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I now have comprehensive, authoritative content from three major textbooks. Here is the full note on semen analysis:
Semen Analysis
Introduction
Semen analysis is the most important and cost-effective first step in the evaluation of male infertility and reproductive dysfunction. Because it evaluates sperm production and reproductive tract patency, a normal result often makes further investigation unnecessary. It is also used to select sperm donors for therapeutic insemination and to monitor outcomes after procedures such as vasectomy and varicocelectomy.
However, semen analysis is not a direct measure of functional fertility. An abnormal result simply indicates a likelihood of reduced fertility - of all semen parameters, count and motility correlate best with actual fertility outcomes.
- Harrison's Principles of Internal Medicine 22E, p. 2702
- Smith and Tanagho's General Urology, 19th Edition, p. 724
Sample Collection
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The patient should abstain from sexual activity for 2-3 days (ideally 48-72 hours) before collection. Shorter abstinence may yield falsely low counts; longer periods (>5 days) increase concentration but reduce motility.
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The sample is collected by masturbation into a sterile plastic container, kept at body temperature, and analyzed within 1 hour of collection.
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A minimum of two samples collected 2-3 weeks apart should be used for a reliable baseline, as semen quality can vary considerably day to day.
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Incomplete specimens should not be analyzed.
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Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 496
Macroscopic Examination
Performed after liquefaction, which normally occurs within 15-30 minutes at room temperature. Failure to liquefy suggests inadequate prostatic secretion.
| Parameter | Normal Value | Notes |
|---|---|---|
| Volume | ≥ 1.5 mL | Low volume suggests retrograde ejaculation, ejaculatory duct obstruction, or androgen deficiency |
| pH | 7.2-7.8 | > 8.0 with infection; < 7.0 with urine contamination or ejaculatory duct obstruction |
| Appearance | Gray-white, opalescent | Yellow hue = pyospermia; rust color = seminal vesicle bleeding |
| Viscosity | Low | Increased viscosity may impair sperm motility |
Microscopic Examination
Sperm Concentration
- Normal: > 15 million sperm/mL (total per ejaculate: ≥ 39 million)
- Total count is calculated by multiplying concentration by volume.
Motility
- Total motility: ≥ 40% (all moving sperm, including twitching)
- Progressive motility: ≥ 32% (forward, linear movement)
- Motility grading:
- Grade 4: rapid, straight, minimal lateral movement
- Grade 3: slower, forward progression
- Grade 2: slow with substantial lateral sway
- Grade 1: no forward progression
- Grade 0: no movement at all
- If motility < 30%, a viability stain (eosin Y with nigrosin counterstain) is performed - dead sperm stain red, live sperm remain unstained.
Morphology
- Assessed using Kruger strict criteria: ≥ 4% normal forms is the WHO 2010 lower reference limit
- A score > 4% indicates excellent fertilizing capacity; 0-3% predicts probable inability to fertilize
- The teratozoospermic index (average number of defects per sperm) is a predictor of in vivo and in vitro fertilizing ability
- Key abnormal features: acrosomal cap < 1/3 of head surface, retained cytoplasmic droplet > 1/2 head size, tail < 45 µm long
- Acrosome size has a direct relationship with fertilization and pregnancy rates
Agglutination
- Motile sperm sticking together (head-to-head, tail-to-tail, midpiece-to-midpiece) - suggests an immunologic cause of infertility
- Distinguished from non-specific clumping due to bacteria or debris by the reproducible orientation
Abnormal Semen Analysis - Terminology
| Term | Meaning |
|---|---|
| Oligozoospermia | Sperm concentration < 15 million/mL |
| Asthenozoospermia | Total motility < 40% or progressive motility < 32% |
| Teratozoospermia | < 4% normal morphology |
| Azoospermia | No sperm in the ejaculate |
| Leukocytospermia (pyospermia) | > 1 × 10⁶ leukocytes/mL |
| Necrozoospermia | All or most sperm are non-viable |
In infertile men, approximately 55% have all normal semen parameters (suggesting a non-semen factor), while 26% have low motility, 8% have low count, and 8% have azoospermia as an isolated finding.
Additional Tests
Computer-Assisted Semen Analysis (CASA)
Uses video digitalization and algorithms to objectively assess sperm parameters. While promising, CASA can overestimate sperm counts by up to 30% when there are high numbers of contaminating cells (immature sperm, leukocytes), and can underestimate motility at high concentrations. It has accepted value in research settings.
Leukocyte Analysis
Leukocytospermia (> 10⁶ leukocytes/mL) affects 2.8-23% of infertile men. Detected by peroxidase stain (identifies neutrophils), Papanicolaou stain, or immunocytology. Neutrophils are the predominant cell type. Its clinical significance for subfertility is debated.
Immunologic Tests (Antisperm Antibodies)
- IgA and IgG class antibodies against sperm head or tail antigens indicate immunologic infertility; IgA is most clinically significant
- Detected by direct/indirect mixed agglutination reaction (MAR) test or immunobead assay
- Suggested when agglutination is present or when routine semen parameters are unexpectedly normal in an infertile couple
Fructose Test
Fructose is secreted by the seminal vesicles. Absent fructose in a low-volume azoospermic sample suggests ejaculatory duct obstruction or absence of seminal vesicles. Can also be tested in post-ejaculate urine to detect retrograde ejaculation. Its significance has declined with wider availability of transrectal ultrasonography.
WHO 2010 Reference Limits (Lower 5th Percentile)
Based on semen samples from > 4,500 men in 14 countries whose partners conceived within 12 months:
| Parameter | Lower Reference Limit |
|---|---|
| Semen volume | ≥ 1.5 mL |
| Total sperm number | ≥ 39 million/ejaculate |
| Sperm concentration | ≥ 15 million/mL |
| Total motility | ≥ 40% |
| Progressive motility | ≥ 32% |
| Vitality (live sperm) | ≥ 58% |
| Morphology (normal forms) | ≥ 4% |
- Harrison's Principles of Internal Medicine 22E, p. 2702-2703
- Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 496-497
- Smith and Tanagho's General Urology, 19th Edition, p. 724-725
Note from recent literature (2026): A 2026 umbrella review (Wang QH et al., Asian J Androl, PMID 41527944) confirms that multiple environmental and lifestyle risk factors (obesity, smoking, heat exposure, endocrine disruptors) negatively impact semen parameters, reinforcing the importance of thorough history-taking alongside semen analysis.
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