Abstract

Semen analysis is the cornerstone investigation for evaluating male fertility. According to the WHO 5th Edition, standardized collection, processing, examination, and reporting improve accuracy and reproducibility. Clinical interpretation should always be correlated with patient history, physical examination, hormonal profile, and female partner evaluation. This review discusses laboratory principles, normal reference values, common abnormalities, quality assurance, and applications in IVF/ICSI.

Introduction

Male factor infertility contributes to approximately 40–50% of all infertility cases, either alone or in combination with female factors. Semen analysis remains the first and most informative investigation in the evaluation of a subfertile male. The WHO Laboratory Manual for the Examination and Processing of Human Semen provides internationally accepted standardized guidelines, with the 5th Edition (2010) being the most widely adopted worldwide.

The manual provides lower reference limits derived from fertile men whose partners conceived within 12 months, making the values clinically meaningful. These reference values represent the 5th centile of fertile men, not ideal or average values.

Key Point

WHO reference values are lower reference limits (5th centile of fertile men), not optimal values. A man can be fertile even at the lower limit, and infertile even above it. Semen analysis must always be interpreted in clinical context.

History of WHO Semen Manuals

The WHO has published successive editions of its semen analysis manual, each refining methodology and reference values based on larger evidence bases.

EditionYearKey Change
1st Edition1980First standardized guidelines
2nd Edition1987Expanded morphology criteria
3rd Edition1992Kruger strict morphology introduced
4th Edition1999Further refinement of reference ranges
5th Edition2010Evidence-based reference values from fertile men; standardized motility grading (PR/NP/IM)
6th Edition2021Updated reference values; expanded guidance on advanced tests

The 5th Edition (2010) is still the most widely used in clinical practice globally.

Male Reproductive Physiology

Understanding semen analysis requires a foundation in male reproductive physiology. Semen is a composite fluid produced by multiple accessory glands:

Contributions to Seminal Fluid

  • Testes (2–5%): Spermatozoa + testicular fluid
  • Epididymis: Sperm maturation, motility acquisition
  • Seminal Vesicles (65–70%): Fructose (energy source), prostaglandins, coagulation factors
  • Prostate (25–30%): PSA, zinc, citric acid, liquefaction enzymes
  • Bulbourethral glands (<1%): Mucus, lubrication

Spermatogenesis Timeline

  • Complete cycle: ~74 days (spermatogonium to mature sperm)
  • Epididymal transit: ~12 days
  • Total from stem cell to ejaculate: ~90 days
  • FSH stimulates Sertoli cells (sperm production)
  • LH stimulates Leydig cells (testosterone production)
  • Temperature: Testes must be 2–4°C below body temperature

This 90-day production timeline is clinically important: any insult (fever, medication, lifestyle change) will affect semen quality 3 months later. Repeat semen analysis after 3 months is recommended before concluding consistent abnormality.

Indications for Semen Analysis

  • Evaluation of male partner in an infertile couple (primary indication)
  • Pre- and post-vasectomy assessment
  • Monitoring response to treatment (medical or surgical)
  • Pre-ART assessment (IUI, IVF, ICSI) for protocol selection
  • Screening in occupational/environmental exposure studies
  • Forensic purposes
  • Sperm banking (before chemotherapy/radiotherapy/surgery)
  • Assessment after varicocele repair or hormonal treatment

Minimum Samples Required

At least two semen samples collected 7–90 days apart should be analysed before a diagnosis is made. Single semen analysis has high intra-individual variability of up to 30%.

Patient Preparation

Standardized patient preparation is essential for reproducible and clinically meaningful results. The following instructions must be given to the patient in writing:

Instructions to Patient

  • Sexual abstinence: 2–7 days (ideally 3–4 days) before collection
  • Avoid alcohol 5 days prior
  • Avoid hot baths/saunas for 2–3 months prior
  • No recent fever illness (if present, repeat in 3 months)
  • Inform about all medications (antibiotics, hormones, steroids)
  • Collection by masturbation into a sterile, wide-mouthed container
  • Do NOT use lubricants (most are spermicidal)

Abstinence Impact on Parameters

  • <2 days: Reduced volume and count, better motility
  • 2–7 days: Optimal results (WHO standard)
  • >7 days: Increased volume and count, reduced motility, more dead sperm
  • Abstinence must be recorded and reported with the result

Sample Collection

Proper collection technique is critical. Collection errors are a major source of pre-analytical variability.

  • Method: Masturbation into a sterile, non-toxic, wide-mouthed container at 37°C (body temperature)
  • Location: Ideally on-site at the laboratory (within 5 min walk); if at home, must reach lab within 30–60 minutes and be kept at body temperature during transport
  • Container: Must be pre-tested for sperm toxicity; avoid rubber or latex containers
  • Condom collection: Only specially designed non-spermicidal condoms (NOT standard latex condoms)
  • Labelling: Name, date of birth, date and time of collection, abstinence period
  • Incomplete collection: Note which fraction was lost; first fraction contains most motile sperm

Macroscopic Examination

Macroscopic examination must be performed after liquefaction is complete (usually 15–60 minutes at room temperature).

ParameterNormal Value (WHO 5th Ed.)Clinical Notes
LiquefactionComplete within 60 minutesMost liquefy within 15 min. Failure suggests prostate dysfunction. Analyse after 60 min maximum.
AppearanceHomogeneous, grey-opalescentYellow: possible infection/jaundice; clear: very low sperm count; brown/red: haematospermia
Volume≥1.5 mLLow volume: retrograde ejaculation, ejaculatory duct obstruction, incomplete collection, hypogonadism. High volume: long abstinence.
pH≥7.2Low pH (<7.0) with azoospermia: suggests bilateral absence of vas deferens (CBAVD). Test within 1 hour.
ViscosityNormal (droplet forms)Hyperviscous semen impairs sperm movement. Assessed by allowing semen to fall from Pasteur pipette — normal drops should be ≤2 cm.
ColourGrey-white, opalescentDegree of opalescence correlates with sperm concentration

Microscopic Examination

Microscopic analysis evaluates sperm concentration, motility, morphology, and other cellular elements. Performed on a well-mixed, liquefied sample at 37°C using a heated stage.

Sperm Motility (WHO 5th Ed. Grading)

Motility Categories

  • PR (Progressive Motility): Sperm moving actively forward (straight or in large circles) ≥25 μm/sec
  • NP (Non-Progressive Motility): Motile but not progressing (<25 μm/sec, small circles)
  • IM (Immotile): No movement at all

Lower Reference Limits

  • Total motility (PR + NP): ≥40%
  • Progressive motility (PR): ≥32%
  • Count 200 spermatozoa in 2 replicate preparations
  • Difference between replicates must be <10%

Important Change from 4th Edition

The WHO 4th edition used Grade a/b/c/d motility. The 5th edition replaced this with PR/NP/IM to improve reproducibility and reduce subjectivity.

Sperm Concentration

Assessed using a validated counting chamber (Improved Neubauer haemocytometer or Makler chamber). At least 200 spermatozoa are counted in replicate preparations. Result expressed as millions/mL and total count per ejaculate.

Sperm Morphology (Kruger Strict Criteria)

The WHO 5th Edition uses Kruger’s strict criteria for morphology assessment. Only sperm with a perfectly formed head, midpiece, and tail are considered normal.

Normal Sperm Morphology

  • Head: Oval, 4–5 μm long, 2.5–3.5 μm wide; smooth contour; acrosome covers 40–70% of head area
  • Midpiece: Slender, ≤1 μm wide, 1.5x head length; tightly attached to head
  • Tail: Uniform, thinner than midpiece, 45 μm long; no coils or sharp bends

Common Morphological Defects

  • Head defects: Large/small/round heads, double heads, pin-heads, amorphous, vacuolated
  • Midpiece defects: Bent, absent, asymmetric insertion
  • Tail defects: Short, coiled, multiple, hairpin, broken
  • Cytoplasmic droplets: >1/3 of head size = abnormal (immature)

Vitality (Sperm Viability)

Mandatory when progressive motility <40%. Distinguishes live immotile sperm from dead sperm.

  • Methods: Eosin-nigrosin stain (dead sperm stain pink/red), HOS test (hypo-osmotic swelling — live sperm swell)
  • Normal: ≥58% live (unstained)
  • Clinical significance: If many live but immotile sperm → suspect primary ciliary dyskinesia (Kartagener syndrome)

Leukocytes (Round Cells)

  • Normal: <1 × 10&sup6; white blood cells/mL (peroxidase-positive cells)
  • Leukocytospermia (>1 × 10&sup6; WBC/mL): suggests genital tract infection/inflammation
  • Note: All round cells are NOT leukocytes — must differentiate from immature germ cells using peroxidase staining or immunocytochemistry

WHO 5th Edition Reference Values

These values represent the 5th centile of a reference population of men whose partners achieved pregnancy within 12 months of unprotected intercourse.

ParameterLower Reference Limit (5th centile)
Semen volume1.5 mL (95% CI: 1.4–1.7)
Total sperm count39 million/ejaculate (33–46)
Sperm concentration15 million/mL (12–16)
Total motility (PR+NP)40% (38–42)
Progressive motility (PR)32% (31–34)
Vitality (live sperm)58% (55–63)
Sperm morphology (normal forms)4% (3.0–4.0)
pH≥7.2
Peroxidase-positive leukocytes<1.0 × 10&sup6;/mL
MAR test (motile spermatozoa with bound particles)<50%
Immunobead test (motile spermatozoa with bound beads)<50%
Seminal zinc≥2.4 μmol/ejaculate
Seminal fructose≥13 μmol/ejaculate
Seminal neutral glucosidase≥20 mU/ejaculate

Source: WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th Edition, 2010.

Interpretation of Results

Semen parameter results are interpreted using the WHO 5th Edition reference limits. Multiple terms are used to describe specific abnormalities:

Nomenclature for Semen Abnormalities

Normozoospermia — All parameters normal

Oligozoospermia — Low concentration (<15 M/mL)
Severe oligozoospermia — <5 M/mL
Cryptozoospermia — Sperm present only after centrifugation
Azoospermia — No sperm in ejaculate (including post-centrifuge pellet)

Asthenozoospermia — Low progressive motility (<32% PR)
Teratozoospermia — Low normal morphology (<4%)
Necrozoospermia — Low vitality (<58% live)

Oligoasthenoteratozoospermia (OAT) — All three parameters abnormal
Leukocytospermia — >1 × 10&sup6; WBC/mL
Hyperspermia — Volume >6 mL
Hypospermia — Volume <1.5 mL
Aspermia — No semen produced

Stepwise Interpretation Approach

  1. Check liquefaction and viscosity first
  2. Assess macroscopic parameters (volume, pH, appearance)
  3. Estimate sperm concentration (wet prep screening)
  4. Count concentration using haemocytometer (two replicates)
  5. Assess motility (PR, NP, IM) — heated stage
  6. Prepare stained smear for morphology (Papanicolaou or Diff-Quik)
  7. Assess vitality if motility <40%
  8. Count round cells; if elevated, do peroxidase staining
  9. Consider anti-sperm antibodies if clumping seen
  10. Report all values with reference ranges; correlate with clinical history

Common Semen Abnormalities

Azoospermia: Obstructive (OA) vs Non-Obstructive (NOA)

  • OA: Normal FSH, normal testis volume, blockage in vas/epididymis. Causes: vasectomy, CBAVD, epididymal block, infection.
  • NOA: Elevated FSH, small testes, spermatogenic failure. Causes: Klinefelter syndrome, Y-chromosome microdeletion, cryptorchidism, post-chemotherapy.
  • Key test: FSH level + testicular biopsy/TESA/PESA for definitive diagnosis

Severe OAT Syndrome

  • Sperm concentration <5 million/mL with <2% normal morphology & <32% PR
  • Causes: varicocele (most common, 40%), genetic (Y-deletion, CFTR), environmental toxins, idiopathic
  • Management: treat varicocele if present; ICSI is treatment of choice if motile sperm available

Globozoospermia (Round-Headed Sperm)

  • 100% round-headed sperm without acrosome
  • Cannot penetrate zona pellucida naturally
  • ICSI required; but fertilization rates remain poor
  • Genetic: DPY19L2 gene mutation in 70% of cases

Necrozoospermia

  • High proportion of dead sperm (>42% non-viable)
  • Causes: prolonged abstinence, epididymal pathology, oxidative stress
  • HOS test or eosin-nigrosin differentiates from immotile live sperm
  • If live but immotile → consider PCD (Primary Ciliary Dyskinesia)

Quality Control in Semen Analysis

Quality control (QC) is essential to reduce the high intra-laboratory and inter-laboratory variability in semen analysis. The WHO 5th Edition dedicates an entire section to QC procedures.

Sources of Variability

Intra-individual biological variation (up to 30%) is the largest source of variability. Laboratory-related variability includes technician experience, equipment calibration, staining protocol, and timing of assessment.

Key QC Measures

  • Internal QC: Use of preserved sperm samples or commercial sperm quality controls for daily checks of motility and concentration assessments
  • External QC / Proficiency testing: Participation in inter-laboratory schemes (e.g., NEQAS, ESHRE SIGA)
  • Duplicate counts: All motility and concentration assessments must be performed in duplicate; difference between replicates must meet WHO acceptable sampling error limits
  • Calibration: Haemocytometers, pipettes, and microscopes must be regularly calibrated
  • Training: Minimum 3–6 months supervised training required before independent reporting
  • SOPs: Standard operating procedures for every step; must be documented and followed strictly
  • Temperature control: Heated stage at 37°C mandatory for motility assessment

Role in IVF and ICSI

Semen analysis results directly determine the appropriate ART treatment pathway. The WHO 5th Edition parameters guide protocol selection.

Semen Parameter ProfileRecommended ART Approach
Normal parameters or mild abnormalitiesIUI (with or without ovarian stimulation)
Moderate OAT (concentration 5–15 M/mL, motility 10–32% PR)IVF (conventional insemination)
Severe OAT (<5 M/mL, <32% PR, <4% morphology)ICSI (Intracytoplasmic Sperm Injection)
Azoospermia — obstructiveSurgical sperm retrieval (PESA/MESA) + ICSI
Azoospermia — non-obstructiveTesticular sperm extraction (TESE/microTESE) + ICSI
Morphology <4% (all other parameters normal)IVF or ICSI depending on degree of teratozoospermia
High ASA (>50% MAR positive)Sperm washing + IUI or ICSI

Sperm Preparation for ART

  • Density gradient centrifugation (DGC): Preferred method; separates motile from immotile and dead sperm and leukocytes. Uses 40%/80% gradient.
  • Swim-up: Simpler; best for good-quality samples. Motile sperm swim up into overlying culture medium.
  • Simple wash: Only for very high concentration samples; least effective for removing debris.
  • All preparation must be done in a sterile, IVF-grade laminar flow hood using certified ART media.

Morphology and IVF Outcome

Kruger strict morphology <4% (teratozoospermia) is associated with reduced fertilization rates in conventional IVF. ICSI bypasses the zona pellucida barrier and is preferred in such cases. However, even in ICSI, very poor morphology can affect fertilization and embryo development.

Advanced Sperm Function Tests

When routine semen analysis is normal but fertility remains unexplained, or when IVF/ICSI fails repeatedly, advanced tests provide additional diagnostic information.

Sperm DNA Fragmentation (SDF)

  • Methods: TUNEL, SCSA (Sperm Chromatin Structure Assay), Comet assay, SCD (Sperm Chromatin Dispersion)
  • DFI (DNA Fragmentation Index): >25–30% is clinically significant
  • Elevated SDF → reduced IVF/ICSI success, recurrent miscarriage
  • Causes: oxidative stress, varicocele, infection, chemotherapy
  • Management: antioxidants, varicocele repair, ICSI with testicular sperm

Anti-Sperm Antibodies (ASA)

  • Methods: MAR test (Mixed Antiglobulin Reaction) and Immunobead test
  • Positive if ≥50% motile sperm with bound particles
  • Causes: testicular trauma, vasectomy reversal, infection, cryptorchidism
  • ASA impair sperm motility, cervical penetration, and zona binding
  • Management: IUI (sperm washing) or ICSI

Reactive Oxygen Species (ROS) / Oxidative Stress

  • Sperm are highly susceptible to oxidative stress due to high PUFA content in membranes and limited cytoplasmic antioxidants
  • Methods: chemiluminescence assay, ELISA for 8-OHdG
  • Elevated ROS damages DNA, lipid membranes, and reduces motility
  • Treatment: antioxidants (Vit C, Vit E, Coenzyme Q10, Lycopene)

Other Advanced Tests

  • FISH (Fluorescence in situ hybridisation): Detects sperm aneuploidy; useful in recurrent miscarriage
  • Acrosome integrity: Lectin staining; relevant in fertilization failure
  • Mitochondrial membrane potential: JC-1 staining; reflects energy status
  • SCSA, Comet assay: Detailed DNA integrity assessment

Limitations of Semen Analysis

Critical Limitation

Semen analysis predicts fertility probability, not certainty. Men with normal parameters can be infertile, and men with severe oligospermia can father children naturally. The test evaluates the quantity and basic quality of sperm, not functional competence.
  • High intra-individual biological variability (up to 30%): single analysis is insufficient
  • Does not assess sperm–egg interaction, sperm capacitation, or acrosome reaction
  • Does not evaluate sperm-binding to zona pellucida (zona binding test needed)
  • Morphology assessment is subjective and inter-observer variability is high (even with strict criteria)
  • Reference values are statistical (5th centile), not absolute diagnostic thresholds
  • Does not detect genetic causes (Y-chromosome microdeletion, CFTR mutation) — separate genetic testing required
  • Computer-aided sperm analysis (CASA) improves objectivity but has standardization issues across platforms
  • Cannot predict embryo quality or implantation potential after ICSI

Future Perspectives

The field of male fertility assessment is evolving rapidly, with several promising developments:

  • WHO 6th Edition (2021): Updated reference values with larger multicenter dataset; expanded guidance on CASA and advanced tests; greater emphasis on total sperm count over concentration
  • Artificial Intelligence & CASA: AI-powered automated sperm analysis systems (MiOXSYS, SQA-Vision, SQUARESIX) reduce human bias and improve throughput
  • Sperm proteomics & metabolomics: Identify molecular markers of sperm function beyond morphology and motility
  • Epigenetics: Sperm epigenome reflects lifestyle and environmental exposures; may predict embryo development and offspring health
  • Microfluidics: Lab-on-chip devices for rapid, standardized semen analysis at point-of-care
  • Functional sperm selection for ICSI: PICSI (physiological ICSI using hyaluronan binding), IMSI (high-magnification morphology selection), MACS (magnetic-activated cell sorting for apoptotic sperm depletion)

Conclusion

Key Takeaways

  • Semen analysis is the first-line investigation in male fertility evaluation; at least two samples must be assessed.
  • WHO 5th Edition (2010) provides standardized 5th centile reference limits from fertile men, not optimal values.
  • The key change from 4th to 5th edition: motility grading changed from a/b/c/d to PR/NP/IM.
  • Morphology uses Kruger strict criteria; lower limit is ≥4% normal forms.
  • Always assess at least two samples before concluding a diagnosis, given biological intra-individual variability.
  • Semen analysis guides ART pathway selection: IUI → IVF → ICSI → surgical sperm retrieval + ICSI.
  • Advanced tests (SDF, ASA, ROS, FISH) are indicated in unexplained infertility or recurrent ART failure.
  • Results must always be interpreted in the context of full clinical evaluation of the couple.
About the Author: Dr. Varada Arora, MBBS, MS, is an IVF Specialist with expertise in reproductive medicine at Valencia IVF & Test Tube Baby Centre. This article is intended for educational and clinical training purposes. Individual clinical decisions should always be made in consultation with a qualified healthcare provider.

References

  1. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th ed. Geneva: WHO Press; 2010.
  2. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 6th ed. Geneva: WHO Press; 2021.
  3. Cooper TG, Noonan E, von Eckardstein S, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010;16(3):231–245.
  4. Björndahl L, Barratt CLR, Fraser LR, et al. ESHRE Basic Semen Analysis Courses 1995–1999: immediate beneficial effects of standardized training. Hum Reprod. 2002;17(5):1299–1305.
  5. Agarwal A, Mulgund A, Hamada A, et al. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37.
  6. Esteves SC, Zini A, Aziz N, et al. Critical appraisal of World Health Organization’s new reference values for human semen characteristics and effect on diagnosis and treatment of subfertile men. Urology. 2012;79(1):16–22.
  7. Kasturi SS, Tannir J, Brannigan RE. The metabolic syndrome and male infertility. J Androl. 2008;29(3):251–259.
  8. Cissen M, van Wely M, Scholten I, et al. Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction. PLoS One. 2016;11(11):e0165125.

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