 5. However, the official EAU guidelines have not yet incorporated these findings into formal recommendations, and the 2-7 day standard remains the guideline-endorsed protocol 1.
Accessory Gland Function Assessment
The EAU guidelines include optional biochemical markers 1:
- Semen zinc: ≥2.4 μmol/ejaculate (prostatic function)
- Semen fructose: ≥13 μmol/ejaculate (seminal vesicle function)
- Neutral α-glucosidase: ≥20 mU/ejaculate (epididymal function)
These markers help identify specific glandular dysfunction when clinical suspicion exists 1.
Common Pitfalls to Avoid
- Single abnormal test: Never base clinical decisions on one abnormal semen analysis; biological variability requires confirmation 1, 2
- Ignoring female partner: Always investigate both partners simultaneously, as 50% of infertility cases involve male factors, often combined with female factors 1
- Over-reliance on morphology: Morphology alone has limited predictive value; interpret within the complete semen profile 2
- Delayed examination: Motility assessment loses accuracy if specimen sits too long before analysis 1