Semen analysis
Semen analysis is a laboratory examination of human semen that assesses various physical and microscopic parameters to evaluate male reproductive health, particularly fertility potential.[1] It serves as a primary diagnostic tool in clinical settings for investigating infertility and in research to monitor spermatogenesis or the effects of interventions on semen quality.[1] The test provides insights into sperm production, function, and overall semen composition, helping to identify potential causes of male-factor infertility, which contributes to approximately 50% of infertility cases in couples.[2] The procedure for semen analysis follows standardized protocols outlined by the World Health Organization (WHO) to ensure reliability and comparability across laboratories.[1] A semen sample is typically collected through masturbation into a sterile container after 2 to 7 days of ejaculatory abstinence, either at a clinic or at home with prompt delivery to the laboratory to maintain sample viability.[2] The sample undergoes initial macroscopic evaluation for volume, appearance, viscosity, and liquefaction time (which should occur within 60 minutes), followed by microscopic analysis using phase-contrast microscopy or computer-assisted systems to assess sperm concentration, motility, morphology, and vitality.[3] At least two samples, separated by 1 to 2 weeks, are recommended due to natural variability in semen parameters influenced by factors like recent illness, stress, or lifestyle.[2] Key parameters evaluated in semen analysis include ejaculate volume, sperm concentration, total sperm number, motility, and morphology, with reference values derived from the 5th percentile of fertile men as per the WHO's 6th edition laboratory manual (2021).[1] Normal ejaculate volume is at least 1.4 mL (95% CI: 1.3–1.5 mL), sperm concentration is ≥16 million per mL, and total sperm number is ≥39 million per ejaculate (95% CI: 35–40 million).[3] Total motility should be ≥42% (95% CI: 40–43%), with progressive motility >30% (95% CI: 29–31%), and morphology features >4% normal forms (95% CI: 3.9–4%).[3] Additional assessments cover pH (≥7.2), vitality (>54%; 95% CI: 50–56%), and white blood cell count (<1 million/mL to rule out infection).[3] These values are not strict cutoffs but guides for interpretation, as semen quality exists on a continuum, and abnormal results may prompt further testing such as hormonal assays or genetic screening.[1]Clinical Indications
Reasons for testing
Semen analysis serves as a primary diagnostic tool for evaluating male infertility, particularly in couples experiencing difficulty conceiving after one year of unprotected intercourse. It is routinely recommended as the initial laboratory test in the assessment of male factor contributions to infertility, including cases of subfertility where no obvious cause is identified.[4][5][6] Testing is indicated in scenarios of primary infertility, where couples have never achieved pregnancy, as well as in evaluations following recurrent pregnancy loss to rule out male reproductive issues. Post-vasectomy semen analysis is essential to confirm successful sterilization through the detection of azoospermia or minimal non-motile sperm, typically performed 8 to 16 weeks after the procedure.[4][7][5] Semen analysis also aids in screening for underlying genetic or endocrine disorders that impair spermatogenesis, such as Klinefelter syndrome, which often presents with azoospermia or severe oligospermia due to an extra X chromosome, and hypogonadism, characterized by low testosterone levels affecting sperm production. Abnormal results may prompt further genetic testing or hormonal evaluations.[8][9][10] Beyond fertility contexts, semen analysis finds application in forensic investigations of sexual assault cases, where identification of semen traces on evidence helps confirm sexual contact and enables DNA profiling for suspect identification. In occupational health monitoring, it is used to assess the impact of environmental toxins, such as solvents or formaldehyde, on reproductive function in workers with potential exposure risks.[11][12][13] The standardization of semen analysis began in the 1950s with pioneering work by researchers like John MacLeod, who established initial reference values for sperm parameters based on studies of fertile men, laying the foundation for its clinical use in fertility clinics. The World Health Organization later formalized these methods in its laboratory manual starting in 1980, promoting global consistency.[14][15] These evaluations contribute to broader fertility outcomes by identifying treatable causes of infertility.[4]Relation to fertility and infertility diagnosis
Semen analysis serves as the cornerstone of male infertility evaluation, as outlined in the World Health Organization (WHO) laboratory manual and the American Urological Association (AUA)/American Society for Reproductive Medicine (ASRM) guidelines, providing essential insights into sperm production, function, and overall reproductive potential.[1][16] These standardized assessments help identify male-factor contributions to infertility, which account for 30-50% of all cases, either solely or in combination with female factors.[17] By evaluating key semen parameters, the test enables clinicians to classify infertility risks and tailor subsequent management strategies accordingly. The correlation between semen quality and fertility outcomes is well-established, with normal semen parameters associated with higher natural conception rates, while subnormal findings signal potential barriers such as impaired sperm transport through the female reproductive tract or defective sperm function during fertilization.[18] For instance, men with optimal semen characteristics demonstrate improved time-to-pregnancy compared to those with diminished quality, underscoring the test's prognostic value in both natural and assisted conception scenarios.[18] Abnormalities in semen analysis thus highlight underlying physiological issues that may compromise fertility, guiding targeted interventions to enhance reproductive success. Key diagnostic categories derived from semen analysis include azoospermia, characterized by the complete absence of sperm in the ejaculate; oligospermia, indicating a reduced sperm count; asthenospermia, reflecting poor sperm motility; and teratospermia, denoting a high proportion of morphologically abnormal sperm.[19] These classifications, based on WHO criteria, facilitate the stratification of male infertility severity and inform the likelihood of spontaneous conception or the need for assisted reproductive technologies.[1] Semen analysis results directly guide the integration of complementary diagnostic tests, such as hormonal assays to evaluate pituitary-gonadal axis function or genetic testing for conditions like Y-chromosome microdeletions in cases of severe oligospermia or azoospermia.[16] According to AUA/ASRM recommendations, abnormal semen findings prompt endocrine evaluation, including measurements of follicle-stimulating hormone, luteinizing hormone, and testosterone, to differentiate between hypothalamic-pituitary disorders and primary testicular failure.[16] Similarly, non-obstructive azoospermia often necessitates karyotyping or genetic screening to identify treatable etiologies before proceeding to advanced therapies.[16] Meta-analyses of semen parameters have demonstrated their predictive utility for in vitro fertilization (IVF) success, particularly highlighting the role of sperm motility in outcomes. For example, progressive sperm motility ≥30% is associated with higher live birth rates in IVF cycles.[1][20] These findings emphasize how semen analysis not only diagnoses infertility but also prognosticates assisted reproductive technology efficacy, enabling personalized treatment pathways.[20]Specimen Collection and Preparation
Collection methods
The standard method for semen collection involves masturbation to produce the ejaculate directly into a sterile, wide-mouthed, non-toxic, clean, and leak-proof plastic container, ensuring the entire sample is captured to maintain representativeness for analysis.[1] This procedure can be performed either at a clinic or at home, provided the sample is delivered to the laboratory within one hour of collection while kept at body temperature (20–37°C).[1] A period of sexual abstinence of 2–7 days prior to collection is recommended to optimize semen volume and sperm concentration, with an ideal range of 2–5 days to balance these parameters without compromising motility or increasing sperm DNA damage.[1] Shorter abstinence (e.g., 1 day) results in lower semen volume and sperm concentration but may yield higher motility and reduced chromatin immaturity, while longer periods (e.g., beyond 7 days) increase volume and count at the potential cost of declining motility after 5 days.[21][22] Hygiene protocols are essential to prevent contamination and ensure sample integrity; hands and genitals should be washed with soap and water, rinsed thoroughly, and dried with a disposable towel, followed by urination to clear the urethra.[1] Lubricants, saliva, or any spermicidal substances must be avoided, as they can immobilize or damage sperm, and the entire ejaculate—including the initial fraction rich in sperm—must be collected without loss.[1][23] For individuals unable to ejaculate via masturbation, such as those with spinal cord injuries or ejaculatory dysfunction, alternative methods include penile vibratory stimulation (PVS), where a vibrating device is applied to the glans penis to induce reflex ejaculation, or electroejaculation (EEJ), performed under anesthesia using rectal probe electrical stimulation.[24][25] PVS is preferred as a first-line, non-invasive option when feasible, with success rates up to 80–90% in suitable candidates, while EEJ serves as a salvage method for PVS failures.[26][24] Special considerations apply for home collection or specific conditions; non-spermicidal, semen-compatible (often silicone-based) condoms may be used during intercourse with immediate transfer of the sample to a sterile container, avoiding standard latex condoms which can release toxic substances.[1][27] In cases of obstructive or non-obstructive azoospermia, or when ejaculation is not possible (e.g., anejaculation), surgical sperm retrieval techniques such as testicular sperm extraction (TESE) or microdissection TESE (micro-TESE) can be used to obtain sperm directly from the testes for assessment or use in assisted reproduction, bypassing standard semen analysis.[24][28] Samples collected by any method require prompt handling to preserve viability, as detailed in subsequent protocols.[1]Handling, storage, and transport
Following collection, semen samples must be handled promptly to preserve sperm quality and prevent artifacts in analysis. The World Health Organization (WHO) recommends maintaining the sample at room temperature between 20°C and 37°C immediately after collection, with analysis commencing within 60 minutes, preferably within 30 minutes, to minimize degradation. Delays beyond this timeframe can significantly impair sperm parameters; for instance, studies indicate a progressive decline in motility of about 5-10% per hour at room temperature.[29] Liquefaction, the process by which semen transitions from a gel-like state to a liquid, should be monitored and typically completes within 15-30 minutes at room temperature (20–37°C); if delayed, incubation at 37°C is advised to facilitate this step without compromising viability.[1] For transport, especially in cases of home collection, samples should be delivered to the laboratory within 30-60 minutes using insulated, leak-proof containers to maintain temperature stability between 20°C and 37°C and avoid extremes that could induce cold shock or overheating. Keeping the container close to the body, such as in an inner pocket, helps sustain near-physiological conditions during transit. Contamination must be rigorously prevented by using sterile, wide-mouthed, non-toxic containers made of glass or medical-grade plastic, which are tested for sperm compatibility; exposure to lubricants, soaps, light, or chemicals should be avoided, as these can introduce artifacts or toxicity affecting sperm function. Extended storage is primarily achieved through cryopreservation for purposes like fertility banking, involving gradual addition of cryoprotectants such as glycerol or glycerol-egg yolk-citrate media over 10 minutes at room temperature, followed by freezing in liquid nitrogen at -196°C. Post-thaw viability typically ranges from 40-50%, with motility recovery often around 50% of pre-freeze levels, though outcomes vary by protocol and sample quality. These WHO 2021 standards emphasize documenting any deviations in handling timelines or conditions to ensure reliable parameter stability during evaluation.[1]Semen Parameters
Macroscopic parameters
Macroscopic parameters in semen analysis involve the initial visual and physical evaluation of the ejaculate, which provides preliminary insights into accessory gland function and overall sample quality before microscopic examination. These assessments are standardized to ensure reproducibility and are typically performed shortly after collection. Key parameters include volume, appearance, liquefaction time, viscosity, and pH, each contributing to the detection of potential reproductive tract issues.[1] Semen volume is measured immediately after collection using a wide-mouthed graduated pipette, serological pipette, or by weighing the sample (assuming a density of 1 g/mL), with adjustments for any loss during transfer (typically 0.3–0.9 mL). The lower reference limit is 1.4 mL (5th centile, 95% CI: 1.3–1.5 mL), reflecting contributions from the seminal vesicles (60–70%), prostate (20–30%), and bulbourethral glands. Low volume (<1.4 mL) may indicate incomplete collection, ejaculatory duct obstruction, congenital bilateral absence of the vas deferens (CBAVD), or hypogonadism, while high volume can suggest inflammation of accessory glands.[1] Appearance is assessed visually after liquefaction, with normal semen described as homogeneous and grey-opalescent, varying slightly with sperm concentration (e.g., more opaque with higher counts). Abnormal colors include yellow (suggesting jaundice or urine contamination), red or brown (indicating hematospermia from blood in the ejaculate), or transparent (possibly low sperm count). These observations can signal underlying pathologies such as infection, trauma, or systemic conditions, warranting further investigation.[1] Liquefaction time refers to the process by which coagulated semen becomes fluid, observed by tilting the container at room temperature or 37°C; complete liquefaction should occur within 60 minutes post-ejaculation, often within 15–30 minutes. Delayed or incomplete liquefaction (>60 minutes) is evaluated for clots and may result from elevated seminal vesicle proteins or prostate dysfunction, potentially interfering with subsequent motility assessments; in such cases, mechanical dispersion or enzymatic treatment may be needed.[1] Viscosity is evaluated post-liquefaction by allowing semen to drop from a pipette or glass rod; normal viscosity results in discrete drops forming without long threads (<2 cm), facilitating accurate pipetting. High viscosity, characterized by sticky homogeneity or threads >2 cm, can impair sperm motility evaluation and concentration measurements, often linked to seminal vesicle or prostate issues, and may require dilution for analysis.[1] pH is measured on the liquefied sample (30–60 minutes post-ejaculation) using pH indicator strips (range 6–10) or a pH meter, with the normal range being 7.2–8.0, influenced by prostatic secretions (acidic) and seminal vesicle fluids (alkaline). A low pH (<7.2, or <7.0 in azoospermic samples) may indicate prostate dysfunction, infection, ejaculatory duct obstruction, or CBAVD, while high pH can reflect contamination or metabolic alterations.[1]| Parameter | Normal Range | Measurement Method | Common Abnormalities and Implications |
|---|---|---|---|
| Volume | ≥1.4 mL (5th centile, 95% CI: 1.3–1.5 mL) | Pipette or weighing (1 g = 1 mL) | Low: obstruction, CBAVD; High: inflammation |
| Appearance | Grey-opalescent | Visual inspection | Yellow: jaundice; Red-brown: hematospermia |
| Liquefaction | Complete in ≤60 min | Observation at room temp/37°C | Delayed: prostate/seminal vesicle dysfunction |
| Viscosity | Discrete drops; <2 cm threads | Drop test from pipette/rod | High: impairs analysis; seminal vesicle issues |
| pH | ≥7.2 | pH strips or meter | Low: prostate dysfunction, infection |