Spermiogram analysis (scientific approach)

The  spermiogram  is the analysis of the  seminal fluid  aimed at evaluating the quality of the spermatozoa, through the verification of the form, number and motility. This examination is the main tool for assessing male fertility. A spermiogram is also necessary to verify the successful outcome of a vasectomy. The reliability of the examination depends on its correct execution, which involves:

• complete abstinence before the examination between 3 and 5 days (a shorter period decreases the concentration of the spermatozoa, a longer period decreases the motility);
• suspension of pharmacological therapies (hormones, anti-inflammatory drugs, antibiotics, steroids);
• sample collection by manual masturbation, in good hygiene conditions and in wide-mouth containers;
• sample collection preferably at the laboratory where the analysis is performed; if this is not possible, the sample can be collected at home in a sterile urine container and taken to the laboratory within an hour in a vertical position, taking care to keep it warm, preferably in a pocket near the body.

If for religious reasons masturbation is prohibited, the collection of the liquid can also be performed after a complete sexual intercourse. In this case a special silicone or polyurethane condom must be used (latex can damage the sperm). The collected sample is initially subjected to a “macroscopic” evaluation for the determination of some physico-chemical characteristics, in particular its appearance, volume, pH, viscosity and fluidization. Subsequently, a microscopic evaluation is performed to determine the concentration, motility and morphology of the spermatozoa. The examination can be performed according to the international standard of the World Health Organization (WHO 1999, then revised as WHO 2010)), which specifies tools, preparatory methods and “normal” reference values ​​of the measured parameters

Physico-chemical features

Color and appearance The color of the seminal fluid is normally opalescent gray. In a report of a spermiogram it will be possible to read also other terms such as lactescent, pioide, brown (blood). A milky appearance, especially if accompanied by a reduced volume and an acidic pH, may reflect damage to the seminal vesicles, while the yellowish pioide appearance is indicative of urinary contamination, the presence of granulocytes, a clear prevalence of the component of seminal vesicles or rarely of the presence of bilirubin.

Volume The volume of the ejaculate (under normal conditions from 1.5 to 5 mL) is a marker of the functionality of the seminal vesicles as these produce from 50% to 80% of the liquid component of the semen. The prostate contributes 15% – 30% and the remainder is supplied by the accessory urethral glands and the deferent.

Ph The seminal fluid normally has an alkaline pH , which oscillates between 7.5 and 8.0, and is the result of the basic secretion of the seminal vesicles and the acid secretion of the prostate. A  pH lower than 7 is an expression of dysfunction of the seminal vesicles, a pH above 8.0, in the presence of leukocytes, indicates the presence of an infection.

Fluidification As soon as it is released, the sperm coagulates and then liquefies. The  liquefaction  is completed within 30 minutes at body temperature (37 ° C). The coagulation takes place through the work of prostatic enzymes that act on the fibrinogen produced by the seminal bladders, going to create a dense knit very packed. A missed coagulation of the sperm, as well as a low volume, indicates a dysfunction of the bladders, or their agenesis. The enzymes that determine the liquefaction are secreted by the prostate.

Viscosity Viscosity should not be confused with fluidification . While fluidification is a transient dynamic process of dissolving a clot, viscosity is a permanent feature of a given seminal fluid. It is normally evaluated by slowly draining the seminal fluid from a pipette: a non-viscous seminal fluid forms a wire less than 2 cm. Excessive viscosity prevents normal movement of spermatozoa.

Presence of chemicals In the seminal fluid, some chemicals are normally found such as:

• fructose , normally in concentrations between 120 and 450 mg / dL. Fructose is produced by seminal bladders under androgenic stimulation. A lower fructose content than normal values ​​may indicate an infection of the seminal vesicles or even an insufficient secretion of androgens;
• citric acid , normally produced by the prostate. A low concentration suggests a prostatic pathology;
• zinc  (or 106368 ), normally from 25 to 400 μg / dL.

Microscopic features

The sperm count takes into account, in addition to their number, their morphology and motility .

Sperm concentration Indicates the number of spermatozoa present in 1 mL of ejaculate. The evaluation is carried out with the aid of counting chambers such as: Fast Read , Modified Neubauer, Burker, Thoma; while the recommended one is Makler’s room . According to the latest World Health Organization’s seminology manual, normal values ​​in terms of sperm count per milliliter are equal to or greater than 15 million.

It is necessary to consider that in 2011 the new WHO parameters were published (WHO 5th) and are as follows:

• Volume ejaculated> 1.5 mL
• Total sperm count> 39 million / ejaculate
• Sperm concentration> 15 million / mL
• Total Motility> 40%
• Progressive motility> 32%
• Vitality> 58%
• Morphology> 4%
• pH ≥ 7,2
• Leukocytes <1.0 million / mL
• MAR test percentage of sperm with adhered particles <50%

Motility
The tail allows the spermatozoon a “flagellar” movement which must be  anterograde , ie it must allow it to move forward. Within 2 hours of ejaculation, the percentage of spermatozoa that maintain a flagellar motion is evaluated which is described as rapid rectilinear motion (A), slow rectilinear motion (B) or irregular motion (C). In normal conditions, the percentage of spermatozoa with a straight A + B motion must be greater than or equal to 50%, of which 25% with fast straight motility. Motility depends physiologically on the duration of abstinence (decreases after the fifth day) and can be influenced by the storage temperature of the sample, by incomplete liquefaction or by the increase in viscosity.

Morphology
The morphological evaluation of spermatozoa must be performed with special histological staining ( Papanicolau ,  Giemsa , DiffQuick ( or  319001)). Each single spermatozoon is composed of a 4.0-5.5 micron long head, 2.5-3.5 micron wide and composed of 40% to 70% from an acrosomal region. Many morphological alterations of the head of a sperm can be observed (pointed, elongated, amorphous, round, double head, with reduced, absent or asymmetric acrosome). The intermediate section connecting the head with the flagellum has a length of 6.0-7.5 microns and a thickness of 1 micron; you can observe insertions of the asymmetrical intermediate section, or thickening or thinning. Finally, the flagellum has a length of about 45 microns (like ten heads), a width of 0.5 microns and can appear broken, hub, wound, swollen or double. It is also important to note the presence of cytoplasmic residues at the intermediate tract level. The presence of abnormal shapes should not exceed 70% of the total. A too high altitude (teratospermia) compromises fertility. The presence of spermatozoa gathered together to form “clusters” is called agglutination . A greater than 10% share of sperm gathered in agglomerates is an indication of ongoing inflammatory or immunological processes.

Presence of other cells
Other types of cells can be found in sperm: the presence of immature forms (sperm precursors) above 3% may indicate phenomena of cellular suffering due to exposure to heat, varicocele, prolonged fever, exposure to drugs, toxic or radiation. The presence of blood cells (red blood cells, lymphocytes or neutrophils) is generally a sign of infection or inflammation of the seminal pathways.

Interpretative value of the spermiogram

For the purposes of assessing the fertility of an individual, the spermiogram can not be considered as a single and definitive examination. In the presence of negative parameters, on the same sample of collected seminal liquid, spermiocoltura is performed with an eventual antibiogram, to verify the presence of infections. The exams (spermiogram and spermiocoltura) are sometimes repeated after 7 or 20 days to check if the shape and number of spermatozoa are inconstant over time, or the presence of permanent infertility. Many individuals with normal spermiogram may not be fertile and, conversely, men with low sperm concentrations may be able to procreate. Therefore, if necessary,

The physiological variation of the seminal fluid over time makes it necessary to perform the examination on at least three samples before making a diagnosis on the fertility of the subject examined. In fact, its production can be influenced by numerous factors, such as fever, medication or other substances (alcohol, cannabinoids); furthermore, since the complete maturation of a spermatozoon involves an average time of three months, the modifications of a spermiogram can reflect events that have already happened and sometimes even exceeded (for example, sexually transmitted diseases, bacterial or viral).

In addition, quantitative variations of up to 20% are not infrequent when different examiners count the same sample (motility and sperm form are usually assessed subjectively by a technician). The finding of an “abnormal” or “normal” finding is not synonymous with infertility or fertility: only azoospermia or complete immobility of spermatozoa are certain causes of infertility, while the presence of any number of mobile spermatozoa can produce conception, although obviously the chances gradually become less scarce as their concentration decreases.

Terminology of some seminal variables

Normal ejaculate normozoospermia , according to the reference values

Oligozoospermia sperm concentration lower than the reference values

Astenozoospermia motility below the reference values

Oligoastenozoospermia motility and sperm concentration lower than the reference values

Teratozoospermia morphology lower than the reference values

Oligoastenoteratozoospermia alteration of all variables

Cryptozoospermia absence of spermatozoa in the ejaculate but presence of spermatozoa in the centrifuged

Azoospermia absence of spermatozoa in the ejaculate (and therefore also in the pellet post-centrifugation)

Ejaculate necrozoospermia characterized by the total death of the spermatozoa present

Hypoposia reduced volume of the seminal fluid

Excessive hyperplastic volume of the seminal fluid

Importance of the spermiogram automation

What has been said so far concerning the count, motility and morphology until a few years ago, took place exclusively through a microscope reading which is to be considered completely “subjective” as it is linked to the operator performing the test.

In recent years, the choice of automating the spermiogram has been consolidated in many laboratories to overcome this type of problem. That is, to make this test “objective” and ” standardized ” through automatic analyzers; a bit ‘as happened several decades ago when the first models of counterparts appeared in the laboratories of analysis which allowed the operators to pass from a subjective reading done in Burker’s Room, to an automatic tool that would standardize the reading.

Another advantage that the technology offers is that it allows the operator to process much larger sample sizes, at least 300 microlitres, compared to the microscope reading which “requires” about 10 microliters.

And yet, while normally under the microscope there are no more than 200 cells, an automatic tool allows you to “count” millions of sperm cells in just a few minutes.

On the market there are essentially two types of automatic instruments with different sample reading principles. A first type is called the CASA System and is based on an analysis of images. In practice, images are captured by a video monitor connected to a PC. The results, subject to the settings of the CASA System and to the frame rate, pixel and video output format, can vary the results. The accuracy and precision are, therefore, dependent on the observer and his experience that the nature of the calibration, subjective, can lead to inter and intra-instrument differences.

Another limitation is the amount of sample tested which is 10 microliters which, on a 2-5 mL sample, results in a tested fraction of 0.2-0.5 microliters. Finally, a few hundred sperms are normally counted.

A second type of tool is that of the Israeli company MES, QWIK CHECK GOLD , SQA-Vgold and SQA-Vision , essentially has three different operating principles depending on whether we are talking about:

• Counts : automatic pre-calibration based on electro-optical system (spectrophotometry) with a CV of less than 2%. The tested sample is 330 microliters with a reading of millions of spermatozoa.
  • Motility : automatic system based on objective electro-optical method (modulation of the signal caused by the movement of spermatozoa detected by the instrument), with CV less than 3%. The tested sample is 20 microliters with thousands of sperms reading.
  • Morphology : automatic system. Calculated parameter (proprietary algorithm) based on the correlation between progressive motility and normal morphology, with CV less than 3%. The tested sample is 50 microliters with thousands of sperms reading.