9.3.3 Semen analysis

Semen analysis remains the most important diagnostic tool for the study of male infertility to date. For this reason, and because of the ease of carrying out this analysis, examination of seminal fluid should be among the first diagnostic steps in cases of suspected infertility, prior to subjecting the man’s partner to long and complex diagnostic tests. The efficacy of an examination of seminal fluid depends on the experience and ability of the seminologist, who must first undertake a subjective analysis of fundamental parameters such as motility and morphology. Moreover, laboratories specialized in such analyses may apply different criteria to the evaluation of sperm parameters, making it extremely difficult to compare tests carried out in different laboratories (1).

In an attempt to resolve these problems of inconsistency, and in order to standardize laboratory techniques, a committee of experts from the WHO established guidelines for semen analysis in 1980 (an updated version was published in 1999) (2).

In recent years, numerous other methods of semen analysis capable of providing in-depth diagnostic information on the fertilising capacity of spermatozoa have become available. The computer-aided sperm analysis (CASA) system is a technique for sperm analysis designed to provide objective data on sperm motility (3). Because of persisting difficulties in software set-up (4), it should not be used for routine analysis, but rather as a research tool. At the same time, significant advances have been made in the study of sperm morphology through the use of scanning and transmission electron microscopes (5). Finally, within the past decade several tests capable of evaluating the integrity of sperm components, such as the membrane, acrosome, DNA, and nuclear protein, have been developed and put into use. These more complex and costly analytical tools should be considered of secondary or tertiary importance, and are to be carried out in specific cases only after standard semen analysis. Standard semen analysis remains the first and fundamental diagnostic tool.

Semen analysis will be inaccurate unless certain rules are followed prior to sample collection. The period of sexual abstinence before taking a sample should be between 2 and 7 days, because of the necessary epididymal period of sperm maturation and length of stay of mature sperm in the caudal tract of the epididymis. This period also includes any ejaculation, not only sexual intercourse, a detail often not mentioned by the patient, and not caught during specific questioning by laboratory staff. The effect of abstinence on sperm parameters is extremely important. Too short an interval may reduce semen volume and sperm concentration. Conversely, a longer period of abstinence may result in a reduction of motility and an increase of abnormal forms. If this period is not standardized, misleading information on the semen quality can result (6).

Masturbation with ejaculation of the sample into a sterile container, such as those used for urine, is the recommended procedure for collection of semen samples. Where masturbation proves difficult, coitus using nonmedicated condoms is recommended. Interrupted intercourse should not be considered, as this method tends to lose part of the ejaculate and makes it difficult to distinguish between the man and his partner’s epithelial cells, white cells, and red blood cells; and can moreover cause bacterial contamination. The sample should ideally be obtained at the site of the laboratory; however, for psychological reasons it can be collected at home and delivered within 60 min after the ejaculation, provided it is not being collected for legal reasons or for cryopreservation. However, the sample must be processed within 60 min after ejaculation in order to evaluate the time and nature of liquefaction. Microscopic evaluation must be carried out after a complete liquefaction of the sample. The sample should not be exposed to excessive fluctuations in temperature.

The patient should be asked to provide information regarding any physical or psychological pathologies from which he may have suffered during the three preceding months, and concerning the use of medication, fevers, viral or bacterial infections, antibiotic therapy, and local or general anaesthetic. Any of these may influence semen characteristics.

Semen analysis should cover a minimum number of seminal and sperm parameters, without which the analysis loses all real value. It is essential for the clinician to have correct and complete determinations of these parameters, in order to interpret and integrate these results with the clinical data available, and to be able to classify the patient as potentially fertile, or infertile.

The normal semen volume is ≥ 2.0 ml. A total absence of ejaculated semen is termed azoospermia. Reduced semen volume (<1.0 ml) is frequently indicative of obstructive pathology of the ejaculatory ducts or of a secretory defect of the seminal vesicles for functional or anatomical reasons. In rarer instances, and usually accompanied by other clinical signs, it can indicate reduced production of testosterone. Hyperspermia can be associated with inflammatory pathologies and/or infections of the seminal vesicle and/or the prostate.

Seminal pH is alkaline (normal variation 7.2–8.0) and results from the combination of the alkaline secretions of the seminal vesicles and the acidic secretions of the prostate. Measurement is carried out using simple indicators of pH with a range from 5.5 to 9.0. Alkaline pH (≥8.0) can indicate inflammatory pathologies. Acidic pH (<7.0) is even more informative, and is often associated with obstructive pathologies of the ejaculatory ducts, or with congenital or acquired hypotrophy or atrophy of the seminal vesicles.

The physiological appearance of semen is opalescent ivory. This becomes milky when the ejaculate derives exclusively from the prostate, as occurs in cases of genital tract obstruction. A yellowish appearance can indicate a high number of white blood cells (pyospermia) (7), and is often associated with acute or subacute infections of the male genital tract. Pink, intense red, or red-brown colours indicate the presence of blood (haematospermia). Haematospermia can be caused by microvascular lesions induced by trauma, inflammations and infections of the male genital tract, duct obstructions, cysts, and neoplastic pathologies (8).

Immediately after ejaculation, human seminal fluid undergoes a process of coagulation, which transforms the liquid into a gelatinous coagulate in which spermatozoa are imprisoned. This physiological process enables the semen to remain fixed to the cervix and to form an interface with the cervical mucus in the vaginal posterior fornix. Immediately after coagulation, the process of liquefaction begins, and is completed in 10–60 min. The assessment process ascertains whether liquefaction is complete or not, and whether it occurs within an appropriate physiological time span. When this is not the case, it becomes more difficult to analyse sperm parameters (concentration and motility) accurately.

Since coagulation and liquefaction depend on factors of vesicular and prostatic origin, an alteration of these processes can indicate pathologies of these structures. Disturbed liquefaction is often found in infection or inflammation of the accessory glands, and can explain infertility, if only in part.

The evaluation of seminal viscosity provides information on rheological characteristics common to all biological fluids. An increase in viscosity makes microscopic analysis difficult and hinders the assessment of sperm parameters. Hyperviscosity can indicate pathologies of the accessory glands, while reduced viscosity often occurs in cases of serious oligozoospermia or in azoospermia.

The number of spermatozoa can first be evaluated under a light microscope prior to any specific preparation. Analysis should begin by using an automatic pipette to place 10 µl of semen on a slide, having mixed thoroughly to ensure that the cells are evenly distributed. It is advisable to prepare at least two drops of 10 µl on the same slide and take an average of the two separate preparations. A dilution is made with immobilising solution immediately following the preliminary observation; in general a 1:20 dilution factor is appropriate, but it can vary from 1:10, when the concentration is less than 20 million/ml, to 1:50 when the concentration is greater than 100 million/ml. A count is then carried out in a Makler, Burker, Thoma, or Neubauer improved chamber, the lattermost being recommended by WHO.

Fertile subjects between the ages of 20 and 40, who are normal from anatomical, urological, andrological and endocrinological standpoints, may show great variation in the number of spermatozoa in different ejaculates. However, this variation can be much lower than that found in the literature provided that sample collection is carried our properly, and the physical and psychological conditions of the subject do not vary. The minimum concentration for potential natural fertilisation should be approximately 20 million/ml. Concentrations below this number are indicative of oligozoospermia. Cryptozoospermia (i.e. ‘hidden sperm’) means spermatozoa found only after centrifugation of semen sample. The absence of spermatozoa in the ejaculate is called azoospermia. Semen samples without spermatozoa in a first examination should be centrifuged at 200 g for 15 min and the seminal plasma recentrifuged at 3000 g for 10 min; both pellets should be evaluated to confirm the absence of sperm.

Sperm motility is evaluated using a sample prepared as in the previous section. Motility should be measured after liquefaction or at fixed time points after ejaculation (1–2 h), never at ejaculation, since the process of coagulation physiologically slows or blocks motility. At least 20 microscope fields should be evaluated per sample, and at least 100 sperm cells in each field. It is important to consider not only the percentage of motile cells, but also the type of motility. This is classified as: rapid progressive, with a velocity of at least 25 µm per second (grade A on the WHO scale); slow or sluggish progressive (grade B on the WHO scale); nonprogressive (grade C on the WHO scale); or absent (grade D on the WHO scale).

In normal subjects, one hour after ejaculation, the percentage of sperm with motility of grades A + B should be at least 50%, with at least 25% grade A. Values below these are indicative of asthenozoospermia.

Many endeavours have been made to find an objective method to read sperm kinetics. Laser light scattering was one of the first attempts. In this method low energy lasers are used to measure the frequency of light diffused by the targeted sperm sample. Time-lapse photography and multiple exposure photography techniques are based on the fact that moving sperm leave a track or print on a photographic image. Spectrophotometric methods are based on the absorption of ultraviolet light, while in video cinematography the sperm trajectories are projected onto a screen and their speed is measured.

The methods that employ image analysis use computer readings of digitized sperm tracks, projected onto a monitor. These systems were proposed to analyse sperm kinetic parameters such as velocity, linearity, amplitude lateral head and beat cross frequency. These methods have received much attention from researchers in the field, and have been used in parallel with microscope reading.

Mature human spermatozoa, observed under a light microscope, show an oval head composed of two parts, the nucleus and the acrosome, and covered by the plasma membrane. The head is connected to a long, thin flagellum or tail, which in turn is divided into a midpiece of 5–6 µm, a principal piece measuring 45 µm, and a terminal piece of 5 µm. Along the tail runs the axoneme, a bundle of nine double fibrils surrounding two single central fibrils. The neck is a short intermediate section, measuring only 1 µm, between head and tail (2, 9).

Sperm morphology is evaluated using fresh semen or stained smears. The May–Grünwald/Giemsa, Papanicolau, Shorr, and Diff-Quick techniques are equally satisfactory for the study of sperm morphology, the two lattermost being recommended by WHO. The maximum percentage of atypical forms should not exceed 70%, above which the sample would be considered as teratozoospermic.

Few classifications have been proposed to describe sperm morphology. The WHO 1999 guidelines (2) suggest the following scheme for classifying sperm typologies.

The study of untreated semen or stained smears allows nonsperm cells in the ejaculate such as the following to be identified, both in physiological and pathological conditions.

In order to collate the data regarding the various seminal parameters, a number of authors have proposed formulae providing an index of fertility. Currently, none of these formulae are in use, given that they do not provide useful clinical indications.

The evaluation of semen samples can also include certain biochemical indices of seminal fluid, deriving from the combined secretory action of the accessory glands of the male genital tract (10). Fructose, which is produced in the seminal vesicles under the stimulation of androgens, has an important role in the metabolism and therefore in the motility of spermatozoa. The secretion of the seminal vesicles has an alkaline pH and in general makes up more than 60% of the ejaculate.

Epididymal secretions include l-carnitine, α-glucosidase, and glycerylphosphorylcholine. A pump mechanism selectively filters l-carnitine from the blood at the epididymal level. Given carnitine’s essential role in lipid mechanisms, it definitely has an important place in the complex metabolic mechanisms of spermatozoa.

Prostatic secretion, with an acidic pH between 6.4 and 6.8, comprises about 15–20% of the ejaculate and is rich in citric acid, zinc, acid phosphatase, magnesium, and polyamines. Citric acid seems to have its own role in the coagulation and liquefaction of semen, and in the maintenance of osmotic equilibrium.

During ejaculation, the secretions of various accessory glands and the testicular fraction are not released in a random or disordered fashion, but according to a very precise sequence. The first fraction is predominantly prostatic, followed by an epididymal and testicular fraction (rich in sperm), and finally a fraction that derives from the seminal vesicles.

Biochemical analysis can make a notable contribution to the differential diagnosis of azoospermia. Thus in secretory azoospermia, which involves normal androgenic production, there are no important modifications in the secretions of the accessory glands. In subjects with obstruction of the ejaculatory ducts, there is a relatively high concentration of citric acid. Subjects with obstruction of the deferent ducts have extremely low levels of free carnitine or α-glucosidase, while fructose and citric acid levels remain normal.

Many sperm function tests can be used in parallel with sperm analysis to establish a complete picture of the fertilizing capacity of sperm and direct the clinician towards treatment or assisted reproduction (11, 12).

Sperm–cervical mucous interaction tests (the in vivo post-coital test (PCT) and the in vitro assay of sperm migration in cervical mucous) have been used for many years as assays of couples’ potential fertility. A simple test can therefore provide valuable information about the condition of cervical mucous, and related hormonal functions, and also sperm survival after intercourse. Sperm selection methods, such as swim-up, are widely accepted as efficient ways to test the ability of sperm to migrate out of seminal plasma to another medium of different composition and density.

In order to evaluate this essential and complex sperm function, the concentration of specific acrosome enzymes can be studied. Acrosine is a trypsin-like enzyme specific to the acrosome which is derived from a precursor (proacrosine). Its glycoproteinase action is essential for penetration of the zona pellucida (ZP). In addition to study of the intra-acrosomal enzymes, there are a number of tests that evaluate the status of the acrosome using probes that target specific acrosomal structures. These tests can be used on untreated spermatozoa or on spermatozoa after incubation with in vitro inducers of acrosome reaction (ionophores, follicular fluid, and progesterone) (13, 14). They can be useful in cases of severe abnormalities of head morphology, or in the setting of unexplained infertility in patients with poor IVF pregnancy rates.

For a number of years, andrologists have tried to create an in vitro biological test capable of establishing the ability of spermatozoa to fertilize the human oocyte. The IVF era increased the need for such a test, in order to have a predictive index before recommending an expensive form of therapy. The basic conclusion to date is that the only reliable indicator of success is the real IVF interaction of the oocyte with the spermatozoa.

Apart from the above in vitro biological tests, several artificial models have been proposed. One of the most widely used is the hamster test or sperm penetration assay (SPA). This test uses ZP-free hamster eggs, enzymatically deprived of cumulus and ZP, and the capacitated spermatozoa of the patient under examination (15). After the in vitro acrosome reaction, the spermatozoa are able to penetrate the vitelline membrane of the oocyte (due to the absence of the species-specific ZP) and to initiate the process of nuclear decondensation. Difficulties of interpretation (failure of SPA, but fertilization in patients undergoing IVF) and increased knowledge of the importance of the sperm-ZP protein interaction make the study of this functional step mandatory.

ZP proteins are one of the acrosome interaction inducers, via their activation of a series of secondary messenger pathways involving various protein kinases. In mammalian models, some ZP proteins have been characterized and used to prepare antiserum for immunological contraception.

Spermatozoa are the carriers of the male genetic material. Under in vivo and IVF conditions, nuclear decondensation leads to a separation of the chromosomal fibres; this enables DNA arrangement in the oocyte equatorial plane during metaphase of the first mitotic spindle. Sperm chromatin is highly condensed in the sperm head due to the presence of disulphide (S₂) bonds. These S₂ bonds result from replacement of histones by the more basic protamines, which have high levels of arginine and cysteine. Protamine content can be studied by aniline blue staining. The nuclei of mature spermatozoa are impervious to denaturing agents that induce sperm membrane permeability, such as SDS. DTT and EDTA are used as chelating agents. Normal sperm chromatin is extremely resistant to denaturation by chemical and physical agents. To study this resistance, the DNA is assessed using a fluorescent dye—acridine orange (AO)—after acid-denaturing stress (citric acid). AO can differentiate between intact double-stranded DNA and denatured single-stranded DNA, based on their relative fluorescent properties. When AO intercalates with double-stranded DNA, it fluoresces green; interaction with single-stranded DNA (or RNA) results in red fluorescence. This can be evaluated both by fluorescent microscopy and by flow cytometry (16). Sperm chromatin structural assay (SCSA) is a flow cytometric assay that assesses the susceptibility of sperm chromatin DNA to in-situ acid denaturation, and can reveal the percentage of cells with DNA damage.

There are other tests currently employed in the evaluation of DNA integrity. Single-cell gel electrophoresis (SCGE), or the COMET assay, is a method for detecting DNA damage at the level of the individual cell. SCGE is based on negatively charged fragments of DNA being drawn through an agarose gel in response to an electric field, and detects both single- and double-stranded DNA breaks. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) uses terminal deoxynucleotide transferase (TdT), which catalyses polymerization of the fluorescein-labelled nucleotides at the DNA 3′ hydroxy terminal. TUNEL is another method for assessing single- and double-stranded DNA breaks (17). The study of sperm DNA integrity is valuable in assessing the damage that andrological diseases, drugs, or other pathological conditions may cause to spermatozoa (18, 19).

These tests are used in cases of distinct hypomotility, in order to differentiate spermatozoa that are immotile but alive from those that are dead.

The most frequently employed tests are the eosin test and the hypo-osmotic swelling test (HOS).

The eosin test utilizes a vital staining system (eosin Y alone or in combination with nigrosin) which distinguishes between the live (unstained) spermatozoa and the dead (stained) cells. The dead cells, with damaged plasma membranes, are permeable during the vital staining.

The HOS uses a light microscope to evaluate the percentage of spermatozoa with swelling of the tail after incubation in a hypo-osmotic solution. This test is based on the fact that spermatozoa with an intact membrane, when suspended in a hypo-osmotic solution (below 150 mOsm/l), allow the passage of water molecules across the plasma membrane to achieve osmotic equilibrium. They consequently swell, especially at the level of the tail, and show specific morphologic changes. Conversely, dead cells allow the passage of water freely in both directions, and do not show swelling.

Since they can distinguish dead from vital cells, these tests have garnered renewed interest in the context of intracytoplasmic injection.

The newest tests in the study of male fertility are represented by the microarray and proteomics. Recent investigations, concerning sperm mRNA content, have described the relevance of the sperm mRNA stock in fertilization and early embryo development, in several species (20). Microarray technology can yield information on the expression levels of thousands of mRNAs in a single experiment, enabling the analysis and comparison of complete sperm expression profiles.

Alternatively, proteomic studies have identified sperm chromatin proteins with fertility roles, which have been validated by molecular studies in model organisms or correlations in the clinic. Sperm rely on testis-specific protein isoforms and post-translational modifications for their development and function; therefore, sperm-specific processes are ideal for proteomic explorations that can bridge the research laboratory and fertility clinic (21).

The aim of the seminological laboratory, as of any type of laboratory performing analyses, is to obtain results that are as error-free as possible, and which can therefore be used in the diagnosis and treatment of the infertile patient. All laboratory tests have an intrinsic error rate. Errors can occur at random, making them difficult to anticipate, or they can be systematic, resulting from a difference between the analysis itself and the value obtained. Effective quality control, therefore, aims to reveal laboratory errors and to ensure that all procedures, analytical ones included, are adequate to provide the best results. This need is even more pressing for the seminological laboratory. Despite the considerable progress made in the standardization of semen analysis (WHO, 1999), evaluations of sperm parameters continue to demonstrate marked differences both within and between laboratories (22, 23). The demand for regular internal and external laboratory quality control systems has become so great that, for the first time, the latest edition of the WHO manual includes a lengthy paragraph on techniques for carrying out correct quality control. The fundamental rules of good quality control must provide for: daily surveillance and correlations of results within samples; weekly analysis of replicate measurements of the main semen variables by different technicians; monthly analysis of the mean results of tests; quarterly participation in an EQA scheme, and the annual calibration of counting chambers and other equipment (2, 24–27).