The normal volume of ejaculate after 2–7 days of sexual abstinence ranges from 2 to 6 ml. However, there are other possibilities as follows (Vasan 2011; Bornman and Aneck-Hahn 2012):

The main component of semen is a coagulated alkaline fluid that comes from the seminal vesicles. This fluid along with the sperm from the vas deferens empties through the ejaculatory duct. Prostatic fluid, the second largest component of seminal volume, generally has a relatively acidic pH of 6.5 and combines with the seminal fluid and sperm in the urethra. Prostatic fluid does not traverse the ejaculatory ducts. Normal semen pH is in the range of 7.2–8.2 and it tends to increase with time after ejaculation. Changes in pH of semen are usually due to inflammation of the prostate or seminal vesicles. A low volume sample with measured pH below 7.0 indicates obstruction of the ejaculatory ducts.

Liquefaction of semen depends on coagulation of proteins found in the seminal fluid as well as the liquefaction by prostate-specific antigen, a proteolytic enzyme, secreted by the prostate. This process may take up to 60 min. If complete liquefaction does not occur after 60 min, it should be noted. Exact liquefaction time is of no diagnostic importance unless > 2 h elapse without any change. The clinical significance of abnormalities in liquefaction is still controversial (Keel 1990). Failure to liquefy is usually a sign of inadequate secretion of the proteolytic enzymes – fibrinolysin, fibrinogenase, and aminopeptidase – by the prostate (Amelar 1962). On the other hand, absence of coagulation may indicate ejaculatory duct obstruction or congenital absence of seminal vesicles.

Viscosity measures the resistance of the seminal fluid to flow. High viscosity may interfere with determination of sperm motility, concentration, and antibody coating of spermatozoa. Normally, semen coagulates upon ejaculation and usually liquefies within 15–20 min. Semen that remains a coagulum is termed non-liquefied, whereas that which pours in thick strands instead of drops is termed hyperviscous. Importantly, liquefaction should be differentiated from viscosity, as abnormalities in viscosity can be the result of abnormal prostate function and/or the use of an unsuitable type of plastic container. Viscosity of semen is noted after liquefaction, although the clinical significance of hyperviscous semen is controversial. There is no correlation between seminal hyperviscosity and semen cultures, leukocytes, or presence of sperm antibodies; however, worse outcomes after in vitro fertilization (IVF) with seminal hyperviscosity have been observed (Munuce et al. 1999; Esfandiari et al. 2008). Sperm processing prior to intrauterine insemination (IUI) can be considered, if there is a clinical concern for hyperviscosity.

Concentration of sperm in unstained preparations of fresh/washed semen sample is determined using, preferably, a phase-contrast microscope with volumetric dilution and hemocytometry. Sperm count is typically reported as concentration (millions of sperm per milliliter) as well as total sperm count (sperm concentration × ml of semen) in the ejaculate. Normozoospermia, oligozoospermia, and azoospermia are diagnosed based upon total sperm count. Azoospermia refers to the absence of sperm in the seminal plasma. Prior to the diagnosis of azoospermia, the sample should be centrifuged and the pellet examined for the presence of sperm. Oligozoospermia (also often called oligospermia) refers to seminal plasma concentration < 15 million/ml. This finding can accompany a variety of defects and has implications for the type of assisted reproductive options that can be utilized, as there are significant reductions in pregnancy rates (Smith et al. 1977).

The efficient passage of spermatozoa through cervical mucus is dependent on rapid-progressive motility, that is, spermatozoa must have a forward progression of a minimum of 25 μm/s (Björndahl 2010; Lindholmer 1974). Reduced sperm motility can be a symptom of disorders related to male accessory sex gland secretion and the sequential emptying of these glands. Rapid and slow-progressive motility is calculated by the speed at which sperm moves with flagellar movement in a given volume as a percentage (range 0–100 %) by counting 200 sperms and classified as follows:

Supravital staining differentiates between live and dead sperm and is assessed when sperm motility is < 50 %. A large proportion of vital, but immotile, sperm may indicate structural defects in the sperm tail (World Health Organization 1999) or Kartagener’s syndrome. A high percentage of immotile, nonviable (dead) sperm may indicate epididymal pathology (World Health Organization 2010). Antisperm antibodies (ASA) may also be present, if the immotile sperms are dead (Björndahl et al. 2010a).

The staining of a seminal smear (Papanicolaou Giemsa, Shorr, and Diff-Quik) allows the quantitative evaluation of normal and abnormal sperm morphological forms in an ejaculate. Smears can be scored for morphology using the World Health Organization (WHO) classification or by Kruger’s strict criteria classification (Menkveld et al. 1990). WHO method classifies abnormally shaped spermatozoa into specific categories based on specific head, tail, and mid-piece abnormalities, which is based on the appearance of sperm recovered from postcoital cervical mucus or from the surface of zona pellucida (>30 % normal forms). In contrast, Kruger’s strict criteria classifies sperm as normal only if the sperm shape falls within strictly defined parameters of shape, and all borderline forms are considered abnormal (>14 % normal forms).

Capacitation is a series of biochemical and structural changes that spermatozoa go through to undergo acrosome reaction (AR) and be able to fertilize. The process takes place in the female genital tract but can be induced in vitro by incubating spermatozoa with capacitation-inducing media. It is thought to have a role in preventing the release of lytic enzymes until spermatozoa reach the oocyte (Tesarik 1989). One of the signs of capacitation is the display of hyper-activation by spermatozoa. At the present time, the clinical value of sperm capacitation testing remains to be determined.

The interaction between spermatozoa and the zona pellucida is a critical event leading to fertilization and reflects multiple sperm functions (i.e., completion of capacitation as manifested by the ability to bind to the zona pellucida and to undergo ligand-induced AR) (Oehninger et al. 1994; Liu and Baker 2003; Consensus workshop on advanced diagnostic andrology techniques. ESHRE (European Society of Human Reproduction and Embryology) Andrology Special Interest Group 1996). The most common sperm-zona pellucida binding tests currently utilized are the hemizona assay (or HZA) and a competitive intact-zona binding assay (Quintero et al. 2005; Fénichel et al. 1991). The HZA which uses non-fertilized oocytes is useful to determine the cause, in couples who have failed to fertilize during regular IVF. As the binding is species-specific, human zona must be used, thus limiting the utility of these assays (Fénichel et al. 1991; Cross et al. 1986). The induced AR assays appear to be equally predictive of fertilization outcome and are simpler in their methodologies. The use of a calcium ionophore to induce AR is currently the most widely used methodology (Henkel et al. 1993; Katsuki et al. 2005).

This assay is also called as sperm capacitation index or zona-free hamster oocyte penetration assay. The concept of the sperm penetration assay was introduced by Yanagamachi (1972; Yanagimachi et al. 1976). It yields information regarding the fertilizing capacity of human spermatozoa by testing capacitation, AR, sperm/oolemma fusion, sperm incorporation into the ooplasm, and decondensation of the sperm chromatin during the process. However, penetration of the zona pellucida and normal embryonic development are not tested. The spermatozoa penetration assay (SPA) utilizes the golden hamster egg, which is unusual in that removal of its zona pellucida results in loss of all species specificity to egg penetration. Thus, a positive SPA does not guarantee fertilization of intact human eggs nor their embryonic development, whereas a negative SPA has not been found to correlate with poor fertilization in human IVF (Yanagimachi et al. 1976). The acrosin assay, an indirect measure of sperm’s penetrating capability, measures acrosin, which may be responsible for penetration of the zona pellucida and also triggering the AR (Rogers and Brentwood 1982). Measurement of acrosin is thought to correlate with sperm binding to and penetration of the zona pellucida (Cross et al. 1986; Cummins et al. 1991).

Mammalian fertilization involves the direct interaction of the sperm and the oocyte, fusion of the cell membranes, and union of male and female gamete genomes. Although a small percentage of spermatozoa from fertile men also possess detectable levels of DNA damage, which is repaired by oocyte cytoplasm, there is evidence to show that the spermatozoa of infertile men possess substantially more DNA damage that may adversely affect reproductive outcomes (Evenson et al. 1999; Zini et al. 2001). There appears to be a threshold of sperm DNA damage which can be repaired by oocyte cytoplasm (i.e., abnormal chromatin packaging, protamine deficiency) beyond which embryo development and pregnancy are impaired (Ahmadi and Ng 1999; Cho et al. 2003).

Reactive oxygen species, (ROS) also referred to as free radicals, are formed as a by-product of oxygen metabolism. Contaminating leukocytes are the predominant source of ROS in these suspensions (Aitken et al. 1989a, b). They can be eradicated by enzymes (e.g., catalase or glutathione peroxidase) or by nonenzymatic antioxidants, such as albumin, glutathione, and hypotaurine, as well as by vitamins C and E. Small amounts of ROS may be necessary for the initiation of critical sperm functions, including capacitation and AR. On the other hand, a high ROS level produces a state known as oxidative stress that can lead to biochemical or physiologic abnormalities with subsequent cellular dysfunction or cell death. Significant levels of ROS can be detected in the semen of 25 % of infertile men, whereas fertile men do not have a detectable level of semen ROS (Aitken et al. 1989b, 1991; Agarwal et al. 2006). Sperm ROS can be measured by using cellular probes coupled with flow cytometry by the detection of chemiluminescence (Marchetti et al. 2002). Briefly, this is done by incubating fresh semen or sperm suspensions with a redox-sensitive, light-emitting probe (e.g., luminol) and by measuring the light emission over time with a luminometer. The clinical value of semen ROS determination in predicting IVF outcome remains unproven, but identifying oxidative stress as an underlying cause of sperm dysfunction has the advantage for suggesting possible therapies. Administration of antioxidants has been attempted in several trials with mixed results. Currently, there are no established semen ROS cutoff values that can be used to predict reproductive outcomes (Agarwal et al. 2005, 2008).

Sperm proteomics, an experimental technique, used extensively in several branches of medicine, may identify some of the molecular targets implicated in sperm dysfunction (Aitken and Baker 2008). Sperm proteomics allows comparison of protein structure of normal and defective spermatozoa (Aitken 2010).

The TZI is an indication of the number of abnormalities present per abnormal spermatozoon. According to the 1992 WHO manual (World Health Organization 1992), each abnormal spermatozoon can have one to four abnormalities, viz., a head abnormality, a neck/mid-piece abnormality, a tail abnormality, or the presence of a cytoplasmic residue. These abnormalities can occur as a single defect or in a combination of two, three, or all four abnormalities simultaneously. The classification of spermatozoa for the TZI is recorded simultaneously, on a five-key laboratory counter, with the recording of spermatozoa as normal or abnormal, in specific classes. The total number of abnormalities recorded are added together and divided by the total number of abnormal spermatozoa, i.e., 100 minus the percentage of morphologically normal spermatozoa.

Sperm acrosomal morphology was evaluated by light microscopy at ×1250 oil magnification based on acrosomal size and form as well as staining characteristics (Menkveld and Kruger 1996). Results were expressed as the AI (% normal acrosomes). For the evaluation of acrosome morphology, the same principles as for the evaluation of normal sperm morphology according to strict criteria are applicable. For an acrosome to be regarded as normal, the acrosome must have a smooth normal oval shape, with the same dimensions as for a normal spermatozoon. Acrosomes must be well-defined and comprising about 40–70 % of the normal-sized sperm head. The post-acrosomal part of the sperm head can be abnormal, but the rest of the spermatozoon must be normal; thus no neck/mid-piece and tail abnormalities and no cytoplasmic residue may be present. If the spermatozoon is classified as normal, the acrosome must always be classified as normal. The acrosome evaluation can be performed simultaneously with the routine morphology evaluation and the TZI, with the use of two laboratory counters. As with the normal sperm morphology, at least 100 spermatozoa are evaluated. The repeatability of the AI is be determined and should be within acceptable limits.

Reference intervals are the most widely used tool for the interpretation of clinical laboratory results. Reference interval development has classically relied on concepts elaborated by the International Federation of Clinical Chemistry Expert Panel on Reference Values during the 1980s. These guidelines involve obtaining and classifying samples from a healthy population of at least 120 individuals and then identifying the outermost 5 % of observations to use in defining limits for two-sided or one-sided reference intervals. Pre-2010 WHO guidelines were based on data obtained from laboratories that used different methodologies and examined different male populations, not supported by standardized methods or without the definition of fertile population. The male population studied included men without proven paternity, patients of human reproduction clinics that sought treatment, semen donors, and vasectomy candidates. Semen donors can be fertile and vasectomy candidates are very likely to be fertile, although there is no data about how long it took for their partners to get pregnant (Cooper et al. 2010). The cutoff point of 20 × 10⁶/ml was suggested as the lower normal value for sperm concentration in an ejaculate (World Health Organization 1999). However, there are studies indicating sperm concentrations of subfertile men to be less than 13.5 × 10⁶/ml (Guzick et al. 2001) and 31.2 × 10⁶/ml for fertility status (Nallella et al. 2006). Therefore, caution must be exercised with interpretation of the semen analysis based upon the reference values as men may be infertile with “normal” semen parameters or alternatively can be fertile with markedly “abnormal” semen profiles. There is likely no upper limit of semen morphology, motility, or count as pregnancy rates appear to generally increase with increasing numbers as well as improved sperm morphology and motility (Garrett et al. 2003).