Abstract
Computer-assisted semen analysis (CASA) is an automated and objective method to evaluate several sperm parameters, either in conjunction with or instead of routine manual semen analysis. CASA systems have undergone a complete metamorphosis since the initial systems were developed to track sperm motion four decades ago [see 1, 2 for detailed history]. Various innovations in bioengineering, software algorithms and computational power have led to more than 14 CASA systems (Figure 2.1) currently available across the globe for commercial use in evaluating both human and animal spermatozoa [3, 4, 5]. Although CASA was initially introduced as a research tool and not commonly used for semen analysis in the clinical setting [1], more and more fertility clinics are investing in and switching to automated systems.
2.1 Introduction
Computer-assisted semen analysis (CASA) is an automated and objective method to evaluate several sperm parameters, either in conjunction with or instead of routine manual semen analysis. CASA systems have undergone a complete metamorphosis since the initial systems were developed to track sperm motion four decades ago [see 1, 2 for detailed history]. Various innovations in bioengineering, software algorithms and computational power have led to more than 14 CASA systems (Figure 2.1) currently available across the globe for commercial use in evaluating both human and animal spermatozoa [3, 4, 5]. Although CASA was initially introduced as a research tool and not commonly used for semen analysis in the clinical setting [1], more and more fertility clinics are investing in and switching to automated systems.
The major difference between CASA and manual semen analysis is inherent in the subjectivity of evaluations. In routine manual analysis, both inter-individual and -laboratory variation have been reported, mainly due to subjective scoring of sperm characteristics by laboratory staff and lack of quality assurance (e.g. methodology standardization and adherence to international guidelines) [2, 6]. Modern CASA systems have been designed to objectively and quantitatively measure several aspects of sperm structure and function, aiming to provide high levels of intra- and inter-laboratory consistency [7]. However, CASA systems are not ready-to-use devices and its usefulness in semen analysis rely on numerous factors discussed below [4, 8]. Despite these differences, it is important to note that both manual and automated semen analysis for humans are based on the same “gold standard”, namely the World Health Organization’s (WHO) laboratory manual [9, 10], as guideline for semen assessment. Several studies have compared values scored or generated for sperm concentration, motility and morphology between manual and CASA analysis, with contradictory results being reported [8, 11–14]. Vendors should provide proof that their CASA systems have been validated [2] and internal quality control should be performed by each laboratory [15].
Any semen analysis typically includes evaluation of sperm concentration, motility and morphology and these three parameters will thus be addressed in this chapter. Since sperm morphology is the focus of Chapter 5, CASA of this parameter is only briefly touched on in the sections below. One of the limitations of a standard semen analysis is that it can only determine basic semen quality and cannot predict or be used to diagnose unexplained (idiopathic) infertility as is the case in around 30 percent of males affected by infertility [6]. The CASA measurement of these sperm parameters includes a much more detailed assessment of individual spermatozoon’s attributes and thus results in more comprehensive biological information that might be directly correlated to sperm quality and fertility potential. In addition, high-end CASA systems now include modules that allow for less routinely evaluated parameters such as vitality (membrane integrity), DNA fragmentation and acrosome integrity, which could elucidate possible reasons for sperm dysfunction [4, 6].
2.2 Principle of Computer-Assisted Semen Analysis
2.2.1 Sperm Concentration and Motility
Most CASA systems use phase contrast optics to establish a centroid for each spermatozoon by registering the location of its head in two dimensions (x- and y-coordinates). Once the sperm heads have successfully been detected, both sperm concentration and motility can be assessed (Figure 2.2). Although sperm motility is essentially a result of flagellar bending that propels the spermatozoon forward, using the sperm head to measure motility is currently accepted as a representation of flagellar motion [16]. Sperm head trajectory and movement are therefore assessed by tracking the sequential position of the head using serial digital imaging. Various sperm motility percentages (Table 2.1) as well as kinematic parameters (Figure 2.3A) are subsequently calculated. It should be noted that alternative technologies used for motility analysis are employed by certain CASA systems [2]. The possibility of including high-throughput real-time flagellar capture and three-dimensional sperm motion tracking into CASA motility analysis in future would open up new avenues for clinical semen analysis [17].
Figure 2.2 Sperm motility analysis at 50 frames per second illustrating correct identification and tracking of individual spermatozoa. Red tracks = rapid-progressive swimming sperm, green tracks = rapid-swimming sperm, blue tracks = medium-progressive swimming sperm, yellow circles = static sperm, turquoise blocks = hyperactivated sperm. The detailed kinematics measured for each spermatozoon are indicated in Figure 2.3.
Figure 2.3 Motility and morphology analysis of individual spermatozoa. A) Centroid-based tracking of the sperm head results in eight kinematic parameters directly measured or calculated as a fraction. VCL = curvilinear velocity, VAP = average path velocity, VSL = straight-line velocity, LIN = linearity, STR = straightness, WOB = wobble, ALH = amplitude of lateral head displacement, BCF = beat cross frequency. B) Sperm morphology is assessed by incorporating various sperm morphometry parameters. Top sperm = normal morphology, bottom = abnormal morphology, yellow = acrosome, blue = head, red = vacuole, green = midpiece.
Table 2.1 Typical Sperm Motility and Morphology Parameters Assessed by Computer-Assisted Semen Analysis
| Motility | Morphology |
|---|---|
| Percentages | Morphology |
| Total motility | Normal (%) |
| Progressive motility | Abnormal (%) |
| Fast progressive [Type A*] | Head defects (%) |
| Slow progressive [Type B*] | Midpiece defects (%) |
| Non-progressive [Type C*] | Tail defects (%) |
| Immotile [Type D*] | Cytoplasmic droplets (%) |
| Rapid# | Acrosome coverage (%) |
| Medium# | Tetrazoospermia index |
| Slow# | Defortmity index |
| Sperm mucous penetration | Multiple anomalies index |
| Hyperactivation |
| Kinematics | Morphometry |
|---|---|
| VCL (µm/s) | Head length (µm) |
| VAP (µm/s) | Head width (µm) |
| VSL (µm/s) | Head area (µm2) |
| LIN (%) | Head perimeter (µm) |
| STR (%) | Head elipticity |
| WOB (%) | Head elongation |
| ALH (µm) | Head roughness |
| BCF (Hz) | Head regularity |
| Midpiece width (µm) | |
| Midpiece area (µm2) | |
| Midpiece insertion angle |
* According to 4th ed. of WHO manual [10]
# Sperm swimming speed classes defined according to CASA sort function [3]
VCL = curvilinear velocity, VAP = average path velocity, VSL = straight-line velocity, LIN = linearity, STR = straightness, WOB = wobble, ALH = amplitude of lateral head displacement, BCF = beat cross frequency.
2.2.2 Sperm Morphology
Both wet preparations and dry preparations of stained spermatozoa can be accommodated by CASA systems to assess sperm morphology. Automated sperm morphology analysis (ASMA) generally uses algorithms to detect similar sperm component abnormalities as recommended by the WHO [10] for manual analysis [18]. Whereas the latter method depends on a trained set of human eyes to decide whether a sperm component is normal or abnormal, CASA employs several quantitative sperm morphometry parameters (Table 2.1) in order to qualify sperm morphology (Figure 2.3B) [3]. In addition, certain CASA systems automatically determine indices of multiple sperm defects, such as the multiple anomalies index (MAI), the teratozoospermia index (TZI) and the sperm deformity index (SDI) [10].
2.3 Equipment and Methods
Sperm concentration, motility and morphology are measured with different modules in the CASA software. A digital or video camera is mounted on a microscope, fitted with a temperature-controlled stage and phase contrast as well as bright field objectives (Figure 2.1). Semen or a sperm preparation is loaded into disposable, chambered slides (concentration and motility) or stained sperm smears are mounted (morphology) prior to analysis. Specific analysis properties are pre-set as recommended for standardized human sperm analysis [3].
2.4 Reference Values and How to Calculate Cut-Off Values
Central to accurate automated semen/sperm analysis is the employment of standardized protocols and analysis properties. Any deviations from recommended guidelines [10, 15] will result in unreliable data generated (see Section 2.6 below). In turn, incorrect data will be compared to reference values for sperm parameters (Table 2.2) and might influence the clinical interpretation of the analysis results (see Section 2.7 below). Apart from employing the WHO manual’s reference values for sperm concentration, total number of sperm in the ejaculate, percentage total and progressive motility as well as normal sperm morphology [10], several other sperm parameter reference values can be incorporated in CASA systems (Table 2.2).
Cut-off values for specific sperm kinematic parameters are used to assess features of sperm motility which are virtually impossible with manual analysis. For instance, evaluation of sperm subpopulations portraying rapid-, medium- and slow-swimming speed, mucous penetration and hyperactivation characteristics are already incorporated in some systems or can be manually added by the technician using cut-offs incorporated into a “sort” function [6, Figure 2.2]. CASA sperm morphology assessments are traditionally based on minimum and maximum cut-off values for sperm head, midpiece and tail morphometry parameters as well as percentage acrosome coverage [3]. In certain CASA systems additional sperm motility or morphology features of interest can be assessed (e.g. subfertile population) by establishing cut-off values by using visual pattern recognition and receiver operating characteristic (ROC) curve analysis [1, 19] or 90–95 percent confidence intervals [3, 19].
2.5 Advantages and Disadvantages of Computer-Assisted Semen Analysis
2.5.1 Advantages of Computer-Assisted Semen Analysis Systems
Since the development of the earliest CASA systems it was reported that automated analysis is (or has the potential to be) more accurate, consistent (precise) and repeatable than manual and subjective semen analysis [4, 8]. Due to the use of algorithms to measure sperm parameters with CASA, there is reduced potential for human error and less analytical variance, although operator subjectivity is not completely eliminated [14]. CASA also provides a lower turnaround time per individual analysis and provides rapid results [8, 14] that can immediately be made available in a report to the doctor or patient (Figure 2.4). This also implies that when the same sample (e.g. morphology slide) is used for CASA analysis on similar systems in different laboratories, there should be no significant differences in the generated results [20]. These advantages of CASA can, however, only be achieved by employing well-trained technicians and optimized CASA settings (see disadvantages below) [8].
Figure 2.4 CASA motility report including different sperm motility classifications and subpopulations. These user-friendly results can be presented in various formats and can be extracted for alternative use. Semen parameters with measured values less the lower reference limits recommended by WHO [10] are highlighted in red in the report (sperm concentration in this example).
Modern CASA systems are much more user-friendly and allow for input and adjustments to be made by the technician. Not only can one change the configuration settings for different hardware (e.g. microscope and camera) and consumables (e.g. slides and staining method) available, in many systems, the initial analysis can be viewed again for verification of accuracy. For example, for motility analysis the “playback” option of the software can be used to identify and remove incorrect sperm tracks or add uncaptured spermatozoa. It is of particular importance that the analysis of curvilinear velocity (VCL) and average path velocity (VAP) are correctly assessed, since these two parameters are used to calculate most other kinematic parameters.
A further indisputable advantage of CASA is the vast amount of data that is generated which can both be used for conventional clinical interpretations and be incorporated into multivariate statistical analyses to predict fertility potential. Apart from the array of motility and morphology/morphometry parameters CASA generates (Table 2.1), the detailed analysis of individually measured spermatozoa can also be accessed (Figure 2.3) and extracted for further analysis [4]. By evaluating additional and more detailed sperm parameters, CASA allows for the detection of subtle changes in sperm motion and morphometry that cannot be identified by conventional, manual sperm motility analysis.
The most recent advancement in human CASA is the fully automated systems where minimum input and no interaction during the analysis are required by the technician. Two examples of such systems are the LensHooke Semen Quality Analyzer (Bonraybio Co., Ltd, Taichung City, Taiwan) and the SCA SCOPE (Microptic S.L., Barcelona, Spain). The LensHooke system only requires loading of the sample in a disposable counting chamber to measure pH, sperm concentration, morphology and motility (percentage total, progressive and non-progressive motility). The SCA SCOPE provides a more comprehensive device that includes all analysis modules available for the Sperm Class Analyzer (SCA) CASA system (motility and concentration, morphology, vitality, DNA fragmentation and leukocytes) and assesses more than 100 parameters. The development and use of such automated systems will possibly eliminate variations in CASA results inflicted by the user.
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