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Human sperm stained for semen quality testing in the clinical laboratory.
A semen analysis (plural: semen analyses), also called seminogram, or spermiogram evaluates certain characteristics of a male's semen and the sperm contained therein. 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.
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 7 days to 3 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 on 2010.
Reasons for testing
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. 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
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. Conversely, men with poor semen analysis results may go on to father children. In NICE guidelines, mild male factor infertility is defined as when 2 or more semen analyses have 1 or more variables below the 5th percentile, and confers a chance of pregnancy occurring naturally through vaginal intercourse within 2 years similar to people with mild endometriosis.
Different methods used for semen collection are masturbation, condom collection and epididymal extraction, etc. The sample should never be obtained through coitus interruptus for several reasons: Some part of ejaculation could be lost, bacterial contamination could happen and the acid vaginal pH could be detrimental for sperm motility. The optimal sexual abstinence for semen sample obtaining is from 2–7 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 in a sterile plastic recipient (never in a conventional preservative since they have chemical substances as lubricants or spermicides that could damage the sample) and be handed in the clinic for it to be studied within the following hour.
Examples of parameters measured in a semen analysis are: sperm count, motility, morphology, volume, fructose level and pH.
Sperm count, or sperm concentration to avoid confusion with total sperm count, measures the concentration of sperm in a man's ejaculate, distinguished from total sperm count, which is the sperm count multiplied with volume. Over 15 million sperm per milliliter is considered normal, according to the WHO in 2010. Older definitions state 20 million. A lower sperm count is considered oligozoospermia. A vasectomy is considered successful if the sample is azoospermic (zero sperm of any kind found). Some define success as when rare/occasional non-motile sperm are observed (fewer than 100,000 per millilitre). 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.[unreliable medical source?]
The World Health Organization has a value of 50% 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. A man can have a total number of sperm far over the limit of 20 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 motility of sperm are divided into four different grades:
- Grade a: Sperm with progressive motility. These are the strongest and swim fast in a straight line. Sometimes it is also denoted motility IV.
- Grade b: (non-linear motility): These also move forward but tend to travel in a curved or crooked motion. Sometimes also denoted motility III.
- Grade c: These have non-progressive motility because they do not move forward despite the fact that they move their tails. Sometimes also denoted motility II.
- Grade d: These are immotile and fail to move at all. Sometimes also denoted motility I.
Regarding sperm morphology, the WHO criteria as described in 2010 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.
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.
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). 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.
According to one lab test manual semen volumes between 2.0 mL and 5 mL are normal; WHO regards 1.5 ml as the lower reference limit. Low volume may indicate partial or complete blockage of the seminal vesicles, or that the man was born without seminal vesicles. In clinical practice, a volume of less than 2 mL in the setting of infertility and absent sperm should prompt an evaluation for obstructive azoospermia. A caveat to this is be sure 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 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 with the human immunodeficiency virus (HIV) produce lower semen volume.
Semen normally has a whitish-gray color. It tends to get a yellowish tint as a man ages. Semen color 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. Presence of blood in semen (hematospermia) leads to a brownish or red colored ejaculate. Hematospermia is a rare condition.
Semen that has a deep yellow color 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. Other causes of unusual semen color include sexually transmitted infections such as gonorrhea and chlamydia, genital surgery and injury to the male sex organs.
Fructose level in the semen may be analysed to determine the amount of energy available to the semen for moving. WHO specifies a normal level of 13 μmol per sample. Absence of fructose may indicate a problem with the seminal vesicles.
According to one lab test manual normal pH range is 7.1-8.0; WHO criteria specify normal as 7.2-7.8. 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. A pH value outside of the normal range is harmful to sperm and can affect their ability to penetrate the egg.
The liquefaction is the process when the gel formed by proteins from the seminal vesicles is broken up and the semen becomes more liquid. It normally takes less than 20 minutes 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.
After liquefaction, the viscosity of the sample can be estimated by gently aspirating into a wide-bore (approximately 1,5 mm of diameter) plastic disposable pipette, allowing the semen to drop by gravity and observing the length of any thread. A normal sample leaves the pipette in small discrete drops. If viscosity is abnormal, the drop will form a thread more than 2 cm long. High viscosity can interfere with determination of sperm motility, sperm concentration, detection of antibody-coated spermatozoa and measurement of biochemical markers.
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 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  and/or by detection of DNA fragmentation revealed by the presence of double-strand breaks detected by the TUNEL assay.
Total motile spermatozoa
Total motile spermatozoa (TMS) or total motile sperm count (TMSC) 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.
The NICE guidelines also include testing vitality, with normal ranges defined as more than 75% of sperm cells alive.
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. Cutoffs may vary, but an example cutoff is over 1 million white blood cells per milliliter of semen.
- Aspermia: absence of semen
- Azoospermia: absence of sperm
- Hypospermia: low semen volume
- Hyperspermia: high semen volume
- Oligozoospermia: Very low sperm count
- Asthenozoospermia: poor sperm motility
- Teratozoospermia: sperm carry more morphological defects than usual
- Necrozoospermia: all sperm in the ejaculate are dead
- Leucospermia: a high level of white blood cells in semens
Factors that influence results
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. 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 2 to 4 weeks must be allowed between each analysis.[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.. If produced at home the sample should be kept as close to body temperature as possible as exposure to cold or warm conditions can effect sperm motility
Volume can be determined by measuring the weight of the sample container, knowing the mass of the empty container. 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.[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. 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.
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