Spermatozoa were first described by Leeuwenhoek in the 17th century but it was not until 1928 that the sperm count was found to be associated with fertility potential (17). Since that time a variety of sperm tests and semen parameters have been developed with the hope of clarifying whether or not a man could impregnate his partner.
MacLeod (1942), MacLeod and Gold (1953), Eliasson (1971) and Hellinga (1949,1976) have led the scientific basis of conventional analysis of spermatozoa and the techniques recommended by them are still considered the reference for more advanced methods(6).
Semen analysis comprises a set of descriptive measurements of spermatozoa and seminal fluid parameters that help to estimate semen quality (4).
Conventional semen analysis includes measurement of particular aspects of spermatozoa such as concentration, motility and morphology and of seminal plasma. Quantification and identification of non-spermatozoidal cells and detection of antisperm antibodies are also part of basic semen analysis (6).
Normal values of semen parameters issued by the World Health Organisation (WHO) in 1992 are generally used as reference values (Table I).
Ideally, each laboratory should set its own normal values, reflecting the specific population analyzed.
Table I. Normal values of semen variables (WHO 1992)
Table II. Nomenclature for semen variables (WHO 1992)
Normal semen is an admixture of spermatozoa suspended in secretions from the testis and epididymus which are mixed at the time of ejaculation with secretions from the prostate, seminal vesicles, and bulbourethral glands. The final composition is a viscous fluid that comprises the ejaculate (25).
Sample collection and deliveryThe following instructions for sample collection and delivery are based on WHO recommendations (24,25). The subject should be provided with clearly written or oral instructions concerning the collection and, if required, transport of the semen sample.
Laboratory technicians should be aware that semen samples may contain harmful viruses (e.g., HIV and viruses causing hepatitis and herpes ) and should therefore be handled with due care.
The semen sample is first evaluated by simple inspection. A normal sample has a grey-opalescent appearance, is homogenous and liquefies within 60min at room temperature under the influence of enzymes of prostatic origin. In some cases, liquefaction does not occur within the normal time period and this fact should be recorded, as it may suggest functional disturbance of the prostate. Normal semen samples may contain jelly-like grains which do not liquefy.
The sample may appear clear if the sperm concentration is too low. It may also appear brown when red blood cells are present in the ejaculate (haematospermia).
The presence of mucous streaks may interfere with the counting procedure and suggests inflammation or abnormal liquefaction.
Samples which do not liquefy need additional treatment such as exposure to bromelin, to make the sample amenable to analysis (6, 25).
The sample should be well mixed in the original container. Incomplete mixing is probably a major contributor to errors in determining sperm concentration.
The consistency, also called viscosity, of the liquefied sample can be estimated by gentle aspiration into a 5-ml pipette and then allowing the semen to drop by gravity and observing the length of the thread formed. A normal sample leaves the needle as small discrete drops, while in cases of abnormal consistency the drop will form a thread of >2 cm (6, 25). Another method to estimate consistency does not use needles and is performed by introducing a glass rod into the sample and observing the thread that forms on withdrawal of the rod. Again the thread should not exceed 2 cm (6, 25).
Increased consistency has the same clinical meaning as abnormal liquefaction, and may be related to prostate dysfunction resulting from chronic inflammation (6).
Very viscous specimens can impair the availability of fertile sperm at the site of fertilization (18).
The major component of the ejaculate volume is made up of secretions from the accessory glands. The bulk of the volume is secreted by the seminal vesicles and between 0.5 and 1 ml originates from the prostate (6). The volume of the ejaculate should be measured either with a graduated cylinder or by aspirating the whole sample into a wide-mouthed pipette by means of a mechanical device (25). The sample volume can also be determined directly in the collection tube by weighing, assuming 1ml equals 1g. (9). Thereby, loss of volume associated with transfer from the collection tube to either another tube or a pipette can be avoided (9).
A low ejaculate volume can reflect abnormalities in accessory sex gland fluid synthesis or secretion (18). It can also be indicative of a physical obstruction somewhere in the reproductive tract (18), or may occur in cases of incomplete or (partially) retrograde ejaculation (6).
Large volumes are sometimes found in association with varicocele or after relatively long periods of sexual abstinence (6).
The pH is determined by acidic secretions of the prostate and alkaline secretions of the seminal vesicles. It should normally be in the range of 7.2-8.0 (25).
Recently, one author has shown (8) that the mean pH values are consistently well above 8.0 regardless of the method of analysis and the time of examination and has suggested that the range of normal values needs to be revised further.
To test pH, pH paper range 6.1 to 10.0 is used. Whatever type of pH paper is used for this analysis, its accuracy should be checked against known standards before the use in routine semen analysis (6, 25).
If the pH exceeds 8.0, infection should be suspected with decreased secretion of acidic products by the prostate, such as citric acid (101). Abnormal pH may also be recorded in cases of incomplete ejaculation. Extremely acidic pH (<6.5) is found in cases of agenesis (or occlusion) of the seminal vesicles.
Initial microscopic investigation
During the initial microscopic investigation of the sample, estimation of motility and concentration of spermatozoa is performed. The presence of cells other than spermatozoa and of agglutination of spermatozoa are determined.
In recent years, a number of techniques for objective assessment of movement characteristics of human spermatozoa have been introduced by using computer-assisted semen analysis (CASA) systems. For the purpose of conventional analysis, a simple classification system which provides the best possible assessment of sperm motility without resorting to complex equipment is recommended.
A fixed volume of semen (not more than 10 m l ) is delivered onto a clean glass slide and covered with a 22x22 mm coverslip (25). It is important that the volume of semen and the dimension of the coverslip are standardized so that the analyses are always carried out in a preparation with fixed depth (i.e., 20m l). This depth allows full expression of the rotating movement of normal spermatozoa (25). The preparation is then examined at a magnification of x400-600. An ordinary light microscope can be used for unstained preparations, particularly if the condenser is lowered to disperse the light. However, a phase-contrast microscope is preferable.
The weight of the coverslip spreads the sample for optimal viewing. The freshly made, wet preparation is left to stabilize for approximately one minute. Motility estimation can conveniently be carried out at a room temperature between 18 and 24°c. At temperatures outside this range, some alteration in sperm motility will occur and this must be standardized in the laboratory.The microscopic field is scanned systematically and the motility of each spermatozoon encountered is graded a, b ,c or d (25) according to whether it shows:
Spermatozoa graded (a) are supposed to display rapid progressive motility along a linear track, covering a distance of at least 20 m m (half the length of a spermatozoon) per second (6).
At least 100 spermatozoa are classified in this way. Visual field close to the border of the coverslip should be avoided.
It is advisable to repeat the procedure on a second drop of semen processed in the same way.
Estimation of sperm concentration
The concentration can be estimated roughly during the initial examination in order to determine the dilution procedure to be used and to indicate whether centrifugation may be required to prepare an adequate smear for morphologic analysis.
Cells other than spermatozoa
The ejaculate usually contains cells other than spermatozoa. These include polygonal cells from the urethral tract. If many of these are present, and they are covered with bacteria then it is probably that the sample was obtained by coitus interruptus and the cells originate from the vagina (6). Spermatogenic cells and white blood cells (WBC), which are often referred to as "round cells", are present in almost every semen sample. By conventional light microscopy or sperm staining techniques it is not possible to reliably differentiate WBC from immature germ cells in semen. In contrast, the cytochemical peroxidase method reliably identifies granulocytes, the most prevalent WBC type in semen (23). The method is cheap, fast and easy to perform. The gold standard for the detection of all WBC populations in semen is immuno-cytology using monoclonal antibodies (23). However, it is expensive and time-consuming, thus remaining a research tool at present. For clinical purposes, the peroxidase method is ideally suited to detect granulocytes.
The method (11) aims at the counting of peroxidase-positive round cells in a haemocytometer. The working solution is prepared by combining 1ml of saturated NH4Cl solution, 1ml of 5% of Na2 EDTA solution, 9ml of orthotoluidine solution and 1 drop of H2O2. This solution is mixed before use and can be conserved for 24h after preparation. The procedure consists of mixing 0.1ml of semen with 0.9ml of the working solution to achieve a total volume of 1ml. This mixture is shaken for 2 min. It is then left for 20-30 min at room temperature and mixed again by shaking. The mixture is now transferred onto a haemocytometer chamber for leukocytes and the number of peroxidase-positive cells which stain brown is counted. Peroxidase-negative cells remain unstained and are counted in the haemocytometer chamber. The differentiation of round cells into either peroxidase-positive polymorphonuclear granulocytes or peroxidase-negative spermatogenic cells or lymphocytes is of clinical relevance. The presence of an excessive number of peroxidase-negative mostly spermatogenic cells suggests pathology at the level of the seminiferous epithelium with inadequate spermatogenesis and premature release of spermatids spermatocytes or, rarely, spermatogonia. The pathological meaning of the presence of an elevated number of WBC is still a matter of dispute. Some reports have demonstrated that leukocytospermia appears to be of no diagnostic value to identify men with actual microbial infections (22). Also, measurement of seminal leukocytes in routine semen analysis appears to be of little prognostic value with regard to male fertilizing potential (21).
Others hold the view that the presence of an elevated number of WBC may be associated with infection or inflammation of the accessory glands (6) and that the unfavorable effect on spermatozoa of hydrogen peroxide secreted by peroxidase-positive WBC has clearly been proven (6).
A comprehensive approach that considers other clinical and laboratory findings seems to be more reliable in detecting male accessory gland infection (4).
Agglutination of spermatozoa means that motile spermatozoa stick to each other, head to head, midpiece to midpiece, tail to tail, or mixed, e.g. midpiece to tail. The adherence of either immotile or motile spermatozoa to mucus threads, to cells other than spermatozoa, or to debris is not considered agglutination and should not be recorded as such (6, 24, 25).
The presence of agglutination is suggestive of, but not sufficient evidence to prove the existence of an immunological factor of fertility (6, 24, 25). The extent of agglutination may be important but even the presence of only a few groups of small numbers of agglutinated spermatozoa should be recorded. In case of agglutination, sperm culture must be performed in order to exclude infection with e.g. Escheria coli. Sperm agglutination could be used also as indication for antisperm antibody testing of infertile men (6, 10).
Further microscopic examination
Vital staining of the spermatozoa allows quantification of the fraction of living cells independently of their motility (4). Live and dead sperm are distinguished by adding one drop of eosin y stain to one drop of semen at room temperature(one to two minutes) and smearing the mixture on a microscopic slide (18). 100 spermatozoa are classified as either colored orange-red, if the stain has passed through the membrane and therefore the cell is considered dead, or non-stained, the cell than being considered alive (6).
This staining technique makes it possible to differentiate spermatozoa that are immotile but alive from those that are dead (25). Reduced percentage of motility with a high percentage of viable sperm may reflect structural or metabolic abnormalities of sperm that are derived from abnormalities in testicular function or antimotility factors in the seminal plasma (18).
This technique also provides a check on the accuracy of the motility evaluation, since the percentage of dead cells should not exceed the percentage of immotile spermatozoa (25).
Hypo-osmotic swelling (HOS) test
The hypo-osmotic swelling (HOS) test measures sperm membrane integrity by examining its ability to swell when exposed to hypo-osmotic media, and has been claimed to be relevant to fertilizing ability (4). The rationale of the test is based on the assumption that an undamaged sperm tail membrane permits passage of fluid into the cytoplasmic space causing swelling and the pressure generated leads to curling of tail fibers, while the damaged or chemically inactive membrane allows fluid to pass across the membrane without any accumulation and accordingly no cytoplasmic swelling and curling of the tail occur.
The HOS test should not be used as a sperm function test but may be used as an optional, additional vitality test (25). It is simple to perform and easy to score and gives additional information on the integrity and the compliance of the cell membrane of the sperm tail (25).
Counting the spermatozoa
The concentration of spermatozoa should be determined using the haemocytometer method (25).
In this procedure a 1:20 dilution from each well-mixed sample is prepared by diluting 50 m l of liquefied semen with 950 m l diluent. The latter is prepared by adding 50 g of sodium carbonate (NaHCO3), 10ml of 35% (v/v) formalin and, optionally, 0.25 g of trypan blue or 5ml of saturated aqueous gentian violet to distilled water and making up the solution to a final volume of 1000ml. The stain needs not to be included if a phase-contrast microscopy is used. If the preliminary examination of the semen indicates that the concentration of spermatozoa present is either excessively high or low, then the extent to which the sample is diluted should be adjusted accordingly. For samples containing less than 20x10 6 spermatozoa/ml, a 1:10 dilution should be used; for samples containing more than 100x10 6 spermatozoa/ml, a 1:50 dilution may be appropriate. Both chambers of the haematocytometer are scored and the average count is calculated, provided that the difference between the two counts does not exceed 1/20 of their sum (i.e., less than 10% difference). If the two counts are not within 10%, they are discarded, the sample dilution re-mixed and another haemocytometer prepared and counted.
An optional procedure for determining sperm concentration employs specialized counting chambers, e.g. Makler chamber.
The total number of spermatozoa per ejaculate reflects spermatogenesis and is related to the duration of sexual abstinence (4).
Perhaps the most widely utilized semen parameter is sperm count. Men with <20x10 6 spermatozoa per ml are typically deemed sub-fertile, and men with counts <5x10 6 spermatozoa/ml are often considered infertile (17).
Other authors have confirmed that in patients with sperm counts <20x10 6/ml the fertility potential is significantly impaired (1). However, it must be emphasized that patients with sperm counts <20x10 6 are not infertile. It simply takes them a substantially longer period of time to achieve pregnancies (1).
Analysis of the morphological characteristics of spermatozoa
Sperm cells represent a unique population in which up to 50% (up to 70% according to WHO criteria 1992 and up to 86% according to strict criteria) of the cells can have morphological defects in normal fertile individuals (4). Although the morphological variability of the human spermatozoon makes differential sperm morphology evaluation very difficult, observations on the selection of spermatozoa recovered from the female reproductive tract (especially in post coital cervical mucus) helped to define the appearance of a normal spermatozoon. The normal head should be oval in shape. Allowing for the slight shrinkage that fixation and staining induce, the length of the head should be 4.0-5.5 m m, and the width 2.5-3.5 m m. The length-to-width ratio should be 1.50 to 1.75. There should be a well-defined acrosomal region comprising 40-70% of the head area. There must be no neck, midpiece or tail defects and no cytoplasmic droplet more than one-third the size of a normal sperm head. This classification scheme requires that all borderline forms be considered abnormal (25).The following categories of defects should be scored.
The traditional feathering technique (whereby the edge of a second slide is used to drag a drop of semen along the surface of the cleaned slide) may be used to make thin smears of spermatozoa. The Papanicolaou smear for staining of spermatozoa is the method most widely used in andrology laboratories. In our practice we have tried simpler methods: Meyer’s haematoxiline, Harris haematoxiline and Giemsa. We have reported that these methods are not as elegant as the Papanicolaou method but allow the classification of the spermatozoa in the main groups with the same accuracy (15).
Sperm morphology gives information for the function of the reproductive tract and is a predictor of man’s fertility potential.
Physical sperm aberrations may occur during the production of sperm or during storage in the epididymus. In cases of teratozoospermia, one should start first by excluding the presence of monomorphic genetic syndromes such as globozoospermia, microcephaly and short tail spermatozoa (4). The increased number of immature spermatozoa may be due to epididymal dysfunction or is a consequence of frequent ejaculations. The increased number of spermatozoa with tapering heads are found in association with varicocele. In a recent study we have reported that the percentage of tapered spermatozoa, spermatozoa containing cytoplasmic droplets and spermatozoa with bent tail are significantly increased in varicocele patients compared to controls (16).
The usefulness of sperm morphology assessment as a predictor of a man’s fertilizing potential has often been challenged due to different classification systems, various slide preparation techniques and problems with reproducibility because of observer variations (12).
According to the literature the importance of sperm morphology as a single and independent predictor of in-vivo and in-vitro fertilization seems to be proven (12).
Testing for antibody coating of spermatozoa
The presence of anti-sperm antibodies in semen can alter the fertilizing ability of the spermatozoa. Being haploid ,sperm cells are immunogenic and display different surface antigens from their diploid counterparts (4). Under normal circumstances, they are protected from the man’s immune system by a basal membrane constituting the blood-testis barrier. When this barrier is ruptured, sperm cells induce the synthesis of anti-sperm antibodies (4). The presence of sperm antibodies coating the spermatozoa is typical of and is considered to be specific for immunologic infertility (6). Sperm antibodies in semen belong to the immunoglobulin classes IgG, IgA or rarely IgM. There are some data suggesting that IgA antibodies may have greater clinical importance than IgG antibodies. The screening test for antibodies is performed on the fresh semen sample and makes use of either the Immunobead method or the mixed antiglobulin reaction test (MAR test).
Immunobeads are polyacrylamide spheres with co-valent bound rabbit anti-human immunoglobulins. The presence of IgG, IgA and IgM antibodies can be assessed simultaneously with this test. Spermatozoa are washed of seminal fluid by repeated centrifugation and resuspended in buffer. The sperm suspension is mixed with a suspension of Immunobeads. The test is considered positive when 25% or more of motile spermatozoa have Immunobead binding (25).
The IgG MAR test is performed by mixing fresh, untreated semen with latex particles or sheep blood cells coated with human IgG. A monospecific antihuman-IgG antiserum is added to this mixture. The formation of mixed agglutinates between particles and motile spermatozoa proves the presence of IgG antibodies on the spermatozoa (25). The diagnosis of immunologic infertility is probable when 50% or more of the motile spermatozoa have particles adherent. Immunologic infertility is suspected when 10%-50% of the motile spermatozoa have adherent particles.
Sperm antibodies could influence sperm function in a variety of ways. For example, sperm agglutination and immobilizing antibodies might limit the number of fertile sperm cells at the site of fertilization (18). Antibody production against sperm surface macromolecules could interfere with critical physiologic fertilization precursor events, such as capacitation and the acrosome reaction (18). It is also possible that antibodies produced against essential intraacrosomal enzyme systems, such as proacrosin-acrosin could impair sperm penetration through egg investment (18).
Whatever the method of action, sperm antibodies have been shown to impair fertility and may account for up to 10% of couples whose infertility is unexplained.
Infection of the genital tract, varicocele, cryptorchidism, testicular torsion and auto-immune disease are the most frequent conditions associated with antisperm antibodies (19).
There are various biochemical markers of accessory gland function, e.g., citric acid, zinc and acid phospatase for the prostate gland ; fructose and prostaglandins for the seminal vesicles, free L-carnitine , glycerophosphocoline, and alfa-glucosidase for the epididymus (25). A low secretory function is reflected in a low total output of the specific marker(s), which may therefore be used for the assessment of accessory gland secretory function. An infection can sometimes cause a considerable decrease in the secretory function of these glands (25). Fructose determination is also useful in cases of dysgenesis of seminal vesicles and in the rare cases of ejaculatory duct obstruction.
The laboratory diagnosis of male infertility has been a topic to study for numerous decades.
Due to the variety of possible sperm and semen abnormalities, a comphrehensive approach using several tests is generally employed to assess the integrity of semen specimens.
Following coitus, a men must deposit an adequate number of functional spermatozoa into his partner’s vagina for pregnancy to occur. (18). Accordingly, testicular function (endocrine, anatomic, biochemical) must be normal for spermatogenesis to proceed normally.
In addition, sperm must be transported through a functional male reproductive duct system, stored for maturation in the epididymus and mixed with accessory sex gland fluids.
Finally, the sperm containing semen must be expelled into the vagina; that requires an intact nervous system that will induce penile erection and normal ejaculation (18). It is abnormalities in these systems that ultimately result in subfertility or infertility.
Male fertility testing is designed to evaluate the quality and quantity of motile sperms present in the ejaculate as well as the fertilizing potential of an individual’s specimen.
For reasons mentioned above, there is no single test that can absolutely confirm an individual’s fertility or infertility.
It should be emphasized that semen is an exception among biological fluids as its parameters display very wide intra-and inter-individual variations (4).
When parameters from a given semen specimen deviate considerably from large population of tested man that ejaculate is likely to be subfertile (18).
More than one specimen is required to establish that a man consistently produces abnormal semen.
Biological evidence of male sterility is only present in cases of azoospermia or globozoospermia or in the presence of a complete lack of sperm motility with underlying genetic deficiencies such as Kartageners syndrome (13).
Because such cases of male sterility are uncommon, clinicians expect to obtain a clear indication of a men’s fertilizing potential from semen analysis.
The clinical value of traditional semen parameters in the diagnosis of male infertility has recently been the subject of considerable debate.
Some authorities clearly hold the view that, apart from a diagnosis of azoospermia (or very severe oligozoospermia) a basic semen assessment is of little clinical value (2).
The diagnosis of a semen sample as «abnormal» or «normal» using the WHO guidelines is artificial and has little diagnostic value.
It is well documented that a considerable number of fertile men are diagnosed as abnormal if such criteria are used (2).
Nevertheless the several less comprehensive studies have examined the predictive value of traditional semen characteristics for in vivo fertility and concluded that these parameters are predictive of pregnancy outcome (2).
The availability of such data allows the estimation of the likelihood of subsequent conception for a new couple attending the clinic based on the semen parameters (2).
In addition to providing a diagnostic/predictive value for in-vivo conception, there is a plethora of studies documenting the assessment of traditional semen parameters that are clinically useful in the management of a couple requiring ART. Recently it has been shown that below a cut-off value of 1x10 6 total motile spermatozoa present in semen no pregnancies were achieved after IUI (5).
It is also reported (3) that a significantly lower pregnancy rate per cycle is achieved if the proportion of normal spermatozoa in the semen was < 10%. Many IUI programs inseminate patients only if a critical number of progressively motile spermatozoa can be isolated from the semen.
In addition to the above, numerous studies have shown that the measurement of traditional semen variables is of clinical value for predicting the likelihood of success of in-vitro fertilization (IVF). Perhaps the most significant single variable is morpholog, whether estimated using strict criteria or by more traditional methods.
When correctly performed, techniques of conventional semen analysis may give reproducible results for certain sperm characteristics. Internal quality control is mandatory and strict standards for technical accuracy must be applied. In doing so, acceptably low levels of inter and intra-observer variability can be obtained and the results may have clinical relevance.
Edited by Aldo Campana,