13.1 Introduction

Semen analysis is a key step in the evaluation of the fertility potential of the male partner of a couple, as it reflects not only the efficiency of spermatogenesis, but also the integrity of post-testicular structures necessary for sperm transport and anterograde ejaculation. The WHO reference values of sperm parameters are based on the analysis of 3500 men (from 12 countries) in couples with time to pregnancy of one year or less. The current thresholds refer to the one-sided lower reference limit of the fifth percentile and are shown in Table 13.1 together with the 50th percentile [1, 2]. It should be stressed that these reference values are applicable only if the analysis is performed according to the recommended standardized procedures. The WHO manual is freely accessible at the following link: https://www.who.int/publications/i/item/9789240030787. The majority of fertile men is expected to have their parameters within the range of the 50th percentile. However, fertility is a “couple-concept” and even severe alterations of semen parameters (much below the fifth percentile) can be compatible with the induction of pregnancy, provided that the female partner’s fertility status is optimal. Vice versa, exceptionally good sperm count does not always imply natural conception. In fact, there is a substantial overlap of semen examination results between fertile and infertile men [3].

Table 13.1 Principal semen parameters and their distribution corresponding to 5th and 50th percentile according to the WHO sixth edition guidelines [1]

13.2 Semen Analysis: General Concepts

Semen evaluation is divided into two parts: macroscopic and microscopic analysis (Fig. 13.1). It is essential that the laboratory performs the analysis according to the current WHO guidelines and participate at an external quality control (EQC) program. In fact, wide discrepancies in the assessments of sperm count, motility, and morphology can result when comparing semen analysis outcomes from different laboratories [4, 5]. Together with EQC programs, participating in periodical teaching courses improves the experience of the laboratory and allows to maximize the probability of obtaining the established standards of quality [6]. In consideration of the above, when interpreting a semen analysis report, it is important that the clinician ascertains whether a given laboratory participates in EQC programs with success.

Fig. 13.1
figure 1

Principal parameters evaluated during semen analysis

For each part of the analysis, different parameters are considered and depending on their alteration, different clinical conditions can be suspected. In case of normozoospermia, a second analysis is not mandatory. On the contrary, if one or more semen parameters are altered, repetition is necessary in order to rule out potential pre-analytical and analytical factors, together with biological variability. Among pre-analytical factors, inappropriate abstinence time, incomplete collection of the ejaculate, inadequate transport of the semen sample would request a short-term repetition. Analytic variables can derive from casual and systematic errors during the procedures. Endogenous factors such as high fever or medications may interfere with spermatogenesis during its entire length (approx. 72 days), therefore the repetition should take place after the completion of the entire spermatogenic cycle.

13.3 Macroscopic Evaluation of Semen

Macroscopic evaluation refers to the chemical and physical parameters of the ejaculate including fluidification, viscosity, appearance, odor, volume, and pH. The main macroscopic alterations are summarized in Table 13.2.

Table 13.2 Summary of the main semen alterations and related suspected clinical conditions

13.3.1 Liquefaction, Viscosity, and Appearance

Liquefaction of the semen needs 15–30 min, and this process is regulated by the prostatic secretion, which is rich in citric acid acting in synergy with proteolytic enzymes (lysozyme, α-amylase, and β-glucuronidase) and prostatic specific antigen (PSA), a trypsin-like protease, that cleaves the semenogelin proteins. PSA may be altered due to congenital or acquired (for instance, prostatitis) factors.

Viscosity is the characteristic of an ejaculate specimen to exhibit a homogeneous stickiness, which may be reduced or increased due to various conditions reported in Table 13.2. A normally liquefied sample will have an opalescent appearance, with a cream/grey color. This parameter is influenced by the concentration of spermatozoa: a transparent ejaculate might indicate an extreme reduction in the number of spermatozoa, while a highly concentrated specimen will be opaque. High viscosity may indicate prostatitis. A red-brown appearance of the ejaculate should alert the clinician for the presence of erythrocytes in the semen. This condition is known as hematospermia and based on the age of the patient, it can have different etiology: in younger men (<40 years), it could be due to inflammation or urogenital infections, while in older men (>40 years), it could underlie more serious pathologies, such as prostate cancer [7].

13.3.2 Volume and pH

Seminal plasma is composed mainly by the secretions of the accessory glands (about 90%) whereas the contribution of the epididymis and bulbourethral glands are minimal. Semen volume not only expresses the secretory activity of these glands, but also their responses to autonomous nerve stimulation elicited by sexual arousal, which will lead to smooth muscle contractions that empties each gland. Prostate and seminal vesicles are target organs of androgens; therefore, severe androgen deficiency is associated with lower semen volume. In addition, when seminal fluid volume is markedly reduced, the clinician should ask the patient whether the collection was complete.

Both prostate and seminal vesicles contribute to the semen pH with their secretions: prostate produces an acidic fluid, while seminal vesicles produce an alkaline fluid leading to the typical neutral pH, around 7.2–7.4. Changes in pH may indicate different pathological conditions, as shown in Table 13.2, even though this parameter is susceptible to analytical biases as well. The pH typically increases with time after ejaculation, so when a highly alkaline specimen is observed, poor sample handling and delay in analysis should be excluded prior interpretation [1].

13.4 Microscopic Evaluation of Semen

Microscopic evaluation refers to the characteristics of the cellular fraction of semen and includes agglutination, aggregation, sperm concentration, motility, vitality, morphology, and round cells. The main microscopic semen alterations are summarized in Table 13.2.

13.4.1 Agglutination and Sperm Antibodies

Aggregation and agglutination are two different parameters. Aggregation is the adherence of both motile and immotile spermatozoa to mucus strands or non-sperm cells. Agglutination, instead, is the tendency of motile spermatozoa to form clumps and it can be categorized based on the degree of agglutination and the sperm structures involved [1]. This feature could indicate the presence of an immunological cause of infertility, even though the presence of anti-sperm antibodies (ASA) should be confirmed by further testing, such as the commercially available mixed antiglobulin reaction test (MAR test). It is important to notice that ASA and agglutination not always come together. Indeed, sperm agglutination can be caused by other factors rather than autoimmunity, as well as antibodies can be present without sperm agglutination.

ASA have been found in several pathological conditions which may lead to the interruption of the blood-testis barrier, such as testicular torsion, testicular carcinoma, and orchitis [8]. The presence of ASA as an isolated abnormality is seen in less than 5% of infertile males and occurs mainly in association with normal sperm counts [8]. In fact, testing for ASA can be requested in case of unexplained couple infertility with normozoospermic male partner [9]. ASA can interfere with physiological reproduction at many levels, affecting sperm number and motility, the ability to transit through cervical mucus, acrosome reaction, and zona pellucida binding [9, 10].

13.4.2 Sperm Number, Motility, Vitality, and Morphology

Sperm number is a quantitative marker of spermatogenesis, whereas sperm motility, vitality, and morphology are qualitative parameters.

Sperm concentration is defined as the number of spermatozoa per unit volume of semen. As stated above, semen volume mainly originates from the accessory sex glands, hence sperm concentration is influenced by their activity and does not represent a direct measure of testicular sperm output. A much better reflection of the capacity of the testis to produce sperm refers to total sperm count (TSC). The fifth percentile reference value for TSC is >39 millions of spermatozoa.

Three quantitative alterations can be distinguished as follows:

  • Azoospermia: absence of spermatozoa in the ejaculate and in the pellet after centrifugation.

  • Cryptozoospermia: absence of spermatozoa in the ejaculate but present in the pellet after centrifugation.

  • Oligozoospermia: sperm concentration or TSC lower than the fifth percentile value (Table 13.1).

Sperm motility is classified based on the direction and velocity of sperm movement into the following categories: (i) progressive motility (rapid and slow); (ii) non-progressive sperm movement; and (iii) immotile spermatozoa [1]. The reduction in sperm motility, called asthenozoospermia (total motility <42% and progressive motility <30%) can be due to congenital or acquired factors. Especially extreme asthenozoospermia, with or without associated teratozoospermia (see below), is due to different genetic anomalies [11]. Sperm motility defects can be present in the context of a syndrome, known as Primary Ciliary Dyskinesia (PCD). PCD is a rare recessive autosomic disorder characterized by defects of motile cilia and flagella, leading to asthenozoospermia, chronic respiratory tract infections, and situs inversus [12]. DNAI1 and DNAH5 gene mutations account for about 30% of PCD cases, whereas the remaining 70% could be explained by mutations in other 26 genes involved in various ciliary ultrastructural defects [13].

In case the percentage of immotile sperm is higher than 40%, vitality test is indicated in order to distinguish vital and dead cells [1]. The extreme reduction in sperm vitality is known as necrozoospermia.

Sperm morphology assessment is aimed at the evaluation of the shape of spermatozoon, which consists of a head and tail, connected through the midpiece, a thicker part of the tail containing mitochondria. The rest of the tail consists of a principal piece (axoneme or ciliary structure surrounded by outer dense fibers), a fibrous sheath with longitudinal columns and an endpiece.

The evaluation of sperm morphology is the most challenging part of semen analysis since its standardization is more complex than the measurement of the other parameters. Human ejaculates contain spermatozoa with a wide range of different morphological appearances. Sperm morphology is assessed by the “strict” criteria and any slight abnormality of the spermatozoa will classify it to have abnormal morphology. In fact, the large majority of spermatozoa does not fulfil the WHO criteria for being considered as “normal” even in the specimen of fertile men [1]. Consequently, the 50th percentile of reference values would correspond to only 14% of typical forms. In the large majority of patients affected by teratozoospermia (normal forms <4%), head, midpiece, tail, and cytoplasmic residue defects are combined, and different spermatozoa may present different types of abnormalities. These mixed morphological defects are usually related to defective spermatogenesis or epididymal inflammation. The presence of excessive cytoplasm around the midpiece is associated with an increased production of ROS and oxidative stress [14]. The WHO guidelines included the calculation of indices of multiple sperm defects among the extended examinations of morphological abnormalities. However, due to the overlapping values between fertile and infertile men, the application of these indices in the clinical practice is still questionable [1].

The opposite of “polymorphic” teratozoospermia are those rare conditions, where all abnormal spermatozoa bear the same specific anomaly. These monomorphic teratozoospermia cases are related to genetic defects and among them, the two typical monomorphic sperm head defects are globozoospermia and macrozoospermia [15]. Both conditions are associated with functional abnormalities leading to severe impairment of fertilizing ability. For instance, macrozoospermia or macrocephalia is characterized by the presence of spermatozoa with large head and multiple flagella. It is a rare recessive disease due to mutations in AURKC gene. Macrozoospermia is typically associated with a high rate of aneuploidy and polyploidy caused by the nondisjunction of chromosomes or defective cytokinesis during meiosis [16]. It has been reported that sulfasalazine treatment may induce transient increase of large headed spermatozoa in the ejaculate with an improvement in sperm morphology and motility after the cessation of the treatment [17]. Therefore, careful medical history taking is essential to rule out iatrogenic teratozoospermia.

An example of extreme astheno/teratozoospermia (AT) are the sperm tail abnormalities. Multiple morphological abnormalities of the sperm flagellum (MMAF) are a rare form of AT, characterized by a mosaic of sperm cells with absent, short, irregular, and coiled flagellum. Routine semen analysis can easily evidence MMAF through optic microscopy, but the transmission electron microscopy and genetic testing are necessary to define the exact ultrastructural defects and their origin, respectively. Besides the major candidate gene (DNAH1), other gene defects have been identified and currently a total of 18 genetic causes are known for being responsible for 30–60% of MMAF cases [18]. There is a growing evidence that mutation in some of these genes (DNAH17, CFAP65, and CEP135) confers very poor prognosis even for IntraCytoplasmic Sperm Injection (ICSI) pregnancies [18]. It is therefore possible that in the near future, genetic testing in MMAF patients may have also a predictive value for ICSI outcome.

It is well known since many years that sperm centriole alterations are associated with unsuccessful ICSI. Human spermatozoa contain two centrioles: the proximal one (PC), located near the head base, and the distal one (DC), located at the base of the axoneme. Sperm centrioles have many different functions, which are essential not only for flagellum movement but also for normal morphology, cell division, and zygote development [19]. In fact, sperm is the sole contributor of centrioles to the zygote, and is essential for bipolar spindle formation during the first division after fertilization. Therefore, sperm centriole defects may cause failure of embryo development. The spectrum of semen phenotypes associated with centriole defects ranges from azoo/oligozoospermia to astheno/teratozoospermia. For instance, abnormal positioning of centrioles can lead to dysplasia of the fibrous sheath [19]. In addition, centriole defects can also cause acephalic spermatozoa syndrome (ASS). It can be due to the dissociation between PC and DC causing the sperm neck to break, resulting in decapitated sperm heads. To date, seven genes (SUN5, BRDT, PFMBP1, TSGA10, DNAH6, HOOK1, and CEP112) have been found mutated in infertile men with acephalic spermatozoa [20].

13.4.3 How to Interpret the Three Principal Sperm Parameters

Sperm number, motility, and morphology should not be considered individually but in combination. In fact, the main clinical question is, how many progressively motile and normal spermatozoa are present in an ejaculate. For instance, in semen presenting all three parameters corresponding to the WHO lowest reference value, there are 11.7 million of progressively motile and 1.56 million morphologically normal spermatozoa. However, a moderately oligozoospermic man (for instance, 25 million spermatozoa) showing 50% of progressive motility and 7% of typical morphology would have a higher number of total progressive (12.5 million spermatozoa) and normal spermatozoa (1.75 million spermatozoa) in his ejaculate in respect to a man defined as “normozoospermic” based on the fifth percentile of each of the three parameters. Therefore, the clinical consequence of isolated oligozoospermia in this man remains questionable. Similar scenarios can occur when sperm number is very high whereas motility and morphology is slightly below the fifth percentile. On the other hand, it is also clear that when the three parameters are lower than the defined thresholds, i.e., oligoasthenoteratozoospermia (OAT), the odds of subfertility sharply increase [3]. Some authors propose to calculate the total motile sperm count (TMSC) which correlates better with spontaneous pregnancy than individual parameters [21].

13.4.4 Round Cells

Apart from spermatozoa, epithelial and round cells can be observed in the ejaculate. Epithelial cells are derived from the genitourinary tract, while round cells refer to either leukocytes or immature germ cells [1].

Leukocytospermia (LCS) is defined as the presence of more than 1 × 106 leukocytes (white blood cells, WBC) per ml of semen. The relationship between WBCs concentration and semen quality is questioned in men asymptomatic for a genital tract infection [22]. The presence of a high number of WBCs and a reduced ejaculated volume has been proposed as an indicator for infections and inflammations of the genitourinary tract [23]. However, the association of LCS with fertility potential is controversial [22].

13.5 From Semen Analysis to Diagnosis

Semen analysis, together with medical history and physical exam, represents the first step of the diagnostic work-up of infertile men. In some instances, the result of semen analysis is suggestive for specific forms of reproductive impairments, which then will be confirmed by subsequent laboratory and instrumental exams (e.g., additional semen microbiological examinations in case of suspected infections or inflammations; transrectal ultrasound exam in case of suspected distal obstruction; genetic exams in case of monomorphic teratozoospermia, etc.). In this paragraph, we are going to briefly describe informative semen analysis’s outcomes.

13.5.1 General Considerations on Azoospermia/Cryptozoospermia

Azoospermia, affecting about 1% of the general male population, is incompatible with natural pregnancy. It is extremely important that the laboratory performs the analysis of semen sediment after centrifugation, allowing to differentiate between azoospermia and cryptozoospermia. Although the likelihood of spontaneous fertilization remains extremely unlikely even in case of cryptozoospermia, the importance in distinguishing between these two conditions refers to different in vitro fertilization options, i.e., in azoospermia testicular sperm extraction (TESE) must precede ICSI. As stated above, when sperm parameters are abnormal, semen analysis must be repeated. Therefore, the diagnosis of azoospermia must be confirmed in at least two semen analysis (possibly after the whole spermatogenic cycle) because it could be a temporary disorder, or it could alternate with cryptozoospermia.

Azoospermia can be divided into obstructive (OA) and non-obstructive (NOA) forms. In OA, spermatogenesis is unaffected, and the absence of spermatozoa in the ejaculate is due to bilateral distal or proximal obstruction of the urogenital tract. NOA is a symptom that can be associated with three different testis histology: Sertoli cell-only syndrome (SCOS), maturation arrest at different stages of germ cell maturation (MA), or hypospermatogenesis. The differential diagnosis is fundamental as the patient management and treatment options are different [24]. The definition of the etiology is also relevant, since NOA due to primary spermatogenic failure cannot be treated with medical therapy, whereas if the cause is a hypothalamus-pituitary dysfunction, the treatment with gonadotropin is effective in about 90% of cases [25]. In the large majority of OA cases and in primary testicular failure, TESE is the most viable treatment option. Which Parameters May Help Clinicians to Distinguish Between NOA and OA?

In Table 13.3, we report five examples of seminal analysis output that are suggestive for a specific etiology. As stated above, when no spermatozoa is present in the ejaculate, it is important to evaluate the sediment after centrifugation, as their presence is indicative of cryptozoospermia (scenario A).

Table 13.3 Examples of routine semen analysis outputs in azoospermia

When azoospermia is confirmed by examining the sediment, three parameters are useful to distinguish between OA and NOA: semen volume, pH, and the presence of immature germ cells. When semen volume is reduced, different pH may indicate different etiology. For instance, an acidic pH indicates the absence of the seminal vesicles secretions, which together with the absence of spermatozoa could strongly suggest congenital bilateral absence of the vas deferens (CBAVD) associated to the agenesis of seminal vesicles (scenario B). Reduced semen volume with normal pH, instead, can be seen in case of severe hypoandrogenism (scenario C). When semen volume and pH are normal, the analysis of the pellet smears for the presence of spermatogenetic cells is useful [9]. If they are present, a maturation arrest can be suspected (scenario D), while if they are absent, a proximal obstruction can be the cause (scenario E). Which Sperm Parameters are Informative in Quantitative and Qualitative Impairment of Spermatogenesis?

In Table 13.4, we report some examples of oligo/astheo/teratozoospermia. The reduction in sperm concentration, especially <5 × 106/ml, is rarely a stand-alone condition, as it is typically associated with other semen alterations. However, isolated oligozoospermia can be found in case of patients with congenital hypogonadotropic hypogonadism treated with gonadotropins (scenario A). In such cases, all semen parameters are normal except for the sperm number which in the majority of cases remains below the reference value.

Table 13.4 Examples of routine semen analysis output showing quantitative and/or qualitative defects

Regarding combined forms of semen alterations, scenario B reports a man exhibiting oligoasthenoteratozoospermia (OAT) with other anomalies: agglutination is present, increased viscosity, and leukocytospermia. In this particular patient, the cause of OAT could be an inflammation or infection of the urogenital tract.

Asthenozoospermia is the most frequent sperm defect observed in infertile men, with variable degrees of severity. In case of total sperm immotility, it is mandatory to distinguish between immotile live and dead sperm (necrozoospermia). A large proportion of live but immotile cells, as seen in scenario C, may indicate structural defects in the flagellum [26]. On the other hand, when complete asthenozoospermia is due to a high percentage of dead cells, as seen in scenario D, it can be associated with an epididymal pathology [27, 28], adult polycystic kidney disease or it can be observed often in patients with spinal cord injury [28]. A second ejaculate within a short-term period of approximately 60 min has been shown to improve seminal quality as compared with the first ejaculate in patients with epididymal necrozoospermia [29].

Semen analysis outcomes reported in scenario E exhibit alterations of motility and morphology classified as asthenoteratozoospermia. It is important that the laboratory describes whether the atypical forms have different anomalies or if they are the same. In fact, this is a case of polymorphic teratozoospermia in which defects of the tail and the head are present. This scenario is compatible with MMAF (see above). Scenario F reports a specific monomorphic teratozoospermia, called globozoospermia. This condition is characterized by the production of spermatozoa with round head without acrosome, hence they are missing PLCζ, an acrosome phospholipase, essential for the activation of the oocyte. Globozoospermia is a rare autosomic recessive disease and the most prevalent genetic defect observed in men with 100% globozoospermia is the complete deletion of DPY19L2 [16]. In case of complete globozoospermia, ICSI should be followed by artificial oocyte activation [11].

13.6 Value and Limits of the Semen Analysis

Routine semen analysis is a valuable diagnostic test although it has its own limitations. In fact, because infertility involves male and female factors, it will not be possible to predict fertility using parameters from either partner alone, unless there is azoospermia in the man or premature ovarian failure in the woman [30]. While azoospermia undoubtedly causes infertility, the presence of triple defects—reduced sperm number, motility, and morphology—also increases the likelihood of a male factor responsible for couple infertility [3]. On the other hand, in case of normozoospermia, searching for female factors could have more relevance at the initial stages of the diagnostic work-up. Despite many discussions about the clinical importance of semen parameters, semen analysis remains a fundamental step in clinical andrology. This is because it may reveal a series of anomalies, which can guide clinician to further explore the etiology behind these defects. It is therefore important that the report is fully reliable, which implies that semen analysis is performed in specialized laboratories strictly following the WHO guidelines and participating at external quality control programs. Clinicians should also be aware of the fact that semen parameters are susceptible of variations due to pre-analytical, analytical factors, together with intraindividual biological variability. Therefore, when semen alterations are observed, a second evaluation is mandatory.

Although routine sperm analysis is able to diagnose extreme conditions which are typically associated with functional and genetic defects, microscopic examination does not provide direct information on the functional integrity of the spermatozoa. For instance, it is unable to detect DNA fragmentation, ultrastructural defects, the inability of spermatozoa to undergo acrosome reaction, to bind the zona pellucida or to fertilize the egg. For this reason, the new WHO manual also describes a series of test which are part of the so-called extended examinations. Among them, the evaluation of sperm DNA fragmentation is already introduced in many laboratories all over the world; however, its clinical utility remains still controversial [1, 9]. Another section of the WHO manual deals with advanced functional tests which are aimed at assessing the competence of human spermatozoa to fulfil those processes which are essential to conception. These specialized assays are available mainly in research laboratories and are used to assess the effect of environmental and pharmacological compounds on spermatogenesis. However, since their association with the fertilizing potential of the male gamete is promising, it is possible that these assays will be included among the so-called extended examination procedures in the future.

In conclusion, although routine semen analysis is considered a poor indicator of reproductive outcomes, it remains a valuable diagnostic test in couple infertility. It provides information on whether infertility can be related to the male partner and is able to identify alterations which can guide clinicians towards tailored diagnostic exams.