Introduction
Although it might seem that human spermatozoa only have to fulfill a single role, namely, to achieve fertilization, this can only occur if a multitude of associated cell functions operate perfectly. If any of the cell systems fail within an individual spermatozoon, it will be prevented from fertilizing the oocyte and supporting subsequent embryonic development. A glance at the complex biochemical diagrams that aim to represent metabolic pathways leads to the thought that there are also multiple functions that could go wrong and that the probability of finding a flawless spermatozoon must indeed be remote. Nevertheless, fertilization events continue to occur despite the drawbacks, and the process is of universal importance for the continuation of life on this planet. It is relevant to this review that the spermatozoa that eventually reach and fuse with oocytes under natural conditions have almost invariably been subjected to stringent selection [1]. If the selection mechanisms that operate in nature are able to discriminate the quality of spermatozoa, it is reasonable to ask whether scientists in a laboratory can do the same thing. To answer this question requires some understanding of the natural mechanisms behind sperm selection. As this reviewer has been charged with assessing the merits of basic semen analysis, the article will focus on whether much relevant information can be gained by using relatively simple approaches.
The technical evaluation of semen quality is also important for any consideration of basic semen analysis. Clearly, if the technical procedures are performed inadequately, it follows that the biological interpretation of results and hence the validity of any fertility-related diagnosis or prediction are undermined. In the United Kingdom, this was recognized more than three decades ago by the British Andrology Society (BAS), which initiated a national quality assurance scheme for clinical andrology laboratories. When the scheme was established, the technical shortcomings within some centers were striking. Single semen samples were reported so differently by diverse laboratories that the same assessments could have identified individuals as being either a semen donor, that is, with a high total sperm count and good progressive motility, or a candidate for infertility treatment. Anecdotal reports persistently indicate that, even today, some laboratories still fail to use a temperature-controlled microscope stage when carrying out subjective sperm motility analyses. In general, the focus on higher standards has paid dividends [2], and consistent and accurate results are now routinely achieved. It is important that these are maintained through technician training, both in-house and through national organizations, and that standards are overseen through quality assurance schemes.
As a key objective of this review is to revisit “basic” semen analysis and to consider its merits, it will be important here to take account of both the techniques involved in semen assessment and the likely meaning of the results. I will limit my interpretation of “basic semen analysis” to what might be considered as the simplest set of parameters; that is, ejaculate volume, sperm concentration, sperm morphology, sperm viability (or rather plasma membrane integrity), and motility. In fact, none of these parameters can really be regarded as simple, but they were relatively easy to implement at a time when laboratories routinely possessed only a microscope, a warm plate, some pipettes, and some counting chambers.
I also avoid discussing the significance of sperm DNA fragmentation in relation to fertility; however, it is worth pointing out that researchers interested in the development of sperm cryopreservation and storage methods had already recognized in the 1960s that the DNA content of spermatozoa might be affected by ex situ storage [3–5]. At that time, their technology was limited to the use of Feulgen staining, a technique that stains DNA red by the use of Schiff’s reagent and spectrophotometric methods that could be used to quantify the amount of DNA in individual cell nuclei. Technologies for more precise measurements of sperm DNA became available in the late 1980s, but their use was limited to a small number of researchers who began to realize the importance of the developing field [6,7].
Specific Aspects of Basic Semen Analysis
Semen Volume
The measurement of human semen volume is less straightforward than might be expected. Volumetric measurements using pipettes or measuring cylinders have been used for many years [8]; this approach is practical and easy to perform and has previously been recommended for routine use [9]. However, the increased demand for data validation and laboratory accreditation has forced some reevaluation of the optimal methodology for semen volume estimation, and it has been shown that the use of pipettes and cylinders consistently underestimates the real volumes [10]. The degree of underestimation may be 0.2–0.4 mL and occurs because some of the semen is retained within the pipette. The better alternative involves collecting the semen in a container of known weight and estimating the combined weight of the container plus semen, thus permitting the semen weight to be calculated by subtraction. As it is known that the specific gravity of semen is slightly over 1.00 g/mL, the volume can be estimated accurately from the weight. This approach is now recommended by the World Health Organization [11] and supersedes the earlier WHO recommendation [12] to use the volumetric method. An underestimate of 0.2–0.4 mL may seem to be of little significance in terms of patient management, but when the lower reference limit for volume is only 1.5 mL (with 5th and 95th confidence limits of 1.4–1.7 mL) [11], the underestimate could shift a volume assessment of 1.5 mL down to below the reference value. It would also affect the calculation of total sperm number within the ejaculate. Moreover, as pointed out by Matson and colleagues [10], medical laboratories have a responsibility to ensure accuracy and provide an indication of the uncertainties associated with the data they report. The prognostic value of semen volume is nevertheless rather uncertain, as the natural within-subject variation can be as high as 40%. This means that working with values obtained from a single estimate is highly unreliable. This may explain why it has been difficult to identify any correlations between semen volume and pregnancy outcome [13].
The sources of variation in semen volume are not clear, but Schwartz et al. [14] identified abstinence period as a significant positive correlate of semen volume. Variation in semen volume is largely determined by the amount of seminal plasma contained in the ejaculate and, in turn, the amount of fluids that emanated from the accessory sex glands. From a strictly technical point of view, seminal plasma may be seen as little more than a fluid vehicle that facilitates the transfer of spermatozoa into the female reproductive tract. However, extensive research across different mammalian species has shown that seminal plasma contains a complex mixture of proteins whose functions extend far beyond that of a simple fluid. A recent discussion [15] attributed several significant functional roles to seminal plasma, including an influence on sperm capacitation, formation of the oviductal sperm reservoir, and facilitation of sperm-oocyte interactions. As seminal plasma is also a source of antioxidants, it confers protective effects on spermatozoa; however, comparisons of seminal plasma proteins between normal men and infertile patients have demonstrated that infertility can be associated with the presence of inappropriately expressed proteins [16]. While these observations show that seminal plasma analysis may therefore prove useful for the diagnosis of infertility through proteomic analysis, they also demonstrate clearly why a simple estimation of seminal plasma volume may not be a helpful diagnostic indicator of semen quality.
Estimation of Sperm Concentration
Sperm concentration measurements are routinely performed using one of several available designs of counting chambers or disposable slides. The operator usually views these chambers by phase-contrast microscopy and has to count the number of spermatozoa seen within certain parts of a grid. Of the counting chambers themselves, some have inherently higher reproducibility and therefore better precision than others, and it is relevant to consider whether this is important in clinical practice. Christensen and his colleagues [17] compared the accuracy and precision of four types of counting chamber, Makler chambers (Sefi Medical Instruments, Haifa, Israel), Thoma 50 and 100 μm deep chambers (Thoma hemocytometers; Hecht Assistent, Sondheim, Germany), and Bürker-Türk hemocytometers (BT, Brand, Wertheim, Germany). The tests were performed using bull and boar semen and were replicated by including data from more than one technician. Importantly, the counts were calibrated against flow cytometric estimation, which is widely accepted as an independent and objective counting technique. These authors found that the Makler chamber showed highest coefficients of variation (CV; 15%–24%) and consistently underestimated the sperm concentrations by about 25%. This is a significant finding and would lead to serious errors in clinical practice. In the same study, the CVs of other chambers were clustered around 7%–14%. In an earlier study, Mahmoud et al. [18] also tested four different methods for determining sperm concentration and found that the Neubauer hemocytometer produced a CV of about 7%. Where technical differences between individual technicians and between laboratories have been evaluated, the CVs appear to be rather large. A study by Auger et al. [19], which invo...