Semen analysis is a laboratory test performed to assess the function of the male reproductive system and male fertility, and to help choose the right treatment for couples attending an assisted reproduction clinic.
Collecting and transporting the sample
The sample is collected in a room close to the laboratory, after at least 2 days of abstinence since the last ejaculation; 7 days is the maximum recommended period of abstinence. Ideally, analysis of the sample begins within 30 minutes of collection, and should in any case not exceed 60 minutes. If collection at the clinic is not possible, it is important that during transport the temperature of the sample does not fall below 20 °C or rise above 37 °C.
Macroscopic assessment
The analysis begins with macroscopic observation, which includes measuring the viscosity, assessing the appearance of the sample, measuring the volume (the lower reference limit is 1.4 ml), the pH (lower reference limit 7.2) and finally the time needed for it to liquefy.
Microscopic examination
The sample is examined microscopically under a phase-contrast light microscope after being mixed thoroughly. The sample is first scanned and checked for agglutination, aggregation and for the presence of cells other than sperm. The presence of such cells can indicate damage to the testes (immature germ cells), disease of the efferent ducts (ciliary tufts) or even inflammation (white blood cells). Assessment of concentration, motility and morphology then follows.
Sperm concentration and total sperm number
Sperm concentration refers to the number of sperm per unit volume. Although concentration is related to the fertilizing capacity of the semen and to pregnancy rates, it is affected by secretions from the seminal vesicles and the prostate and so is not indicative of testicular function. The total number of sperm in the ejaculate, by contrast, where there is no obstruction, is proportional to the volume of the testes and is a measure of their capacity to produce sperm.
The lower reference limit for concentration is 16 × 10⁶ sperm per ml, while for total number it is 39 × 10⁶ sperm per ejaculate.
Where no sperm are seen after the sample has been centrifuged, the term azoospermia is used. In that case a testicular biopsy (TESE) may be suggested in order to find sperm that can then be used for intracytoplasmic sperm injection (ICSI).
Motility
Sperm movement is assessed using a system of four categories.
- A) Sperm moving with rapid progressive motion (≥ 25 µm/s).
- This analysis relies on an electronic imaging system working together with an advanced algorithm. These systems have developed rapidly over the past 40 years or so, taking advantage of the increase in available computing power, of new algorithms and of the use of fluorescent DNA stains. Their purpose is to increase the objectivity and accuracy of semen analysis, since they largely remove the human factor from the process. As well as measuring sperm concentration, morphology and motility, these analysers also allow the calculation of kinematic parameters that are absent from the standard semen analysis, such as curvilinear velocity (VCL) and straight-line velocity (VSL). Each analyser is also linked to software, allowing the data to be organized and analysed statistically. Their disadvantages include unreliable measurements for samples with a low (< 2 × 10⁶/ml) or very high concentration (> 50 × 10⁶/ml), and for dirty samples containing many cells that are not sperm. Although more research is needed to demonstrate their benefits in andrology laboratories, these systems are useful for laboratory quality control.
- B) Sperm moving with slow progressive motion (5 to < 25 µm/s).
- D) Immotile sperm.
Motile sperm attached to cells, to cell debris or to immotile sperm on top of one another are recorded as aggregation. Agglutination refers to motile sperm sticking together by their heads, their tails or in a mixed fashion. The presence of agglutination, although not enough on its own for immune factors to be considered a likely cause of infertility, does demonstrate the presence of antisperm antibodies and calls for further tests.
The lower reference limits are 30% for progressive motility (rapid and slow) and 42% for total motility (progressive and non-progressive).
Morphology
A spermatozoon consists of the head, the neck, the midpiece and the tail. These are the parts on which the assessment of morphology focuses. A spermatozoon is considered morphologically abnormal when it deviates from the standard in any of these regions. The lower reference limit for normal forms is 4%. Increased proportions of abnormal forms are associated with disease of the epididymis and with errors during gametogenesis. Abnormal sperm also have lower fertilization rates, and a correlation has been observed between abnormal forms and DNA damage. Where the proportion of normal forms is low, a DNA fragmentation test may therefore be suggested in order to choose the right treatment.
Computer Aided Sperm Analysis (CASA)
C) Sperm moving without progression (< 5 µm/s).
Artificial Intelligence Sperm Analysis (AISA)
Artificial intelligence is defined as the ability of machines to learn and to display intelligence. Introducing this technology into semen analysis promises to automate, optimize and standardize the process completely. The first stage in creating such a system is building a database of semen analyses, photographs and video. These data are used to train the system, which acquires the ability to predict the semen parameters (motility, concentration, morphology, pH) accurately. Such systems have been used to overcome the limitations of CASA, and some studies demonstrate their usefulness as a diagnostic tool in clinics. Finally, efforts are being made to use this technology to select the most suitable spermatozoon for ICSI instead of the clinical embryologist. These efforts are, however, at an early stage.