Andrology Lab Corner*: Reflections on CASA After 25 Years (2004)

Like the Journal of Andrology, computer assisted sperm analysis (CASA) systems did not arise de novo. Although both are celebrating 25th anniversaries, the Journal evolved over several years and drew on predecessors. In contrast, today's CASA systems represent third-generation devices for visualization and analysis of sperm motion. Modern CASA evolved some 300 years after the first-generation device was placed into use. This device was the light microscope, which von Leeuwenhoek used to first visualize sperm in 1678. The concept and principles underlying such a device did not change until shortly before World War II, when European opticians developed phase-contrast optics. Such second-generation devices were first used by pioneering andrology labs in the mid-1950s, and phase-contrast microscopes remain the primary instruments for observation of living sperm. Phase-contrast optics are integral to every contemporary CASA system because they enable high-contrast visualization and edge detection of each translucent cell. Important publications on quantifying sperm motion appeared between 1940 and 1970 (summarized by Boyers et al, 1989), and provided a foundation for CASA systems. However, the approaches in these studies were not at all automatic, and most used manual cartography. By the early 1970s, convergence of technology and government priorities set the stage for development of CASA. Federal and private investments in tracking rockets and diverse objects on the macroscale increased significantly. Computer technology, user friendliness, and cost began to improve exponentially. Video cassettes replaced the original reel-to-reel technology. As a result, computerized video image digitization, recognition, and quantification technologies began to emerge, with substantial cost savings over prior methodologies. Applications to the microscope followed and primitive CASA systems emerged. Here we consider the motives of individuals and companies pioneering CASA, and comment upon whether their expectations were realistic (with the benefit of hind-sight) and met. We note the positive and negative impacts of CASA in sperm biology, clinical medicine, and epidemiology. We also reflect on the role of high-technology devices in the practice of andrology. We restrict our considerations to measurement of sperm motion, although current CASA systems can measure number of sperm per unit volume and can be modified to capture data appropriate for morphologic classification of each sperm examined. CASA refers to an automated system (hardware and software) to visualize and digitize successive images of sperm, process and analyze the information, and provide accurate, precise, and meaningful information on the kinematics of individual cells, and also population summary statistics, that is, mean values. Early systems required operator intervention, but preferred systems would require the operator only to insure that the system is functioning properly, place the sample into the instrument, and examine/store output data. Underlying concepts of CASA are illustrated in Boyers et al (1989). Beginning in the 1940s and continuing for decades, a few university scientists recognized the need to obtain objective data (ie, bias-free) on percentage of motile sperm and, ideally, velocity of movement of spermatozoa. They were driven by the desire to establish standards useful to retrain or train individuals making subjective evaluations in a commercial setting (ie, animal genetics companies), and for objective data to enhance research on sperm function. Routine use in clinical andrology was not an immediate goal, although some clinicians had recognized limitations of visual observations of sperm motility. It was believed that if precise and accurate data on sperm movement could be obtained, this information could be used to predict the potential fertility of a male or select a “best procedure” for sperm preparation. As early as the 1950s, it was appreciated that electronic technology could be developed or adapted to the measurement of sperm motion (Van Demark et al, 1958). Several different technologies were developed to infer estimates of average velocity of sperm in a suspension without actually identifying the swimming trajectory or measuring velocity of individual cells. These approaches included assessment of disruption of light passing through a pinhole by moving sperm heads (van Duijn and Rikmenspoel, 1960), analysis of scattering of light from a laser directed at a sperm suspension (Dubois et al, 1974), and use of an early image-analysis computer to count fluctuations in sperm numbers in a fixed volume (Katz and Dott, 1975). However, these all were indirect methods that did not identify and track individual sperm cells. In the late 1940s, Lord Rothschild introduced the use of time-exposure photomicrographs, using dark-field illumination, to create images of the swimming trajectories of spermatozoa, which could be manually analyzed to determine swimming velocity (Rothschild and Swann, 1949; Rothschild, 1953). During the 1960s and 1970s, this technique was used in a number of contexts, including analysis of bull (Elliott et al, 1973) and human (Janick and MacLeod, 1970; Overstreet et al, 1979) sperm. This approach has been modernized by use of a digital camera (JL Schenk and RP Amann, personal communication). There also were a number of studies that identified sperm trajectories via frame-by-frame projection of cine films (eg, Rikmenspoel, 1957; Katz et al, 1978) obtained micrographically. However, these studies and similar ones using videotapes still required extensive manual work for raw data acquisition as well as subsequent analysis. These manual cartographic studies established two important points: 1) human observers were biased when estimating percentage of motile sperm; and 2) information on pattern and velocity of sperm motion indeed was of biological significance and possibly clinical utility. Acceptance of these conclusions provided motivation for seeking automated cartographic analysis of sperm trajectories. In 1973, Jecht and Russo reported that a motion-analysis system developed for the National Aeronautics and Space Administration at the Jet Propulsion Laboratory could track human sperm. Videotape interfaced a microscope with the analysis system, and operator input was obligatory. Although there was no comparison of multiple samples or a follow-up publication, this paper included concepts still used today (eg, determination of sperm centroids, linear and angular velocities, and linear and angular displacements). At this time, Amann at Penn State recognized the need for automated quantitative measurement of percentage of motile sperm. He approached commercial bull studs in Pennsylvania with a proposal to make computerized measurements, and his colleague Hammerstedt sought assistance with the requisite computer programming. With additional local and federal funding, plus efforts of a dedicated student and several technicians, the first presentation of a system designed to track sperm motion was made at the Third International Conference on the Spermatozoon in Woods Hole, Mass, in 1978 (Amann, 1979) and it utilized software developed by Liu and Warme (1977). Because real-time video-capture boards and high-speed recording hardware cost >$200 000, the Penn State team recorded primary data on motion picture film (a step back from Jecht and Russo, 1973). The film was projected frame-by-frame on a screen so that a video camera could capture each stationary image over several seconds (reasonably priced reel-to-reel video recorders introduced image distortion) and move data for digitization and storage on a 33-cm diameter hard disc (1.2 mb). The computer (16 kb memory) required 3 minutes to analyze a sample. Output data based on 4 or 5 frames were considered adequate to gave meaningful data on percentage motile sperm and velocity. This system was subjected to comprehensive validation (Amann and Hammerstedt, 1980), and that paper set a standard for validations of other systems. As summarized later, this system was the first using computerized cartography, rather than manual cartography, to provide output data for production of training/educational aids or publish linkage with fertility of individual males. Schoevaret-Brossault (1984) introduced a similar approach with human sperm, and his system analyzed 30 frames and provided more comprehensive output on sperm movement characteristics. The first system enabling direct transfer of video information from a microscope into a video-capture board, followed by automatic image processing and data output, was described by Katz et al (1985). The heart of this system was an Expert Vision™ system developed by Motion Analysis Corporation for study of macroscale (human ambulation) and microscale (marine microorganisms) movements. The authors emphasized that there was useful information in measures of vigor and pattern of sperm motion (eg, curvilinear velocity, average path velocity, and linerarity) as well as percentage of motile sperm. Profit was the goal of commercial developers of CASA systems, and this was predicated, primarily, on sales to clinical human or processing human or animal sperm for use in The pioneering Expert system Analysis and several of systems, were not over systems. The first commercial CASA system developed for of sperm motion was the system which and a number of in the several a of publications described use of the system with and sperm (eg, et al, and et al, et al, At this time, it began to be recognized that CASA data had potential in of and, later, on sperm et al, the Expert and systems used phase-contrast microscopes with real-time video and provided analysis after image The commercial system developed for of sperm motion was the a in a introduced in The for development of this system was quantification of in sperm of storage in a in development as by a with a This system had including an system and video and automated of the sample to The and dark-field optics of the system were in the by and phase-contrast optics. on a video screen the of of the human an making or the of of the objective in when they would not a image to the video to with a fixed of all are to The and systems both provided meaningful output data et al, although and were and direct of could be the of in these systems, there still was a need for user to the instruments the for software to track sperm from a used in that (eg, et al, These the from an (hardware and software) in automated capture and processing of image data were considered by Boyers et al and most remain by of the and systems were in Amann with validation and data for bull or sperm with the systems. In and Motion Analysis Corporation the of sperm analysis. introduced the system in and the for use with an microscope and of the to provide images and, more accurate and precise image to enable tracking of sperm; automated classification of sperm and use of and sperm, so that these could be from other In with development of commercial systems, developed systems in the (eg, et al, in to the system, at other commercial CASA systems are in use. The system developed for sperm analysis early in the is the only system to detection of the sperm as a to if an indeed is an or to of the of trajectories or a of The was introduced in the and it evolved from software developed for use with The system has evolved to classification of each sperm as motile or and also or to a personal communication). of the Journal of included several Although it is that we an of a CASA presentation at an of the there were such in publications reported use of the system with human sperm (Katz and and the system with sperm and The for volume was the first to and sperm motility. goal of or early of CASA systems was to obtain data of to establish standards for and of making subjective There is on of this The system described by Liu and Warme was used to a with of bull sperm with average percentage of motile sperm and average velocity for technicians, plus other information for Although this was by bull they were with the or that of or was or not the This negative was by bull and a after of a more and comprehensive in the using a CASA system Amann, personal of aids is to the that animal genetics companies and are not required to in sperm analysis to a With to human sperm a on was in and production been personal communication). This is to an and for sperm in a disc federal on and Laboratory This human to use digital video images of human sperm with or observations on the of CASA on for of the early goal of use of CASA could be Although use of CASA in clinical andrology was not an immediate goal, this was the CASA systems would their current use if each was and by companies were driven by the goal of systems are in in human andrology and in with an in has in in the and in personal with most in human and for evaluations in animal genetics companies (eg, of CASA in human analysis be because most evaluations are in clinical and not in andrology we of an andrology without a CASA system is not of CASA systems in has been although there has been into and as well as It is that the clinical and the processing human and approach the and the European and with possibly at has their goal of from sales of CASA systems. The for CASA was acquisition of objective data to enhance research on sperm of a “best procedure” for sperm or to predict the potential fertility of a the and of CASA were established in it was to use CASA to measure of or processing on of sperm function. This use is important for in which fertility is (eg, or not (ie, number of such studies been authors consider in the percentage of motile sperm with the velocity and most linear movement to be There is no biological to this In CASA we whether a mean or a of the mean is it was that precise and accurate data on sperm motion could be obtained, of potential fertility of a on the of CASA a of the first to data from CASA with for individual the system described by Liu and Warme (1977). of the were used for CASA in Amann and Hammerstedt, and for of fertility of was established by after of numbers of sperm from in different approach has more than between CASA data and fertility were not than based on other visual or subjective measures of sperm et al, et al, evaluations of other of the with a system, after only between the fertility and percentage of motile sperm or curvilinear velocity et al, However, multiple each including by at or after the gave of The paper included a study of the between percentage of after commercial with from of Although data were based on and a of percentage no was This in of between CASA data and fertility data was in to the used to measure as by the authors and also in Amann (1989). The other linkage of CASA and fertility for or are not because with the benefit of it is that it was to or of of sperm motion to be of by individual that be via analysis. The underlying is that of of and sperm can a of an adequate is a but is to insure potential of a or population of cells. sperm for different of sperm motion in the has because it information on of other and (Amann, Amann and Hammerstedt, and such linkage precise measurement of fertility and detection of in potential of sperm in a sample. 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CASA system sperm motion and sperm be a by information on percentage of sperm, but such a system is not However, CASA evaluations of and with with of as is et al, et al, using a study et al, analyzed of from for which fertility in commercial was fertility was standard the study mean were as and all other as It was that of were when only sperm were considered only when all sperm were commercial CASA system be adapted to this of a CASA system to use the rather than the system in their This is provided the is and Such de of although for the measure made by instruments with different hardware or software are not As and of clinical analysis some or the the of this establish for each CASA system or of each the CASA is the that it a for sperm motion. CASA can not and not as a in to or of motion, because no a at stage in of sperm in the that sperm at different in their from the to the (eg, et al, Katz et al, sperm in by at after into the of on were motile 5 minutes and at time, of the sample on of the sample (eg, Katz and Amann, or of the most bull sperm in plus or in in a when in a plus most sperm without the is similar and the of motion can be by back and Amann and are after in and in the percentage of motile sperm and also percentage of motile sperm. of sperm a on the information from CASA. CASA can and as the for standard images and visual of sperm for This is by data to images of samples with of motion in to both motile and velocity as well as number of sperm in the of Modern from of appropriate data This is of as in other there is a of (eg, of which CASA is a is the of a and The and of sperm, and of their between from the until into an make sperm a for based The of sperm analysis (eg, Lord Rothschild, recognized and sought to and technology to and analyze sperm motion. of followed from the to technologies that would all of and In the we of to and biology, medicine, and (eg, are developed to process and the of information in biological and However, as and more we need to to with the biological and clinical is not is biological clinical and commercial of federal and During the early of CASA, were or to the into which this technology would to be to be development of CASA was based on the that sperm was to and clinicians human or animal the of clinicians the work of and The of in and sperm has sperm motion, and to a role in clinical than it It is that the of CASA is more as a to in of than as a to into biological CASA has not to a linkage between in sperm, and their via integral and into in sperm motion. However, there is at biological the of CASA This is in and epidemiology. Here there a biological to use the of CASA to in human or animal sperm reflect or important from the of CASA is that as technology is adapted to a there be of the different and The in each to the and of the other of is that the technology the and medicine, not By the it is upon and clinicians to to to some the actually is measuring or of the of as well as The a role in the by and and In by the National of on use of CASA with human and sperm et al, et al, by federal or such as can a role in of potential of technology, and in of the and of that technology. it is to create technology and more CASA instruments that would most limitations of current instruments (eg, sperm motion, sperm and a CASA a clinical However, development of of device be by of an of and did a the technology and of the time, in seeking to and CASA. We all and provided and information important for use of CASA in However, we for and conclusions By were to a as this is not a comprehensive and we to that their important to the of were