Semen Analysis


Because the tissue composition varies with fraction, mixing all four fractions during ejaculation is required to create a decent semen specimen.

Composition of semen

  • Spermatozoa:    5%
  • Seminal Fluid:    60% – 70%
  • Prostate fluid: 20% – 30%
  • Bulbourethral glands: 5 %


Normal semen is clear and has a musty odor. Increased white turbidity indicates the presence of white blood cells (WBC) and reproductive tract diseases. If required, specimen culture is conducted prior to the semen testing. Before microscopic inspection, WBCs must be isolated from immature sperm (spermatides). The leukocyte esterase strip reagent check can be used to screen WBCs. RBCs (red blood cells) with varying degrees of redness are uncommon. The yellow coloration is caused by sperm oxidation, the buildup of samples after extended abstinence, and medication.


As fresh sperm becomes coagulated and therefore should be liquefied within 30 to 60 minutes of processing, monitoring the collection time is critical for determining sperm liquefaction. The liquefaction process must be completed before specimen testing may proceed. If the specimen has not liquified after 2 hours, proteolytic enzymes such as alpha-chymotrypsin may be utilized to finish it. Liquefaction issues can be caused by a lack of prostatic enzymes, which should be investigated.


Semen is normally 2 to 5 mL in volume. This can be determined by pouring the material into a calibrated sterile graduated container at 0.1-mL intervals. Following long durations of abstinence, there is a decrease in volume. Reduced volume is more commonly associated with infertility, and it may suggest abnormal processing of one of the semen-producing organs, particularly the seminal vesicles. It is also necessary to consider an inadequate specimen collection.


The viscosity of a specimen corresponds to its fluid consistency, which may be related to its liquefaction. Materials which haven’t completely liquefied agglomerate together due to high viscosity. The normal sperm specimen may be easily taken into a pipette and ejected in droplets that are not clumped or stringy. As they are removed from the pipette, regular droplets form a thin chain. This term refers to viscous droplets with strings longer than 2 cm in length. The Viscosity Test has a scale of 0 (watery) to 4 (sticky) (gel-like). Viscosity can also be categorized as minimum, moderate, or solid. Higher viscosity and partial liquefaction restrict sperm motility.


Normal semen pH is alkaline, with a pH range of 7.2 to 8.0. Increased pH in the reproductive tract suggests infection. To test the pH of sperm, add a semen specimen to the pH surface of a urinalysis reagent strip and compare the color to the manufacturer’s chart. pH test paper can also be used.

Sperm Concentration/Count

Even when only one spermatozoon fertilizes, the total sperm count in a sperm specimen is a good fertility predictor. Sperm concentrations of more than 20 million per milliliter are considered normal, with threshold values ranging from 10 to 20 million per milliliter considered borderline. The total eyaculate sperm count is calculated by multiplying the sperm content by the volume of the specimen. It is considered normal to have a total sperm count of more than 40 million per ejaculate (20 million per milliliter, around 2 mL).

Sperm Motility

Although sperm delivered to the cervix can push through the cervical mucosa to the uterus, fallopian tubes, and ovum, the existence of sperm capable with forward movement is vital for fertility. Typically, clinical laboratory evaluation of sperm motility was a retrospective test that included analyzing an undiluted specimen and measuring the fraction of motile sperm as well as the motility rate.

The World Health Organization assigns grades a, b, c, and d. According to the interpretation, 50% or more of the sperm in categories a, b, and c should be mobile within 1 hour, and 25% or more should demonstrate progressive motility (a and b). The presence of a large proportion of immobile sperm and sperm aggregates requires further testing to assess sperm viability or sperm agglutinin concentration.

Sperm Motility Grading

  Grade WHO Criteria
4 A Rapid, Straight-line motility
3 B Slower speed, some lateral movement
2 B Slow forward progression, noticeable lateral movement
1 C No forward progression
0 D No Movement

Sperm Morphology

Infertility is caused by the presence of a significant amount of nonmotile sperm as well as the production of sperm that is morphologically incapable of fertilizing. The head, collar, midpiece, and tail forms of sperm are all evaluated. Poor ovum penetration is associated with defects in head morphology, whereas abnormalities in the back, midpiece, and tail limit motility.

The normal sperm has an oval-shaped head that measures approximately 5 μm length by 3 μm width and a flagellar neck that measures around 45μm in thickness. The acrosomal cap, which is positioned at the tip of the head and contains enzymes, is essential for ovum penetration. The acrosomal shield will protect approximately half of the head and two-thirds of the sperm nuclei. The neckpiece is made up of the head, which is joined to the tail, and the midpiece. The midpiece is the thickest part of the tail because it is surrounded by a mitochondrial sheath that allows the tail to move.

Normal sperm morphology levels vary depending on the technique of measurement, ranging from standard forms more than 30% when using normal criteria to standard forms larger than 14% while using strict standards.

Normal Spermatozoa Structure (Source: Urinalysis and BodyFluids)
Abnormalities of sperm and tails (Source: Urinalysis and BodyFluids)

Other Examinations of Semen


It is the major sugar in sperm, and decreased seminal fructose levels are associated with androgen insufficiency. The sperm count and fructose levels are inversely related. Low fructose production is caused by insufficient testosterone or seminal insufficiency in the vesicles. The resorcinol technique is easy and affordable. According to this concept, the fructose in an acidic solution heated with resorcinol creates a crimson precipitate. The fructose must be transformed to hydroxymethyl furfural before it can condensate into the real resorcinol precipitate.

Antibodies to Spermatozoa

Antibodies can be produced by either a man or a female. In laboratory tests, the significance of spermatozoal antibodies in infertility is well established, but there is still a lot of doubt concerning human spermatozoa. Many clinical correlative investigations have used agglutination tests for sperm in the past.


  1. Concise Book of Medical Laboratory Technology Methods and Interpretations by Ramnik Sood
  2. Urinalysis and Body Fluids 5th Ed by Strasinger SK and Di Lorenzo MS

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