Abstract
Semen analysis is a fundamental part of male fertility investigation. Samples are produced by masturbation, ideally in a private room nearby to the andrology laboratory or submitted within one hour if produced off-site. The ejaculate should be collected in a wide-mouthed, clean, glass or plastic container, from a batch confirmed to be non-toxic to spermatozoa. Patients should be given clear instructions about collection of their sample. To ensure consistency and reliable interpretation of results, there should be a minimum of two days and maximum of seven days abstinence (1), and the complete sample should be collected. Loss of the first portion of the ejaculate, which mainly comprises sperm-rich prostatic fluid, may significantly affect sperm count and concentration.
1 Semen Analysis
Semen analysis is a fundamental part of male fertility investigation. Samples are produced by masturbation, ideally in a private room nearby to the andrology laboratory or submitted within one hour if produced off-site. The ejaculate should be collected in a wide-mouthed, clean, glass or plastic container, from a batch confirmed to be non-toxic to spermatozoa. Patients should be given clear instructions about collection of their sample. To ensure consistency and reliable interpretation of results, there should be a minimum of two days and maximum of seven days abstinence (1), and the complete sample should be collected. Loss of the first portion of the ejaculate, which mainly comprises sperm-rich prostatic fluid, may significantly affect sperm count and concentration.
The World Health Organisation (WHO) Laboratory Manual for the Examination and Processing of Human Semen (fifth edition) was published in 2010 and includes standardised methods for semen analysis, sperm preparation and cryopreservation, as well as laboratory quality assurance and quality control (1). Routine assessment of semen includes visual and microscopic inspection, viscosity, volume, pH, presence of round cells or anti-sperm antibodies, sperm count, motility and morphology.
Semen volume is almost entirely made up of secretions from the accessory organs, mainly the prostate and seminal vesicles. Volume is best measured by weighing the sample in its container, then subtracting the weight of the empty container. Volume can be calculated from the sample weight, using the assumption that the density of semen is 1g/ml (2). Semen volume may also be measured using a volumetric pipette, although this is only accurate to the degree of markings on the pipette itself. Viscous samples or presence of bubbles in the semen may cause inaccuracies in volume measurement.
Semen pH is measured using colorimetric pH test strips, and reflects contributions from accessory gland secretions. Seminal vesicle secretions are alkaline and prostatic secretions are acidic. Consensus opinion suggests that semen pH should be no lower than 7.2.
Sperm count is assessed using a haemocytometer (a thick glass microscope side with a rectangular indentation that creates a chamber). Semen is diluted in a known concentration of water to render sperm immobile. By counting the number of cells in a specified volume, the overall concentration can then be calculated.
Sperm motility is assessed by analysis of at least 200 sperm. Motility is described as progressively motile (PR), non-progressively motile (NP) and immotile.
Vitality testing is used to determine if immotile sperm are alive or dead and is indicated when sperm motility is less than 40%. There are two approaches commonly used. The dye exclusion assay relies on the ability of live sperm to resist absorption of certain dyes, which penetrate and stain nonviable sperm. Trypan blue and Eosin Y stains are commonly used. After staining, spermatozoa are smeared on a glass slide and air dried, so cannot be used in treatment. The hypo-osmotic swelling (HOS) test also evaluates the functional integrity of the plasma membrane of the spermatozoa. Under hypo-osmotic conditions, influx of fluid causes the sperm tail to coil and swell, and thus identifies live cells. This method does not damage or kill spermatozoa and can be used to identify viable immotile sperm for intracytoplasmic sperm injection (ICSI) (3).
Morphology is arguably the most contentious element of diagnostic semen analysis. The variable morphology of human spermatozoa makes standardised assessment very challenging, however, evidence supports a relationship between in vivo fertilisation and the percentage of normal forms in a sample (strictly defined and/or using computer-aided assessment of morphology) and therefore justifies attempts to assess and record. Optionally, Teratozoospermia index (TZI) can be calculated by recording individual abnormalities identified in head, neck and midpiece, tail (principle piece) and excess cytoplasm (4). The total number of abnormalities recorded is divided by the number of spermatozoa with one or more defects to calculate TZI, and is significant if >1.5.
Anti-sperm antibodies (ASAs) may cause cell death, immobilisation of spermatozoa or create agglutinated clumps of moving sperm, thus impeding passage through cervical mucus, defective zona binding and fertilisation. Agglutination specifically refers to motile spermatozoa sticking to each other. ASAs may be IgG or IgA, although IgA are acknowledged to be of greater clinical significance. There are two direct tests for ASAs: the mixed agglutination reaction test (MAR test), which is performed using fresh semen, and the immunobead-binding assay (IB), which uses prepared sperm. There are currently no reference values for antibody-bound spermatozoa in the MAR test of semen from fertile men. Pending further evidence, the WHO manual consensus opinion recommends >50% motile spermatozoa with adherent particles to be considered clinically significant.
The WHO laboratory manual also includes revised reference values for human semen characteristics (5). These values were derived from semen analysis characteristics from men with a known time to pregnancy (TTP) of less than 12 months. Retrospective and prospective semen analysis data was collected and included 1953 samples from 5 studies, performed in 8 countries across 3 continents. Inclusion criteria were stringent and laboratory methods standardised, resulting in creation of a cohort of fertile men representative of a global population. The data was used to generate one-sided lower reference limits (5th centile), which are used as current criteria to define male fertility (Table 7.1).
| Parameter | Lower reference limit |
|---|---|
| Semen volume (ml) | 1.5 |
| Total sperm number (106 per ejaculate) | 39 |
| Sperm concentration (106 per ml) | 15 |
| Total motility (PR + NP, %) | 40 |
| Progressive motility (PR, %) | 32 |
| Vitality (live spermatozoa, %) | 58 |
| Sperm morphology (normal forms, %) | 4 |
| Other consensus threshold values | |
| pH | >7.2 |
| MAR test (motile spermatozoa with bound particles, %) | <50 |
If the result of the first semen analysis is abnormal, a repeat confirmatory test should be offered, ideally after three months to allow for a cycle of spermatogenesis. However, if a significant abnormality is identified (azoospermia, severe oligozoospermia), then the repeat test should be undertaken sooner. The nomenclature describing semen analysis abnormalities is shown in Box 7.1. Descriptive terms are used in combination where more than one abnormality is present.
| Azoospermia | No sperm in ejaculate |
|---|---|
| Oligozoospermia | Total number / concentration of sperm below reference limit |
| Asthenozoospermia | % progressive motile sperm below reference limit |
| Teratozoospermia | % morphologically normal sperm below reference limit |
| Oligoasthenozoospermia | Total number / concentration of sperm and % progressive motility below reference limit |
| Oligoteratozoospermia | Total number / concentration of sperm and % normal morphology below reference limit |
| Asthenoteratozoospermia | % progressive motile sperm and % normal morphology below reference limit |
| Oligoasthenoteratozoospermia | Total number / concentration of sperm, % progressive motility and % normal morphology below reference limit |
2 Sperm Function Tests
Although semen analysis represents the cornerstone of male fertility investigation, it is a quantitative, rather than qualitative, test and does not evaluate sperm function. The journey of the human sperm in vivo includes penetration through viscous cervical mucus and negotiation of the uterus and oviduct to the site of fertilisation. Mammalian sperm also undergo a series of cellular changes, termed capacitation, to acquire the ability to fertilise. Only capacitated spermatozoa can penetrate the cumulus, undergo acrosome reaction, penetrate the zona pellucida, bind to the oocyte and achieve fertilisation.
Sperm function tests aim to examine various functional attributes of spermatozoa. However, lack of standardised methods, or available laboratory kit, generally limits their widespread use. Initial promise commonly fades because wider use reveals a more subjective endpoint than anticipated, or inadequate sensitivity and/or specificity, or that it is relevant to only a few selected cases and therefore of limited utility. Furthermore, cut-off values tend to be unclear and may be affected by measurement uncertainty. Moreover, sperm function tests may not help clinical management or improve success, or may simply identify a problem that has no effective treatment.
The post-coital test examines functional sperm motility by examining the ability of spermatozoa to penetrate cervical mucus. Timing is critical, due to the varying composition of the cervical mucus during the course of the menstrual cycle. The couple are advised to have intercourse around the predicted time of ovulation, and then attend the fertility clinic 9–12 hours later. Cervical mucus is aspirated for analysis, and presence or absence of motile sperm within the mucus is determined. Post-coital testing is no longer practised widely, mainly due to logistical difficulties, but also because it has no predictive value on pregnancy rate.
An alternative approach, based on the Kremer penetration test, involves the use of an artificial viscous media to replicate cervical mucus at the correct menstrual cycle phase. One per cent methylcellulose is used as an accepted surrogate for cervical mucus or cumulus complex (6). This sperm penetration assay is not widely used for clinical purposes, but remains a key tool in the research laboratory for assessing sperm function.
Computer aided sperm analysis (CASA) uses negative phase contrast to identify and track the motility and kinematics of individual spermatozoa, in either semen or prepared sperm samples. CASA systems have evolved greatly over the past 40 years, although the basic concepts for identifying sperm and their motion characteristics have changed little. Each system comprises a high power microscope, with image capture and computer analysis software, and uses mathematical algorithms to compute detailed movement variables. Kinematics measured by CASA systems are listed below, and illustrated in Figure 7.1.
1. VCL, curvilinear velocity (Pm/s): Time-averaged velocity of a sperm head along its actual curvilinear path.
2. VSL, straight-line velocity (Pm/s): Time-averaged velocity of a sperm head along the straight line between its first and last detected position.
3. VAP, average path velocity (Pm/s): Time-averaged velocity of a sperm head along its average path.
4. ALH, amplitude of lateral head displacement (Pm): Magnitude of lateral displacement of a sperm head about its average path, either expressed as a maximum or an average.
Note that different CASA instruments compute ALH using different algorithms, so values may not be comparable between systems.
5. LIN, linearity: The linearity of a curvilinear path, VSL/VCL.
6. WOB, wobble: A measure of oscillation of the actual path about the average path, VAP/VCL.
7. STR, straightness: Linearity of the average path, VSL/VAP.
8. BCF, beat-cross frequency (Hz): The average rate at which the curvilinear path crosses the average path.
9. MAD, mean angular displacement (degrees): The time-averaged absolute values of the instantaneous turning angle of the sperm head along its curvilinear trajectory.
CASA can also calculate the concentration of sperm within a sample. CASA is useful as it eliminates the variation between individual assessors of semen motility and concentration. However, the computer cannot distinguish between sperm and other cells or debris of a similar size, thus counting them as non-motile sperm. CASA is available in some assisted reproduction technology (ART) centres, but the extent of use and reliance ranges widely; its main use is within a research setting.
