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Semen analysis
Human sperm stained for semen quality testing in the clinical laboratory
MedlinePlus003627
HCPCS-L2G0027

A semen analysis (plural: semen analyses), also called seminogram or spermiogram, evaluates certain characteristics of a male's semen and the sperm contained therein.[1][2][3] It is done to help evaluate male fertility, whether for those seeking pregnancy or verifying the success of vasectomy. Depending on the measurement method, just a few characteristics may be evaluated (such as with a home kit) or many characteristics may be evaluated (generally by a diagnostic laboratory). Collection techniques and precise measurement method may influence results. The assay is also referred to as ejaculate analysis, human sperm assay (HSA), sperm function test, and sperm assay.[citation needed]

Semen analysis is a complex test that should be performed in andrology laboratories by experienced technicians with quality control and validation of test systems. A routine semen analysis should include: physical characteristics of semen (color, odor, pH, viscosity and liquefaction), volume, concentration, morphology and sperm motility and progression. To provide a correct result it is necessary to perform at least two, preferably three, separate seminal analyses with an interval between them of seven days to three months.

The techniques and criteria used to analyze semen samples are based on the WHO manual for the examination of human semen and sperm-cervical mucus interaction published in 2021.[1]

Reasons for testing

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The most common reasons for laboratory semen analysis in humans are as part of a couple's infertility investigation and after a vasectomy to verify that the procedure was successful.[4] It is also commonly used for testing human donors for sperm donation, and for animals semen analysis is commonly used in stud farming and farm animal breeding.

Occasionally a man will have a semen analysis done as part of routine pre-pregnancy testing. At the laboratory level this is rare, as most healthcare providers will not test the semen and sperm unless specifically requested or there is a strong suspicion of a pathology in one of these areas discovered during the medical history or during the physical examination. Such testing is very expensive and time-consuming, and in the U.S. is unlikely to be covered by insurance. In other countries, such as Germany, the testing is covered by all insurances.

Relation to fertility

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The characteristics measured by semen analysis are only some of the factors in semen quality. One source states that 30% of men with a normal semen analysis actually have abnormal sperm function.[5] Conversely, men with poor semen analysis results may go on to father children.[6] In NICE guidelines, mild male factor infertility is defined as when two or more semen analyses have one or more variables below the 5th percentile, and confers a chance of pregnancy occurring naturally through vaginal intercourse within two years similar to people with mild endometriosis.[7]

Collection methods

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Main article: Semen collection

Methods of semen collection include masturbation, condom collection, and epididymal extraction. The sample should never be obtained through coitus interruptus as some portion of the ejaculate could be lost, bacterial contamination could occur, or the acidic vaginal pH could be detrimental for sperm motility. The optimal sexual abstinence for semen sampling is two to seven days. The most common way to obtain a semen sample is through masturbation and the best place to obtain it is in the clinic where the analysis will take place in order to avoid temperature changes during the transport that can be lethal for some spermatozoa. Once the sample is obtained, it must be put directly into a sterile plastic receptacle (never in a conventional condom, since they have chemical substances such as lubricants or spermicides that could damage the sample) and be handed to the clinic for it to be studied within the hour.

There are some situations that necessitate alternative collection methods, such as retrograde ejaculation, neurological injury or psychological inhibition. Depending on the situation, specialized condoms, electrostimulation or vibrostimulation might be used.

Parameters

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The parameters included in the semen analysis can be divided in macroscopic (liquefaction, appearance, viscosity, volume and pH) and microscopic (motility, morphology, vitality, concentration, sperm count, sperm aggregation, sperm agglutination, and presence of round cells or leukocytes). The main three parameters of the spermiogram are the concentration of the spermatozoa in the semen, the motility and the morphology of them. This analysis is important to analyse fertility, but even in a perfectly fertile man is very difficult to find normal spermatozoa. For the average fertile man, only 4% of their spermatozoa are normal in every parameter, while 96% are abnormal in at least one of them.

Sperm count

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Approximate pregnancy rate varies with amount of sperm used in an artificial insemination cycle. Values are for intrauterine insemination, with sperm number in total sperm count, which may be approximately twice the total motile sperm count.

Sperm count, or sperm concentration to avoid confusion with total sperm count, measures the concentration of sperm in ejaculate, distinguished from total sperm count, which is the sperm count multiplied with volume. Over 16 million sperm per milliliter is considered normal, according to the WHO in 2021.[8] Older definitions state 20 million.[5][6] A lower sperm count is considered oligozoospermia. A vasectomy is considered successful if the sample is azoospermic (zero sperm of any kind found). When a sample contains less than 100,000 spermatozoa per milliliter we talk about cryptozoospermia. Some define success as when rare/occasional non-motile sperm are observed (fewer than 100,000 per millilitre).[9] Others advocate obtaining a second semen analysis to verify the counts are not increasing (as can happen with re-canalization) and others still may perform a repeat vasectomy for this situation.

Chips for home use are emerging that can give an accurate estimation of sperm count after three samples taken on different days. Such a chip may measure the concentration of sperm in a semen sample against a control liquid filled with polystyrene beads.[10][unreliable medical source?]

Sperm motility

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Main article: Sperm motility

The World Health Organization has a value of 40%[11] and this must be measured within 60 minutes of collection. WHO also has a parameter of vitality, with a lower reference limit of 60% live spermatozoa.[8] A man can have a total number of sperm far over the limit of >16 million sperm cells per milliliter, but still have bad quality because too few of them are motile. However, if the sperm count is very high, then a low motility (for example, less than 60%) might not matter, because the fraction might still be more than 8 million per millilitre. The other way around, a man can have a sperm count far less than 20 million sperm cells per millilitre and still have good motility, if more than 60% of those observed sperm cells show good forward movement - which is beneficial because nature favours quality over quantity.

A more specified measure is motility grade, where the total motility(PR+NP) and immotile.[11]

Progressively motile- Sperm moving in forward direction is Progressively Motile Non progressively Motile-Those sperms are moving circular motion are Non Progressively Motile Immotile- Those sperms are fail to move or dead sperms.

The total motility reference of 40% can be divided in a 32% of progressive motility and 8% of motility in situ.

Semen samples which have more than 30% progressive motility are considered as normozoospermia. Samples below that value are classified as asthenozoospermia regarding the WHO criteria.

Sperm morphology

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Regarding sperm morphology, the WHO criteria as described in 2021 state that a sample is normal (samples from men whose partners had a pregnancy in the last 12 months) if 4% (or 5th centile) or more of the observed sperm have normal morphology.[8][12] If the sample has less than 4% of morphologically normal spermatozoa, it's classified as teratozoospermia.

Normal sperm morphology is hard to classify because of lack of objectivity and variations in interpretation, for instance. In order to classify spermatozoa as normal or abnormal, the different parts should be considered. Sperm has a head, a midpiece and a tail.

Firstly, the head should be oval-shaped, smooth and with a regular outline. What is more, the acrosomal region should comprise the 40–70% area of the head, be defined and not contain large vacuoles. The amount of vacuoles should not excess the 20% of the head's area. It should be 4–5 μm long and a width of 2,5–3,5 μm.

Secondly, the midpiece and the neck should be regular, with a maximal width of 1 μm and a length of 7–8 μm. The axis of the midpiece should be aligned with the major axis of the head.

Finally, the tail should be thinner than the midpiece and have a length of 45 μm approximately and a constant diameter along its length. It is important that it is not rolled up.

Since abnormalities are frequently mixed, the teratozoospermia index (TZI) is really helpful. This index is the mean number of abnormalities per abnormal sperm. To calculate it, 200 spermatozoa are counted (this is a good number). From this number, the abnormalities in head, midpiece and tail are counted, as well as the total abnormal spermatozoa. Once that task has been done, the TZI is calculated like this:

TZI= (h+m+t)/x

  • x = number of abnormal spermatozoa.
  • h = number of spermatozoa with head abnormalities.
  • m = number of spermatozoa with midpiece abnormalities.
  • t = number of spermatozoa with tail abnormalities.

Another interesting index is the sperm deformity index (SDI), which is calculated the same way as the TZI, but instead of dividing by the number of abnormal spermatozoa, the division is by the total number of spermatozoa counted. The TZI takes values from 1 (only one abnormality per sperm) to 3 (each sperm has the three types of abnormalities).

Morphology is a predictor of success in fertilizing oocytes during in vitro fertilization.

Up to 10% of all spermatozoa have observable defects and as such are disadvantaged in terms of fertilising an oocyte.[13]

Also, sperm cells with tail-tip swelling patterns generally have lower frequency of aneuploidy.[14]

Motile sperm organelle morphology examination

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A motile sperm organelle morphology examination (MSOME) is a particular morphologic investigation wherein an inverted light microscope equipped with high-power optics and enhanced by digital imaging is used to achieve a magnification above x6000, which is much higher than the magnification used habitually by embryologists in spermatozoa selection for intracytoplasmic sperm injection (x200 to x400).[15] A potential finding on MSOME is the presence of sperm vacuoles, which are associated with sperm chromatin immaturity, particularly in the case of large vacuoles.[16]

Semen volume

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According to one lab test manual semen volumes between 2.0 mL and 5 mL are normal;[6] WHO regards 1.4 mL as the lower reference limit.[8] Low volume, called hypospermia, may indicate partial or complete blockage of the seminal vesicles, or that the man was born without seminal vesicles.[5] In clinical practice, a volume of less than 1,4 mL in the setting of infertility is most likely due to incomplete ejaculation or partial loss of sample, asides this, patient should be evaluated for hypoandrogenism and obstructions in some parts of the ejaculatory tract, azoospermia, given that it has been at least 48 hours since the last ejaculation to time of sample collection.

The human ejaculate is mostly composed of water, 96 to 98% of semen is water. One way of ensuring that a man produces more ejaculate[17] is to drink more liquids. Men also produce more seminal fluid after lengthy sexual stimulation and arousal. Reducing the frequency of sex and masturbation helps increase semen volume. Sexually transmitted diseases also affect the production of semen. Men who are infected[18] with the human immunodeficiency virus (HIV) produce lower semen volume.

The volume of semen may also be increased, a condition known as hyperspermia. A volume greater than 6mL may indicate Prostate inflammation. When there's no volume, the condition is named as aspermia, which could be caused by retrograde ejaculation, anatomical or neurological diseases or anti-hypertensive drugs.

Appearance

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Semen normally has a whitish-gray colour. It tends to get a yellowish tint as a man ages. Semen colour is also influenced by the food we eat: foods that are high in sulfur, such as garlic, may result in a man producing yellow semen.[19] Presence of blood in semen (hematospermia) leads to a brownish or red coloured ejaculate. Hematospermia is a rare condition.

Semen that has a deep yellow colour or is greenish in appearance may be due to medication. Brown semen is mainly a result of infection and inflammation of the prostate gland, urethra, epididymis and seminal vesicles.[citation needed] Other causes of unusual semen colour include sexually transmitted infections such as gonorrhea and chlamydia, genital surgery and injury to the male sex organs.

Fructose level

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Semen Fructose Test

Fructose level in the semen may be analysed to determine the amount of energy available to the semen for moving.[6] WHO specifies a normal level of 13 μmol per sample. Absence of fructose may indicate a problem with the seminal vesicles. The semen fructose test checks for the presence of fructose in the seminal fluid. Fructose is normally present in the semen, as it is secreted by the seminal vesicles. The absence of fructose indicates ejaculatory duct obstruction or other pathology. [5]

pH

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According to one lab test manual normal pH range is 7.2–8.2;[6] WHO criteria specify normal as 7.2–7.8.[5] Acidic ejaculate (lower pH value) may indicate one or both of the seminal vesicles are blocked. A basic ejaculate (higher pH value) may indicate an infection.[5] A pH value outside of the normal range is harmful to sperm and can affect their ability to penetrate the egg.[6] The final pH results from balance between pH values of accessory glands secretions, alkaline seminal vesicular secretion and acidic prostatic secretions.[20]

Liquefaction

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The liquefaction is the process when the gel formed by proteins from the seminal vesicles and the prostate is broken up and the semen becomes more liquid. It normally takes between 30 minutes and 1 hour for the sample to change from a thick gel into a liquid. In the NICE guidelines, a liquefaction time within 60 minutes is regarded as within normal ranges.[21]

Viscosity

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Semen viscosity can be estimated by gently aspirating the sample into a wide-bore plastic disposable pipette, allowing the semen to drop by gravity and observing the length of any thread. High viscosity can interfere with determination of sperm motility, sperm concentration and other analysis.[11]

MOT

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MOT is a measure of how many million sperm cells per ml are highly motile, that is, approximately of grade a (>25 micrometer per 5 sek. at room temperature) and grade b (>25 micrometer per 25 sek. at room temperature). Thus, it is a combination of sperm count and motility.

With a straw or a vial volume of 0.5 milliliter, the general guideline is that, for intracervical insemination (ICI), straws or vials making a total of 20 million motile spermatozoa in total is recommended. This is equal to 8 straws or vials 0.5 mL with MOT5, or 2 straws or vials of MOT20. For intrauterine insemination (IUI), 1–2 MOT5 straws or vials is regarded sufficient. In WHO terms, it is thus recommended to use approximately 20 million grade a+b sperm in ICI, and 2 million grade a+b in IUI.

DNA damage

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DNA damage in sperm cells that is related to infertility can be probed by analysis of DNA susceptibility to denaturation in response to heat or acid treatment [22] and/or by detection of DNA fragmentation revealed by the presence of double-strand breaks detected by the TUNEL assay.[23][24] Other techniques performed in order to measure the DNA fragmentation are: SCD (sperm chromatin dispersion test), ISNT (in situ nick translation), SCSA (sperm chromatin structural assay) and comet assay.

Total motile spermatozoa

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Total motile spermatozoa (TMS)[25] or total motile sperm count (TMSC)[26] is a combination of sperm count, motility and volume, measuring how many million sperm cells in an entire ejaculate are motile.

Use of approximately 20 million sperm of motility grade c or d in ICI, and 5 million ones in IUI may be an approximate recommendation.

Others

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The sample may also be tested for white blood cells. A high level of white blood cells in semen is called leucospermia and may indicate an infection.[5] Cutoffs may vary, but an example cutoff is over 1 million white blood cells per milliliter of semen.[5]

Abnormalities

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Factors that influence results

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Apart from the semen quality itself, there are various methodological factors that may influence the results, giving rise to inter-method variation.

Compared to samples obtained from masturbation, semen samples from collection condoms have higher total sperm counts, sperm motility, and percentage of sperm with normal morphology.[citation needed] For this reason, they are believed to give more accurate results when used for semen analysis.

If the results from a man's first sample are subfertile, they must be verified with at least two more analyses. At least two to four weeks must be allowed between each analysis.[27][medical citation needed] Results for a single man may have a large amount of natural variation over time, meaning a single sample may not be representative of a man's average semen characteristics.[medical citation needed] In addition, sperm physiologist Joanna Ellington believes that the stress of producing an ejaculate sample for examination, often in an unfamiliar setting and without any lubrication (most lubricants are somewhat harmful to sperm), may explain why men's first samples often show poor results while later samples show normal results.[medical citation needed]

A man may prefer to produce his sample at home rather than at the clinic. The site of semen collection does not affect the results of a semen analysis.[28] If produced at home the sample should be kept as close to body temperature as possible as exposure to cold or warm conditions can affect sperm motility.

Measurement methods

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Pre-weighted container for semen analysis

Volume can be determined by measuring the weight of the sample container, knowing the mass of the empty container.[29] Sperm count and morphology can be calculated by microscopy. Sperm count can also be estimated by kits that measure the amount of a sperm-associated protein, and are suitable for home use.[30][unreliable medical source?]

Computer assisted semen analysis (CASA) is a catch-all phrase for automatic or semi-automatic semen analysis techniques. Most systems are based on image analysis, but alternative methods exist such as tracking cell movement on a digitizing tablet.[31][32] Computer-assisted techniques are most-often used for the assessment of sperm concentration and mobility characteristics, such as velocity and linear velocity. Nowadays, there are CASA systems, based on image analysis and using new techniques, with near perfect results, and doing full analysis in a few seconds. With some techniques, sperm concentration and motility measurements are at least as reliable as current manual methods.[33]

Raman spectroscopy has made progress in its ability to perform characterization, identification and localization of sperm nuclear DNA damage.[34]

Semen Fructose Test has made progress in its ability to perform characterization, identification and localization of sperm nuclear DNA damage.[34]

See also

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References

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Further reading

[edit]
[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Semen analysis

View on Grokipedia
from Grokipedia
Semen analysis is a examination of human that assesses various physical and microscopic parameters to evaluate reproductive health, particularly potential. It serves as a primary diagnostic tool in clinical settings for investigating and in research to monitor or the effects of interventions on . The test provides insights into production, function, and overall composition, helping to identify potential causes of male-factor , which contributes to approximately 50% of cases in couples. The procedure for semen analysis follows standardized protocols outlined by the (WHO) to ensure reliability and comparability across laboratories. A semen sample is typically collected through into a sterile container after 2 to 7 days of ejaculatory , either at a or at with prompt delivery to the laboratory to maintain sample viability. The sample undergoes initial macroscopic evaluation for volume, appearance, viscosity, and time (which should occur within 60 minutes), followed by microscopic analysis using or computer-assisted systems to assess concentration, , morphology, and . 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 . Key parameters evaluated in semen analysis include ejaculate volume, sperm concentration, total sperm number, , and morphology, with reference values derived from the 5th percentile of fertile adult men as per the WHO's 6th edition laboratory manual (2021). 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). Semen volume and sperm concentration are generally treated as independent parameters in standard semen analysis, exhibiting weak or no significant correlation. Some studies indicate a weak negative correlation, where higher semen volume is associated with lower sperm concentration due to dilution effects, while others show no significant relationship or weak positive associations. Consequently, total sperm count (concentration × volume) is considered more relevant for fertility assessment. 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%). Additional assessments cover (≥7.2), vitality (>54%; 95% CI: 50–56%), and count (<1 million/mL to rule out infection). These reference values for adult men 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.

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. 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. Semen analysis also aids in screening for underlying genetic or endocrine disorders that impair spermatogenesis, such as , which often presents with azoospermia or severe oligospermia due to an extra X chromosome, and , characterized by low testosterone levels affecting sperm production. Abnormal results may prompt further genetic testing or hormonal evaluations. 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. 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. These evaluations contribute to broader fertility outcomes by identifying treatable causes of infertility.

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. 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. 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. 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. 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. 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. 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. 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. Similarly, non-obstructive azoospermia often necessitates karyotyping or genetic screening to identify treatable etiologies before proceeding to advanced therapies. 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. These findings emphasize how semen analysis not only diagnoses infertility but also prognosticates assisted reproductive technology efficacy, enabling personalized treatment pathways.

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. 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). Home sperm test kits provide an alternative for preliminary self-assessment of basic semen parameters without the need for immediate laboratory delivery. These over-the-counter or smartphone-attached devices typically evaluate sperm concentration, indicating whether it is normal or low based on thresholds such as 6 million motile sperm per mL, and some also assess motility by observing sperm movement. Studies show variable accuracy for these basic measures, ranging from 70-98% depending on the product and user operation, but they cannot assess advanced parameters like sperm morphology, vitality, volume, pH, or full WHO reference values such as ≥16 million sperm per mL. Specifically, home sperm vitality testing with a magnifier provides only rough preliminary observations; it cannot accurately assess concentration, morphology, or DNA integrity and is prone to false positives or negatives from sample handling or inexperience. Additionally, results decline over time due to sample degradation, with sperm motility decreasing by approximately 5-10% per hour after ejaculation. While useful for initial insights into fertility potential, home kits have limitations in scope and precision and are not substitutes for comprehensive laboratory semen analysis. A period of sexual abstinence of 2–7 days prior to collection is recommended to optimize semen volume and sperm concentration, as this range allows sufficient time for the seminal vesicles and prostate gland—which produce most of the seminal fluid and have limited storage capacity—to replenish their reserves, requiring several hours to days for full restoration after ejaculation. Consecutive ejaculations in short intervals deplete these reserves faster than they can be replenished, leading to significantly reduced semen volume. An ideal range of 2–5 days balances these parameters without compromising motility or increasing sperm DNA damage. 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. 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. 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. 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. 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. 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. 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. Samples collected by any method require prompt handling to preserve viability, as detailed in subsequent protocols.

Handling, storage, and transport

Following collection, semen samples must be handled promptly to preserve sperm quality and prevent artifacts in analysis. The 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. 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. 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.

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. 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. 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. Persistent changes in semen appearance, or those accompanied by symptoms such as pain, difficulty conceiving, or unusual smell, should be evaluated by a doctor, who may recommend semen analysis for further insights. 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. Rapid liquefaction (very quick or immediate) is not considered abnormal; clinical concerns primarily focus on delayed or incomplete liquefaction exceeding 60 minutes, which may indicate prostate issues, infections, or enzyme deficiencies. Delayed or incomplete liquefaction (>60 minutes) is evaluated for clots and may result from elevated seminal vesicle proteins or dysfunction, potentially interfering with subsequent assessments; in such cases, mechanical dispersion or enzymatic treatment may be needed. Viscosity is evaluated post-liquefaction by allowing to drop from a or glass rod; normal results in discrete drops forming without long threads (<2 cm), facilitating accurate pipetting. High , characterized by sticky homogeneity or threads >2 cm, can impair evaluation and concentration measurements, often linked to seminal vesicle or issues, and may require dilution for analysis. pH is measured on the liquefied sample (30–60 minutes post-ejaculation) using strips (range 6–10) or a , 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.
ParameterNormal RangeMeasurement MethodCommon 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
AppearanceGrey-opalescentVisual inspectionYellow: jaundice; Red-brown: hematospermia
LiquefactionComplete in ≤60 minObservation at room temp/37°CDelayed: prostate/seminal vesicle dysfunction
ViscosityDiscrete drops; <2 cm threadsDrop test from pipette/rodHigh: impairs analysis; seminal vesicle issues
pH≥7.2pH strips or meterLow: prostate dysfunction, infection
This table summarizes the macroscopic parameters for quick reference, based on WHO guidelines.

Microscopic sperm parameters

Microscopic evaluation of semen focuses on the sperm cells themselves, assessing their quantity, movement, structure, and viability using light microscopy techniques. This analysis provides critical insights into sperm production and function, which are essential for fertility assessment. Parameters are typically examined after liquefaction of the semen sample, with observations made under high magnification (e.g., 400x for motility and concentration) using phase-contrast or bright-field optics. Sperm concentration, also known as sperm count per milliliter, measures the number of spermatozoa in the ejaculate and is determined by counting sperm in a diluted sample using an improved Neubauer hemocytometer chamber under phase-contrast microscopy. At least 200 sperm are counted across multiple fields to ensure accuracy, with the result expressed in millions per milliliter. The lower reference limit for concentration is 16 million sperm per mL, based on the 5th percentile from a multinational study of fertile men. Typical concentrations in fertile men range from 15 to 200 million per mL, with averages around 60-80 million per mL. The total sperm number represents the overall count of spermatozoa in the entire ejaculate and is calculated by multiplying the sperm concentration by the semen volume. This parameter accounts for variations in ejaculate size and provides a comprehensive measure of spermatogenic output. Semen volume and sperm concentration generally exhibit weak or no significant correlation. Some studies indicate a weak negative correlation due to dilution effects in higher semen volumes, while others report no significant relationship or weak positive associations. Consequently, they are treated as independent parameters in standard semen analysis, with total sperm count being the more relevant metric for assessing spermatogenic output and fertility potential. The lower reference limit is 39 million sperm per ejaculate, derived from the same fertile population data as concentration values. Typical total sperm counts range from several hundred million to over a billion spermatozoa per ejaculate, and higher counts within normal ranges may aid fertility. Sperm motility evaluates the percentage of sperm exhibiting movement and is classified into categories: rapid progressive (forward movement ≥25 μm/s), slow progressive (5–25 μm/s), non-progressive (movement in place <5 μm/s), and immotile. Assessment involves observing at least 200 spermatozoa in multiple microscopic fields using phase-contrast optics at 37°C to mimic physiological conditions, with total motility as the sum of progressive and non-progressive categories. The lower reference limits are 30% (95% CI: 29–31%) for progressive motility and 42% (95% CI: 40–43%) for total motility. Sperm morphology assesses the proportion of sperm with normal structure, focusing on head, midpiece, and tail features using strict criteria that define normal forms as those suitable for fertilization. Air-dried smears are stained (typically with ) and examined at 1000x magnification, with at least 200 sperm evaluated per sample. The lower reference limit for normal forms is 4% (95% CI: 3.9–4%), emphasizing that even minor defects can impair fertility. This strict evaluation, originally developed by Kruger et al., prioritizes head shape and acrosome integrity. Sperm vitality determines the percentage of live sperm among immotile ones, distinguishing necrotic from viable but non-motile cells. This is typically assessed by supravital staining with eosin-nigrosin, where live sperm exclude the dye (appearing white) and dead sperm take it up (appearing pink) under bright-field microscopy, evaluating at least 200 sperm. While laboratory methods use staining techniques for accurate vitality evaluation, home-based attempts using simple magnifiers provide only rough preliminary observations and are not recommended for precise diagnosis, as detailed in the specimen collection methods section. The lower reference limit is 54% live sperm (95% CI: 50–56%).

Biochemical and advanced parameters

Biochemical parameters in semen analysis evaluate the chemical composition and functional integrity of seminal plasma and spermatozoa, providing insights into accessory gland function and sperm quality beyond routine microscopic assessments. These tests, often performed in specialized laboratories, include measurements of key metabolites and advanced assays for sperm DNA integrity, capacitation, immunological factors, and ultrastructure. Such evaluations are crucial for diagnosing specific causes of male infertility, such as glandular dysfunction or genetic damage, and guiding assisted reproductive technologies (ART) like intracytoplasmic sperm injection (ICSI). Fructose, primarily secreted by the seminal vesicles, serves as an energy substrate for spermatozoa and is a marker of seminal vesicle contribution to ejaculate volume. Normal levels are defined as ≥13 µmol per ejaculate, assessed through enzymatic spectrophotometric methods involving deproteinization and reagents like indole or resorcinol for colorimetric detection at 470 nm. Low fructose concentrations indicate potential seminal vesicle dysfunction, ejaculatory duct obstruction, or conditions like congenital bilateral absence of the vas deferens and partial retrograde ejaculation. White blood cells (leukocytes) in semen are quantified to detect inflammation or infection, with levels exceeding 1 × 10⁶ per mL signifying leukospermia. Detection typically employs peroxidase staining with ortho-toluidine blue on a semen smear examined at ×1000 magnification, or advanced techniques like flow cytometry with CD45 antibodies for confirmation. Elevated counts are associated with genital tract infections, which can impair sperm function and contribute to infertility, though distinguishing leukocytes from immature germ cells requires careful validation. Sperm DNA fragmentation assesses nuclear integrity, a critical factor in fertilization and embryo viability, using assays such as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or sperm chromatin structure assay (SCSA). TUNEL detects single- and double-strand breaks via fluorescent nucleotide incorporation, visualized by flow cytometry or microscopy, while SCSA measures chromatin susceptibility to denaturation with acridine orange staining. A fragmentation index below 30% is considered normal; levels above this threshold correlate with a 20-30% reduction in natural conception rates and poorer outcomes in ART, including increased miscarriage risk. High fragmentation is linked to idiopathic male infertility and recurrent pregnancy loss. The hyaluronan binding assay (HBA) evaluates sperm capacitation and maturity by measuring the proportion of motile spermatozoa that bind to hyaluronic acid, a marker of plasma membrane stability and acrosome reaction readiness. Performed using a commercial slide-based kit where binding is quantified under phase-contrast microscopy, normal HBA scores exceed 80%, indicating sperm suitable for IVF or ICSI selection. Low binding (<60-80%) predicts reduced fertilization rates in ART and is associated with higher DNA damage, making HBA useful for identifying optimal sperm in cases of teratozoospermia. Antisperm antibodies (ASAs) are detected to identify immunological infertility, where immune responses against sperm antigens impair motility and fertilization. The mixed antiglobulin reaction (MAR) test, recommended by WHO, involves incubating motile sperm with latex beads coated in anti-human immunoglobulins; binding >50% of spermatozoa indicates significant immunological interference. Positive ASA results, occurring in 2.6-6.6% of infertile men, correlate with reduced IVF success and are often due to blood-testis barrier breaches from trauma, infection, or . Motile morphology examination (MSOME) provides high-magnification (×6600) assessment of head ultrastructure, particularly nuclear vacuoles, to select candidates for ICSI in severe male factor infertility. Using differential interference contrast optics, MSOME classifies as normal if free of large vacuoles (>50% of head area); abnormal forms show multiple or anterior vacuoles indicative of defects. This technique, foundational to intracytoplasmic morphologically selected injection (IMSI), improves development and rates in teratozoospermic cases by avoiding with genetic abnormalities.

Interpretation and Abnormalities

Reference ranges and normal values

The World Health Organization (WHO) establishes lower reference limits for semen parameters based on the 5th percentile values derived from large-scale studies of fertile adult men whose partners achieved pregnancy within 12 months, leading to live birth. These limits serve as standardized benchmarks to identify potential deviations in semen quality, though they are not diagnostic thresholds for infertility. The 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen (2021) provides the following key lower reference limits, reflecting data from over 3,500 men across multiple countries: semen volume of 1.4 mL (95% confidence interval: 1.3–1.5 mL), sperm concentration of 16 million per mL (95% CI: 15–18 million/mL), total sperm count of 39 million per ejaculate (95% CI: 35–40 million), total motility of 42% (95% CI: 40–43%), progressive motility of 30% (95% CI: 29–31%), and normal morphology of 4% (95% CI: 3.9–4.0%). As of 2025, these values from the 2021 (6th) edition remain the standard, with a 7th edition in development. There are no official, standardized reference ranges for semen analysis specifically for adolescents during puberty, unlike the well-established WHO reference values for adult males. Semen parameters in adolescents vary significantly depending on the stage of puberty (Tanner stages), with values generally lower than in adults during early and mid-puberty and approaching adult levels in late puberty or late adolescence. In early puberty (Tanner stages 2-3), many adolescents have low or absent sperm (azoospermia or severe oligospermia is common before spermarche, which typically occurs during Tanner stage 4 around ages 13-15). By late puberty (Tanner stage 5, usually ages 15-18+), sperm concentration, motility, and morphology often reach levels comparable to adults, though some studies show slightly lower means in adolescents. Adolescent values can be below these adult lower limits even in normal development, so adult norms should be applied cautiously in pubertal boys, and interpretation requires consideration of pubertal stage, age, and clinical context. These lower limits represent the 5th percentile, while typical values in fertile men are substantially higher. According to WHO reference ranges, sperm concentration typically ranges from 15 to 259 million per mL, and total sperm count from 39 to 928 million per ejaculate, with averages around 60 million per mL for concentration and 209 million for total count based on studies of fertile populations. In cases of higher volume and concentration, total sperm counts can reach several hundred million to over a billion per ejaculation. These reference ranges exhibit variability influenced by factors such as age and . Semen parameters generally decline with advancing age, alongside reductions in and morphology. Ethnic differences also contribute to variability, with studies showing lower average concentrations and in some populations, such as Black men compared to Caucasian or Asian men, though WHO limits apply universally without ethnicity-specific adjustments. The total motile sperm count (TMSC), calculated as TMSC = × concentration × (% total / 100), integrates multiple parameters and is a useful prognostic indicator; a TMSC exceeding 20 million is considered within the normal range for fertility potential. Compared to the 5th edition (2010), the 2021 update incorporated an expanded dataset from 1,953 men in the original cohort plus additional contributions, resulting in minor adjustments to thresholds, such as a slight increase in sperm concentration from 15 to 16 million/mL and total from 40% to 42%, to better reflect contemporary fertile populations. In clinical practice, semen parameters below these lower limits suggest a potential male factor contributing to and warrant further investigation, but normal values do not guarantee due to multifactorial influences on .

Common abnormalities and their implications

refers to the complete absence of spermatozoa in the ejaculate after of at least two separate samples. It affects approximately 1% of all men and 10-15% of those evaluated for . is classified into obstructive and non-obstructive types based on volume and hormone levels; obstructive typically presents with normal ejaculate volume and normal (FSH) levels, indicating a blockage in the reproductive tract despite intact , while non-obstructive often shows normal or low ejaculate volume with elevated FSH levels, reflecting primary testicular failure or severe spermatogenic impairment. Oligoasthenoteratospermia (OAT), also known as oligoastheno-teratozoospermia, is a combined abnormality characterized by low sperm concentration (oligozoospermia), reduced (asthenozoospermia), and a high percentage of morphologically abnormal sperm (teratozoospermia). It represents one of the most frequent semen analysis findings in men with , often linked to genetic factors such as microdeletions in the azoospermia factor (AZF) regions of the , which disrupt and contribute to the severity of the defects. Severity of OAT can be assessed using prognostic systems like the Tygerberg ( strict) criteria for sperm morphology, which categorize outcomes as good (>14% normal forms), fair (4-14%), or poor (<4%), helping to predict potential and guide assisted reproduction decisions. Aspermia is the total absence of ejaculate, distinct from as it involves no production or emission rather than just absent . It arises from conditions preventing semen release, such as into the or congenital aplasia of reproductive structures, and precludes natural conception while requiring alternative sperm retrieval methods for treatments. Abnormalities in semen liquefaction primarily concern delayed liquefaction, where complete liquefaction does not occur within 60 minutes after ejaculation. This may indicate accessory gland dysfunction, including prostate infections, seminal vesicle dysfunction, or deficiencies in liquefying enzymes. In contrast, rapid liquefaction is not considered pathological and is rarely of clinical significance. These abnormalities significantly impair outcomes; for instance, precludes natural conception, while moderate to severe oligozoospermia, asthenospermia, or OAT substantially reduce the probability of natural conception. Conversely, higher sperm counts within the normal range can potentially improve fertility outcomes by increasing the chances of successful fertilization. They inform clinical management by directing towards assisted reproductive technologies like intrauterine insemination (IUI) for mild cases or fertilization (IVF) with (ICSI) for severe defects, where success rates vary based on the extent of impairment. Additionally, associated sperm DNA damage in abnormal samples doubles the risk of in pregnancies achieved via assisted reproduction.

Influencing Factors and Limitations

Physiological and lifestyle factors

Semen quality peaks during the reproductive years, typically between 25 and 35 years of age, when parameters such as count and are at their highest. After the age of 40, there is a notable decline in these parameters, with concentration and total motile decreasing significantly, alongside reductions in progressive . This age-related deterioration is attributed to gradual testicular function impairment and increased in aging germ cells. Hormonal factors profoundly influence semen parameters, with testosterone and (FSH) essential for maintaining . Low testosterone levels, as seen in , disrupt development and lead to significantly reduced counts, often resulting in or . In cases of , success rates can be as low as 68-75%, with mean concentrations around 3-5 million/mL post-treatment, highlighting the severe impact of hormonal imbalance. The frequency of ejaculation directly affects semen composition, as prolonged abstinence greater than 7 days increases sperm count due to accumulation in the epididymis but simultaneously decreases motility owing to aging of stored spermatozoa. Conversely, shorter abstinence periods of 2-3 days optimize overall by enhancing motility while maintaining adequate sperm numbers, aligning with recommendations for balanced parameters in fertility assessments. Abstinence duration remains the strongest predictor of semen volume, with longer periods leading to significant increases, while other factors exert less pronounced effects. Importantly, semen volume does not increase with consecutive or multiple ejaculations in a short time frame; it typically decreases significantly with each subsequent ejaculation. The seminal vesicles and prostate gland, which produce most of the seminal fluid, have limited storage capacity and require several hours to days to fully replenish after ejaculation. Frequent or consecutive ejaculations deplete these reserves faster than they can be restored, resulting in lower volume per ejaculation. Longer abstinence periods (typically 2–7 days) are associated with higher semen volume, which is why fertility tests recommend this range. Claims suggesting increased volume with consecutive ejaculations are not supported by scientific evidence and may stem from misconceptions or anecdotal reports. Lifestyle habits, including , adversely impact semen parameters through nicotine's toxic effects on function. smoking is associated with a reduction in by 3-17%, alongside lower total counts, as evidenced by meta-analyses of thousands of men. Similarly, excessive alcohol intake, defined as more than 14 units per week, correlates with decreased normal morphology, contributing to overall subfertility by altering testicular and hormone levels. Lifestyle factors such as smoking and alcohol indirectly affect semen parameters overall, including potential reductions in semen volume, but their specific impact on volume is less pronounced than that of abstinence duration. Nutritional factors play a key role in supporting , with —often occurring at intakes below 15 mg/day—impairing maturation through reduced testosterone synthesis and heightened oxidative damage in the testes. In contrast, adequate omega-3 fatty acid intake improves by 7-8%, enhancing and reducing in seminal plasma. , characterized by a BMI greater than 30, is linked to lower counts, with reductions of 10-20% observed in affected men compared to those with normal , primarily due to adipose tissue-mediated conversion of testosterone to , which disrupts hormonal balance. This physiological shift not only lowers androgen levels but also promotes , further compromising .

Environmental and methodological factors

Environmental toxins, such as pesticides, have been linked to adverse effects on . pesticides, commonly used in , are associated with reduced count, concentration, total and progressive , and normal morphology in exposed individuals. Studies indicate that occupational exposure to these compounds can lead to significant declines in semen parameters, with inverse associations between metabolite levels and morphology observed in population-based . Air pollution, including exposure to particulate matter and other ambient pollutants, is also associated with decreased semen volume and other parameters. Heat exposure represents another environmental factor impairing . Scrotal from use induces reversible alterations in production, including decreased count and motility, persisting for up to three months post-exposure. This effect is mediated by elevated testicular temperatures that trigger germ cell and disrupt DNA integrity. Factors like temperature and humidity indirectly affect semen parameters overall, but their impact on volume specifically is less pronounced than abstinence duration, which remains the strongest predictor. Certain medications can also influence semen analysis outcomes. Chemotherapy regimens for cancer often cause due to gonadotoxic effects, affecting approximately 50% of treated men, though recovery of occurs in many cases following treatment cessation. Antihypertensive drugs, particularly beta-blockers like and atenolol, are associated with lower , reduced volume, and decreased concentration. Methodological variations in sample collection and handling further impact results. The duration of ejaculatory significantly affects semen volume, with extensions beyond the recommended 2-7 days leading to increases of up to 10% per additional day, potentially altering overall parameter assessments by substantial margins. temperature deviations during analysis can impair viability and ; for instance, incubation at 37°C accelerates loss of progressive movement compared to cooler conditions around 22-27°C. Seasonal environmental changes, particularly in temperate climates, contribute to fluctuations in . Summer heat exposure has been correlated with deteriorations in parameters, including lower and concentration, attributed to impaired during . Research in regions with marked seasonal temperature shifts reports reduced overall during warmer months, potentially by 15% or more in affected cohorts. Inter-observer variability introduces additional methodological challenges in semen evaluation. Assessments of can differ by 10-20% between technicians due to subjective manual counting, highlighting the need for standardized protocols. The World Health Organization's laboratory manual addresses this by providing evidence-based guidelines for consistent examination procedures, including measures to minimize discrepancies across laboratories.

Laboratory Techniques

Sample processing

Upon receipt in the laboratory, the semen sample undergoes initial processing to ensure its suitability for accurate evaluation of sperm parameters. This involves controlled conditions to promote , homogenization, and preliminary assessments, typically performed within 60 minutes of collection to minimize artifacts. is facilitated by incubating the sample at 37°C for 30–60 minutes, allowing the seminal plasma to transition from a gel-like state to a liquid form essential for subsequent analyses. Investigations may begin after 30 minutes if complete has occurred, but if incomplete, incubation extends to 60 minutes; any delays or failures should be noted in the report. In cases of high , gentle mechanical aids such as vortexing or an orbital mixer for 15–30 seconds can be employed to reduce without damaging spermatozoa. Following liquefaction, homogenization ensures uniform distribution of spermatozoa throughout the sample. This is achieved through gentle mixing techniques, including aspiration with a wide-bore (approximately 1.5 mm ) about 10 times, manual swirling for 15–30 seconds, inverting the container 3–5 times, or using a two-dimensional shaker or rotating wheel. Excessive agitation, such as vigorous vortexing, must be avoided to prevent sperm damage or introduction of air bubbles. Initial quality checks include measuring the ejaculate , typically by weighing the sample (assuming a of 1 g/ml), using a or graduated tube, while accounting for potential losses of 0.3–0.9 ml during transfer. assesses macroscopic features such as color, , clumping, , debris, or contaminants to identify any issues that could affect reliability. For advanced evaluations, the sample may undergo optional to concentrate motile spermatozoa, using techniques like swim-up or gradient with recovery rates of 50–80%. is performed at 300–500 g for 5–20 minutes, or specific protocols such as 3000 g for 15 minutes at (20–25°C), isolating the pellet for further use. Preparation for staining involves creating smears for morphology or vitality assessments. For sperm morphology, slides are fixed using agents like 95% for at least 15–30 minutes or a mixture of acetone// for 90 seconds, then air-dried before applying . Vitality staining employs eosin-nigrosin, where 50 µl of well-mixed is combined with an equal volume of stain, allowed to stand for 30 seconds, and air-dried on a slide. Other fixatives, such as 3.7–4% for 30 minutes at , may preserve cellular structures as needed.

Measurement and analysis methods

Semen analysis employs a variety of standardized techniques to quantify key parameters such as concentration, , morphology, and biochemical components, ensuring reproducibility across laboratories. The (WHO) laboratory manual outlines these methods, emphasizing equipment calibration, quality control, and procedural precision to minimize variability. Microscopic examination remains the cornerstone for assessing concentration and . For concentration, semen is diluted (typically 1:20) with a fixative such as formalin-sodium solution and loaded into an improved Neubauer chamber (depth 0.1 mm). Under at 200× or 400× magnification, spermatozoa are counted in designated areas of the grid, typically 5 large squares (each 1 mm²) in the central field for standard counts or the 25 smaller squares (0.04 mm² each) within the central large square for lower concentrations, aiming for at least 200-400 cells across replicates to achieve a below 5%. The concentration in millions per mL is calculated as (number of spermatozoa counted × dilution factor) / volume counted in mm³, where the volume per large square is 0.1 µL. is evaluated by placing a 5 µL aliquot on a pre-warmed (37°C) glass slide with a 20 µm depth coverslip and observing at least 200 spermatozoa across five random fields using at 200× magnification; categories include rapid progressive (velocity ≥25 µm/s), slow progressive (5-25 µm/s), non-progressive (<5 µm/s), and immotile. Computer-assisted semen analysis (CASA) systems automate and assessments, enhancing objectivity over manual methods. These systems capture video sequences of diluted in standardized chambers at 37°C, tracking individual spermatozoa trajectories with software algorithms. Key metrics include straight-line (VSL), curvilinear (VCL), and path (VAP), all in µm/s, with linearity (VSL/VCL × 100) indicating path straightness; CASA achieves over 95% agreement with manual counts when calibrated properly. For concentration, serves as an optional rapid screening tool, measuring optical density of diluted at 540 nm against a derived from standards, linear up to 200 million/mL. Advanced techniques address DNA integrity and biochemical markers. using the sperm chromatin structure assay (SCSA) quantifies DNA fragmentation by staining fixed spermatozoa with , which fluoresces green for intact double-stranded DNA and red for fragmented single-stranded regions; a flow cytometer analyzes 5,000 events per sample, calculating the DNA fragmentation index (%DFI) as the ratio of red to total (red + green) fluorescence intensity. Biochemical assays for seminal employ enzymatic kits where diluted seminal plasma (1:40) is incubated with and , producing NADPH measurable by UV absorbance at 340 nm against a standard curve to yield concentrations in mmol/L. Antisperm antibodies are detected via the mixed antiglobulin reaction (MAR) test, mixing motile spermatozoa with IgG- or IgA-coated latex beads and scoring binding under ; a threshold of ≥50% bound spermatozoa among 200 assessed indicates clinical . Recent advancements integrate (AI) into morphology assessment, traditionally subjective under . classifiers applied to high-resolution images of stained spermatozoa achieve over 90% accuracy in categorizing normal versus abnormal forms, surpassing manual variability; for instance, convolutional neural networks have reported up to 95% accuracy in human samples since 2020. These AI-enhanced systems, often embedded in CASA platforms, reduce analysis time while improving reliability for parameters like head and tail defects.

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