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Semen Analysis Test

Updated: Mar 11

Reasons for infertility are 40% female issues, 40% male issues and 20% unknown.  To determine if infertility is due to the male a semen analysis is a good starting step.  It should involve: physical characteristics of semen (color, odor, pH, viscosity and liquefaction), volume, concentration, morphology and sperm motility and progression.


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 criptozoospermia. 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.


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.


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 (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]


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 volumen, the condition is named as aspermia, which could be caused by retrograde ejaculation, anatomical or neurological diseases or anti-hypertensive drugs.


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. 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

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]


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]


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]


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 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

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

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.


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