Parameter
WHO 2010 lower reference limits (95% CI)
WHO manual (1999) cutoff points
Semen volume (ml)
1.5 (1.4–1.7)
≥2 ml or more
Total sperm number (106 per ejaculate)
39 (33–46)
≥40
Sperm concentration (106 per ml)
15 (12–16)
≥20
Total motility (PR + NP, %)
40 (38–42)
≥50
Progressive motility (PR, %)
32 (31–34)
≥25
Sperm morphology (normal forms, %)
4 (3.0–4.0)
Undecided
Vitality (live spermatozoa, %)
58 (55–63)
≥50
Biological Variability
The quality of semen is influenced by many biological variables [13]. These include
The completeness of sample collections: Collecting all the successive emissions (fractions) of ejaculated semen is very important because of the varying composition of the first fraction compared to the latter ones. The first fraction of ejaculated semen is sperm-rich. This is followed by the part of the ejaculate that is diluted by the seminal vesicles fluid [14]. Thus, if the first fraction of the ejaculate is lost, the sperm concentration and total sperm count will be lower than what it should be.
The impact of age: With age both the number of sperm produced and the fluid component contributed by the prostate and seminal vesicles may be reduced [15]. This may give the elusion that sperm concentration in older men is maintained when in reality the total number of sperm in the ejaculate is reduced.
Testicular size: The size of the testis correlates with the total number of spermatozoa in the ejaculate [16]. This is reflective of the level of spermatogenesis in the seminiferous tubules [17]. An extreme clinical example of this association between testicular size and sperm count is encountered in cases of hypogonadotropic hypogonadism and in anabolic steroid abuse cases presenting with azoospermia and small testicular volume due to suppressed spermatogenesis. The testicular volume increases gradually as spermatogenesis resumes in response to treatment with human chorionic gonadotropin (hCG) injections and abstaining from anabolic steroid use, respectively.
Duration of abstinence: The WHO recommends 2–7 days sexual abstinence prior to semen analysis as a means of standardizing semen analysis. Frequent sexual activity prior to semen analysis may result in reduced semen volume, sperm concentration, and total motile sperm count without significant change in sperm motility and normal morphology [18]. On the other hand, there is significant increase in semen volume, sperm concentration, and total sperm count when the abstinence length is increased [19, 20]. However, length of abstinence does not influence pH, viability, morphology, or total motility [17]. A short abstinence period (24 h) is associated with immature sperm DNA, whereas longer abstinence intervals may be associated with sperm DNA fragmentation [14, 15].
The intensity of sexual stimulation: It has been documented that ejaculates recovered from non-spermicidal condoms used during intercourse at home can be of higher quality than those produced by masturbation and collected into containers in a room close to the laboratory [3]. However, for the sake of standardization it is recommended that ejaculates are collected in a well-equipped room next to the laboratory to reduce the impact of prolonged transport time and the exposure to nonoptimal temperature. The place of producing the ejaculate should be noted in the semen analysis report.
Delayed liquidation and viscosity: Delayed liquefaction and abnormal sample viscosity after complete liquefaction may impact the result negatively. Semen samples are kept at a temperature of 20–37 °C while waiting for sample liquefaction that is normally completed within 30 min of ejaculation. Liquefaction is reported as delayed when it is not complete after an additional 30 min standing. If liquefaction is not achieved after 60 min, mechanical and enzymatic techniques are described in the WHO manual to achieve liquefaction. This should be noted in the analysis report because treatments applied to the sample to achieve liquefaction may affect seminal plasma biochemistry, sperm motility, and sperm morphology [4]. High semen viscosity after complete liquefaction can also interfere with determination of sperm motility, sperm concentration, and the detection of antibody-coated spermatozoa. High viscosity can be recognized by the elastic properties of the sample when a semen droplet is stretched for 2 cm or more between two pipettes. Again the clinician needs to take note of this when interpreting the results.
Reversible changes in sperm parameters: Fever, intercurrent viral infections, medication with drugs such as nitrofurantoin, genital tract infection (epididymitis), and exposure to excessive heat may be associated with reversible deterioration in different semen parameters. When the semen analysis result is suboptimal, these factors should be excluded or corrected before making a final judgment on the quality of semen.
In view of the variability in the quality of ejaculates produced by one man it is impossible to rely on a single semen analysis to characterize a man’s semen quality. The analysis of two or more ejaculates may be required to obtain a more valid baseline data to facility a sound view of man’s clinical status.
External Technical Factors that May Influence Semen Analysis Result
The result of semen analysis may be impacted negatively by external factors such as the temperature at which the sample is left standing before the start of the analysis and the time elapsed between ejaculation and the start of the test. This information should be noted in the result report before making a considered judgment of its significance.
Does the Traditional Semen Analysis Identify a Specific Pathology?
The semen analysis results should be correlated with relevant medical and lifestyle issues identified during history taking, clinical examination, and other baseline assessments such as hormone profile and scrotal ultrasound scanning. In cases of unexpected azoospermia additional investigations, such as karyotyping and cystic fibrosis gene screen, may be required. Checking for Y chromosome micro-deletions may be sought in severe oligospermia (sperm count <3 × 106) on repeated testing. If the analysis reveals teratospermia (sperm with normal morphology <3%) karyotyping is recommended because of the association between teratospermia and chromosomal abnormalities both in somatic cells and spermatozoa [21]. The presence of leucospermia (leucocytic count >1 × 106/ml semen) is associated with poor sperm morphology and motility and sperm oxidative stress [22–24].
However, traditional laboratory testing may fail to provide an explanation for the reduced sperm parameters in other cases. This is referred to as idiopathic infertility. Level 2 sperm testing is required in these situations (see later) [25]. But initially let us consider the clinical significance of some of the traditional semen parameter:
The Significance of Semen Volume
The seminal vesicles secretions contribute up to 70% of the normal ejaculate volume. The lower reference limit for semen volume is 1.5 ml (5th percentile, 95% confidence interval 1.4–1.7) [4]. A low sperm volume is more likely to be due to the incomplete collection of the ejaculate. It also may be due to acquired obstruction of the ejaculatory duct. In cases of congenital bilateral absence of the vas deferens (CBAVD) there is dysplasia or absence of the seminal vesicles resulting in the loss of its contribution to the semen volume [26, 27].
According to WHO standards, retrograde ejaculation should be suspected in any case when the seminal fluid volume is <1 ml [4]. The diagnosis is confirmed by finding spermatozoa in the post-ejaculatory urine sediment when all collected urine is centrifuged and pelleted. The sperm is found mostly dead due to the combined effects of osmotic stress, low pH, and urea toxicity [28]. The recovery of high-quality sperm after the induced modification of the urine composition and pH to facilitate its use in the intracytoplasmic sperm injection technique (ICSI) has been described [29, 30].
Occasionally, the orgasm is associated with a miniscule amount of ejaculate or no ejaculate at all (dry ejaculation, aspermia). This happens in a diversity of neurological diseases and subsequent to surgical procedures on the lower urinary tract [31]. It may also be the presentation of retrograde ejaculation. The explanation for the dry ejaculation is mostly apparent from the initial history taking. If not, then retrograde ejaculation needs to be excluded before referring to the urologist or neurologist for further assessment.
The Significance of Semen pH
The balance between the alkaline secretion of the seminal vesicles and the acidic prostatic secretion determines the semen pH. The importance of assessing the semen pH and its physiological reference range has been a matter of intense debate [32]. However, WHO 2010 sets the lower reference value of the pH of liquefied semen at 7.2 [4]. In CBAVD, the semen pH is characteristically lower (pH 6.8) because of the absence of the seminal vesicles’ alkaline secretion. In these cases the scanty seminal plasma is formed mainly from the relative acidic prostatic secretion.
The Value of Semen Analysis in Azoospermic Cases
In the vast majority of azoospermic men, their condition is first identified when they are referred for fertility assessment. In a minority of cases, azoospermia was previously identified including men with cystic fibrosis disease, the majority of Klinefelter’s syndrome and, presumably, after vasectomy. Even in these men semen analysis is required to confirm the azoospermic status. Clinically, cryptospermia has been described after vasectomy [33] and up to 10% of men with Klinefelter’s syndrome have few sperm in their ejaculates [34–36]. In the andrology laboratory, azoospermia is suspected when no spermatozoa are observed in replicate wet preparation examined according to the WHO standards [4]. Azoospermia only can be confirmed if no spermatozoa are found in the sediment when the whole ejaculate is centrifuged and pelleted. Cryptospermia describes the absence of sperm in wet preparation but sperm is then found in the sediment. This additional process of semen analysis should be described in the analysis report sent to the clinician. The benefits of a reliable diagnosis of azoospermia in the field of reproductive medicine are multiple. First, an inaccurate diagnosis may lead to an unnecessary invasive procedure to retrieve testicular sperm to treat the couple using their own genetic material. Second, when azoospermia is confirmed additional genetic testing is required to diagnosis the underlying cause. These include karyotyping for chromosomal abnormalities, testing for Y chromosome micro-deletions, and screening for cystic fibrosis gene mutations. Thirdly, the underlying genetic abnormality may have a detrimental impact on the offspring and the affected couples are required to receive adequate genetic counseling prior to undergoing treatment. Finally, the demonstration of absent motile spermatozoa in ejaculate is required after vasectomy to declare the procedure a success.
If azoospermia is secondary to the testosterone hormone or anabolic steroids abuse, periodic repeat semen analyses are required for up to 24 months after abstaining from taking these drugs to monitor the spontaneous resumption of spermatogenesis in many of these patients [37, 38]. If spermatogenesis has not resumed in an acceptable time frame or abstinence is not tolerated, alternative medications may be considered [37].
When considered together, semen volume and pH can help in determining the differential diagnosis of the cause of azoospermia. In patients with low-volume, acidic, azoospermic samples, the differential diagnosis is CBAVD or bilateral complete ejaculatory duct obstruction (EDO). Azoospermic ejaculates with a normal volume and alkaline pH indicate functional seminal vesicles and patent ejaculatory ducts. The differential diagnosis includes spermatogenic failure or an obstruction at the level of the more proximal vas deferens or epididymis, but does not include CBAVD or bilateral EDO.
Can This Man Achieve a Pregnancy Given His Female Partner’s Fertility Potential?
In its successive five editions, the WHO manual offered increasingly lower cutoff points to assess sperm parameters. The reference limits for sperm count, motility, and normal morphology quoted in the latest edition [4] are significantly lower compared the cutoff points that were thought to be compatible with normal male fertility in the previous editions. As an example, the cutoff point for sperm count quoted in the 1999 WHO manual [8] as 20 × 1066/mL is almost double the 5th centile of the reference limit (12 × 106/ml) quoted by the 2010 WHO manual [4] (Table 2.1). This may cause confusion among concerned clinicians if the andrology laboratory does not indicate which WHO reference limits values are used. Another reason for the confusion is how the same semen analysis result may be classified as not compatible with normal fertility prior to 2010, but is judged as compatible with normal fertility when applying the 2010 lower reference limits.
At this point it becomes apparent that a semen analysis result is not prescriptive but descriptive of a man’s clinical status, with the exception of azoospermia where spontaneous pregnancy cannot happen. This stems from the huge heterogeneity in the characteristics of semen in men compared to other animals. This has led to an extensive overlap of the distributions of semen parameters’ results among fertile and non-fertile men (Fig. 2.1c) [39]. This is contrary to the assessment of other biological features where there is distinct distribution or only a minimal overlap of results among affected and unaffected populations (Fig. 2.1a, b) [39]. When that is the case, the identification of a cutoff point to reliably distinguish affected and unaffected individuals is feasible. On the other hand, the extensive overlap in semen analysis results between affected (infertile) and unaffected (fertile) populations causes significant numbers of both false-positive and false-negative cases [39] compromising the prognostic value of semen analysis.
