Semen analysis is the initial and single most important test in male fertility evaluation. Although it does evaluate a man’s fertility potential, it does not, however, predict with accuracy the likelihood of pregnancy [17]. Patients should be provided with instructions on proper sample collection. Semen analysis should be performed after 3–4 days of sexual abstinence [5], principally because semen volume and sperm count are negatively affected by more frequent ejaculations [18]. In addition, significant reductions in the percentage of sperm motility and normal morphology were detected after 10 days of abstinence [19]. Semen can be collected by means of masturbation into a clean container or by intercourse into special semen collection condoms devoid of substances toxic to sperm. The specimen is preferably collected at the laboratory. If home collection is desired, the specimen should be brought to the laboratory within 1 h of collection and kept at room or body temperature during the transport. To ensure accurate and homogeneous results, samples are better analyzed at laboratories implementing quality control programs for semen analysis and adopting the latest World Health Organization (WHO) protocols for semen testing and reporting of reference values (Table 1.1) [20]. The components of the semen analysis include:

Morphology: (Normal forms >4%, 5th percentile, 95% CI 3–4; Kruger’s strict criteria—Normal forms >14%) Sperm morphology is routinely examined on a semen smear that has been air-dried, fixed, and stained. The Papanicolauo, Shorr, or Diff-Quik smear stains are advocated by the WHO as they provide adequate color to spermatozoa. Scoring is then performed according to the WHO classification [20], or to Kruger’s strict criteria classification [21]. The WHO criteria for normal morphology were originally established after observing spermatozoa recovered from postcoital cervical mucous or from the surface of the zona pellucida. In contrast, Kruger’s strict criteria classify sperm as normal only if their shape falls within strictly defined parameters. Regardless of what standard is used, a sperm is considered normal when it has a smooth oval head, intact and slender midpiece, principal piece without breaks, and a clearly visible acrosome with vacuoles not exceeding 20% of the acrosomal area. When less than 4% of sperm have normal morphology, the term teratozoospermia (or teratospermia) is given. A number of defects are identified and classified according to the part of the sperm affected. Head defects include: oval heads, tapering pyriform and vacuolated heads, absence of acrosome (globozoospermia), double heads, and heads with irregular forms (amorphous). Midpiece defects include: thin, thick, or irregular midpiece and asymmetric midpiece insertion into the head. Finally, tail defects include: tail coils, or 90° bends (hammer head), or breaks. Duplication of the tail into two, three, or even four tails on a single sperm is sometimes observed.

Agglutination: (Normal Absent) agglutination is clumping of sperm. The word comes from the Latin agglutinare meaning “to glue.” It differs from sperm aggregation, which is the adherence of spermatozoa to debris or other elements of the ejaculate. Four grades of agglutination are reported according to the type and extent of interaction.
Extensive agglutination is suggestive of immunologic infertility and warrants detection of anti-sperm antibodies.

White Blood Cells: (Normal <1 million/ml) Leukocytospermia is a term given in the presence of WBCs in semen, which can directly or indirectly contribute to infertility [22]. Direct counting of round cells in a semen sample is highly inaccurate because white blood cells can be difficult to distinguish from immature germ cells using light microscopy. As a result, specialized testing needs to be implemented aiming to differentiate between round cells. Immunocytologic staining of semen samples using monoclonal antibodies is considered the gold standard method in this regard [23]. However, it is not widely performed due to difficulties faced in standardizing the monoclonal antibodies used in staining. Consequently, the WHO recommends peroxidase staining as the best next option to diagnose leukocytospermia [20]. Peroxidases are enzymes that break down hydrogen peroxide liberating oxygen, which oxidizes the benzidine derivate present in the staining solution. As a result, a brown color appears that allows the identification of leukocytes under light microscopy. This test works best with polymorphonuclear granulocytes and macrophages as they are rich in peroxidases. However, it fails to stain lymphocytes that represent about 5% of leukocytes in semen [24].

Additional sperm tests attempt to identify specific functional deficiencies in sperm. While there is currently insufficient evidence to support the routine use of these tests in patient evaluation, on occasion these tests can be helpful for specific situations [5]. Advanced sperm function testing may be considered in men with unexplained infertility, 1 or more abnormal semen parameters on repeated semen samples, recurrent pregnancy loss or failure of intrauterine insemination (See Chap. 2 for further discussion on level 2 testing). The most commonly performed tests include:

The contribution of the hypothalamic-pituitary testicular axis to normal spermatogenesis is well known. As such, an endocrine evaluation is sometimes performed on patients presenting with infertility. Indications for testing include: abnormal semen analysis, specifically when the sperm concentration is less than 10 million/ml; presence of symptoms of hypogonadism; or existence of other clinical findings suggestive of a specific endocrinopathy such as gynecomastia or testicular atrophy [5]. At a minimum, serum follicle-stimulating hormone (FSH) and testosterone levels should be ordered initially and in the presence of abnormal levels, a repeat measurement of total and free testosterone, serum luteinizing hormone (LH), and prolactin levels should be obtained [5]. It is important for the clinician to be aware of the relationship between these hormones to identify the clinical condition. Primary testicular failure is suggested when a low serum testosterone is associated with high FSH and LH levels. Secondary causes of hypogonadism are suggested when both gonadotropins as well as serum testosterone levels are low. A representation of the different hormone findings in various clinical entities is depicted in Table 1.2.