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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 and this was on to clinical human or processing human or animal sperm for use in The pioneering Expert system Analysis and several of 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 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 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 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. As Amann and Hammerstedt, is not a measure of potential of sperm because if a a or living This is by of so sperm that are when are based on (Amann and Hammerstedt, of the original expectations of CASA been and in the has been There is no when and used with appropriate software a CASA system can provide both accurate and precise data on sperm in successive video when image capture is at and the number of sperm in the of is of the path of each can be with appropriate and However, for (eg, path velocity, of motion, of are that provide but not the most accurate or meaningful data. of motile sperm can be established and but the between a or and a motile is set based on rather than on the or for of of the for a of sperm in a sample. between and or and sperm are and of CASA systems detection of in sperm motion and, in a study of or other in processing (eg, on of a sperm However, there has been on mean for each although the for data for sperm per sample are not (van and Rikmenspoel, (eg, et al, be and be more than (eg, et al, et al, in a data for for sperm in the samples are subjected to analysis without that this of a that a is of the are not there is no for that a change of a in a measure of sperm motion is of biological as to the change The is more the of CASA systems the of to on of a in to an Although there is no to this the of is in Amann and Hammerstedt is appropriate in this of analysis to CASA data not this to significance of (eg, et al, The and of a CASA system it a role for of of swimming sperm used to aids for of It has years for this important role to emerge, at for human sperm, and it years for such to use in most human In our of standards percentage of motile sperm and, consider the of a velocity with a path of At as important in visual sperm analysis is of at data a in percentage of motile sperm at as to use of a phase-contrast microscope, and of the to be and use of CASA is in processing human or animal for use in via This more in number of motile to number of from each more important is the of CASA to in sperm of from each if CASA data are not of potential of a of the is than that provided by a human and this biological via to or use. the of CASA has been in the of and studies of from to or Such use is a of early only because of diverse on male did not until when the an important the need to use objective measures of sperm motion, and the et al, early CASA Early the system was for use with sperm and et al and which be meaningful when CASA to of on considerations of approaches and remain of software for the system (eg, et al, et al, with sperm and of detection of sperm with from or other in (eg, in or an Early studies CASA were and data. they provided that CASA could be useful in of and a approach was et al, studies been reported (eg, et al, of CASA in studies of human consider the most to be of CASA systems in of human and of andrology in the However, we consider this use for clinical andrology to be and by of the of and recording data in which possibly 5 of output clinical utility. measures of percentage of motile sperm with a velocity than some and moving and accurate determination of number of of clinical path velocity or percentage of motile sperm also be important (eg, et al, this the and of CASA in of motile sperm and motile sperm over a visual This that a human is via a and and of the observations are in a meaningful The clinical objective of sperm is to that as a of male This a such as are or of the sperm motile or motile sperm when if the male has a with sperm motion as to sperm or This not require a of percentage the World on an and at not motile and of between the is not that the number of sperm of visual or CASA the concept that percentage of motile sperm can in each of several samples obtained at from the individual is by CASA data. of CASA is that not to living sperm early in clinical but not current CASA can of the motile sperm a (ie, appropriate of in the of This is an important because such sperm are more to to an than a motile but cell. 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

Perhaps one of the most exciting and revolutionary scientific discoveries of the past 3 decades has been the development of in vitro fertilization (IVF) to treat human infertility. It is impossible to quantify its effect on numerous families since the first IVF birth in 1978 in Old-ham, England (Steptoe and Edwards, 1978). With increasing clinical utilization of assisted reproductive technologies (ART), scientists and clinicians gain insights into basic gamete and embryo biology and translate that knowledge into improving the process of IVF. Critical analyses of individual steps have improved outcomes. Attention to sperm processing and isolation to increase recovery of motile sperm and reduce sperm damage has improved fertilization rates and embryo development (Mortimer, 1994). Use of intracytoplasmic sperm injection (ICSI) has allowed fertilization even in severe cases of compromised sperm quality or number (Bonduelle et al, 1999). Finally, refinement of embryo culture has led to improved in vitro embryo development and implantation rates (Gardner and Lane, 1998; Pool, 2002). Most scientific attention, however, has focused on methodologies rather than technology development and equipment. Semen is still processed in test tubes regardless of technique, sperm are physically placed with oocytes after processing, and fertilization and embryo culture occur in culture dishes, test tubes, or both with relatively large volumes (Trounson and Gardner, 2000). With the exception of gamete and embryo micromanipulation, no technologic advancements in IVF have reached widespread use. Nevertheless, it is precisely those technologic advancements, rather than procedural or methodology changes, that have had the greatest effect on assisted reproduction. A promising new technology, microfluidics, exists and is becoming increasingly studied. This technology shows promise as an alternative for each step in the IVF process. Microfluidics, based on physical principles of fluid behavior in a microenvironment, has been used widely in chemistry and molecular biology applications (Tomlinson et al, 1995). Currently, microfluidics is gaining interest in studies of cellular behavior and interactions (Shim et al, 2003). In this article, we introduce basics of fluid behavior at the microscale and highlight previous uses of this technology outside of the reproductive sciences. We then describe fabrication of devices and review initial studies that used microfluidics in sperm sorting and microinsemination. Last, we point out some limitations of this new technology and provide speculation on future directions and application of microfluidics in ART. Fluid mechanics is a complex physical and mathematical science; therefore, an extensive technical description and review of fluid physics is beyond the scope and intent of this review. Instead, basic principles will be discussed that govern fluid behavior in a microenvironment, especially those aspects with a specific link to devices and technology currently being developed for IVF. We have purposely avoided including mathematical details, choosing instead to convey a general conceptual sense of fluid mechanics present within microchannels. A comprehensive technical and mathematical description of microfluidic physics can be found in excellent reviews from Beebe et al (2002a) and Brody et al (1996). Fluids at the microscale are subject to forces typically not important at scales present in our everyday lives. Fluid at the scale of our normal environment is turbulent; particles within a stream of fluid move in an unpredictable pattern. Turbulent flow depends on certain fluid characteristics (viscosity, density, and velocity) and the geometry and size of the channel, leading to calculation of a value known as the Reynold's number. As the scale of the channel reaches micrometer levels, the Reynold's number decreases and becomes increasingly dependent on fluid characteristics. Decrease of the Reynold's number below a threshold value leads to fluid flow in a laminar fashion. Simply put, flow within microchannels becomes streamlined and predictable (Figure 1). At the microscale, fluid behavior becomes increasingly governed by viscous forces and surface tension, which can be described as the cohesiveness of the liquid's molecules. . Schematic representation illustrating fluid mixing in turbulent (A) and laminar (B) flow patterns. This dominance by viscous forces results in several interesting phenomena. Flows with a low Reynold's number possess little to no momentum; thus, fluids within a microchannel respond quickly and reliably to changes in external forces. In addition, at the microscale, 2 or more streams of laminar flow in contact with each other do not mix, except by diffusion of molecules across the interface of the streams. The rate of diffusion between the contacting surfaces at the microscale can be very quick, partially because of the relatively short distances needed to cross fluid volumes. Many of these fluid characteristics at the microscale form the principles driving the interest in the use of microchannels for gamete and embryo manipulation. In general, a microenvironment more closely resembles the in vivo conditions of fertilization and development when compared with a culture dish or drop of media. Below, we discuss the theory behind investigating the use of microfluidics in andrology, its testing, limitation, and potential future influence. Interest in microfluidics began with attempts to miniaturize chemical and biological analysis devices in the laboratory (Kricka, 1998). Current designs are often referred to as “laboratory-on-a-chip” or micrototal analysis systems (μTAS) and function by allowing a variety of chemical processes and interactions to occur as fluid flows within their miniature channels and chambers (Weigl and Yager, 1999). Such devices perform all the analytical functions necessary for their purpose, including sample handling, mixing, incubation, sorting, transport, interaction, and detection or signaling within an integrated microfluidic “chip.” Examples include, but are not limited to, immunoassays for antibodies present in serum (Linder et al, 2002) and assays determining enzyme reaction kinetics (Xue et al, 2001; Yakovleva et al, 2002). Additional applications in cellular biology have emerged, such as integrated cell sorting devices working at the microscale (Fu et al, 2002) and microfluidic devices that allow for the study of cellular interactions with substrates or other cells (Shim et al, 2003). Advances in cell biology have been demonstrated with the use of microfluidics and the principle of laminar flow, allowing for selective exposure of subcellular areas of interest to membrane-permeable molecules (Takayama et al, 2001). Such precise delivery of molecules to cellular subdomains illustrates the precision with which microfluidic regulation of fluid flow is capable. Advantages of such laboratory-on-a-chip technology are multiple. First, once designed and tested, the manufacture of such devices is straightforward and inexpensive, allowing them to be disposable (McDonald et al, 2000). Microfluidic analysis devices use very low volumes of samples and reagents and provide for faster reactions and response times (Weigl and Yager, 1999). Miniaturization very importantly allows for integration of multiple processes within a small, self-contained unit (Kricka, 1998). This can be translated into either multiple parallel analyses, consecutive serial processes, or both. The brief overview given here is only intended to familiarize readers with the variety of capabilities of microfluidic technology and is by no means a comprehensive listing of microfluidic applications in sciences. Readers are encouraged to consult more thorough reviews (Khandurina and Guttman, 2002; Verpoorte, 2002). Microfluidics systems were initially fabricated with the use of materials and techniques common in the industry that inspired them—microelectronics (McDonald et al, 2000). Photolithography and etching of silicon and glass was a highly developed technology also readily available to researchers interested in miniaturizing analytical systems, yet costs were a significant barrier. In search of a suitable alternative, polymers have quickly emerged as a material for microfluidic biological device fabrication (McDonald et al, 2000). Compounds such as poly(methyl)methacrylate (Martynova et al, 1997), fluorinated ethylene propylene (Sahlin et al, 2002), and poly(dimethylsiloxane) (PDMS; McDonald and Whitesides, 2002) are cheaper and easier to manipulate than silicon-glass alternatives (Martynova et al, 1997). PDMS in particular has become one of the most actively explored and promising materials thus far, possessing numerous characteristics specifically suitable for biological use. It is nontoxic, transparent, insulating, and permeable to gases (McDonald and Whitesides, 2002). From a fabrication standpoint, PDMS permits submicron fidelity with molding, cures at low temperatures, and can easily seal reversibly to itself and a host of other materials (McDonald et al, 2000). Although PDMS is generally regarded as nontoxic, special consideration must be given to its use with gametes and embryos, which can be very sensitive to their environment compared with transformed cell lines. Before the use of microfluidic devices with sperm, testing confirmed that no negative effects resulted from prolonged exposure to the materials used in their fabrication. Schuster et al (2003) reported that 30 minutes of exposure to PDMS did not alter sperm survival. In addition, Glasgow et al (2001) found that development of 2-cell mouse embryos to the blastocyst stage was unchanged by continuous exposure to numerous photolithography compounds compared with controls. Thus it appears that PDMS-composed microchannels or the materials used in their construction do not confer deleterious effects to gametes or embryos. Numerous efforts have improved methods of semen processing and sperm isolation. Currently, swim-up techniques or density gradient separation are methods of choice (Trounson and Gardner, 2000). Both methods result in adequate recovery of motile sperm, although additional steps might be necessary in poor-quality semen samples (Bourne et al, 1995a,b). However, some researchers have stated concern that these methods could contribute to sperm morphological damage, DNA damage, production of oxygen-free radicals, or multiple injuries (Aitken and Clarkson, 1988; Zini et al, 1999). In addition, these techniques can be labor and time intensive. Ideal sperm isolation would involve a simple, rapid, and atraumatic method to obtain sufficient motile sperm for use in either IVF or ICSI, depending on need and the quality of the original semen sample. Attempts have been made to develop devices for such a purpose. The Wang tube (Wang et al, 1992), a uniquely configured glass tube, allows motile sperm to progress to an upper arm that is then separated for sperm use in intrauterine insemination or IVF. Comparison testing with swim-up and density gradient separation for normozoospermic samples revealed greater motility and morphology with the device (Wang, 1995). Lih et al (1996) have developed and tested a Lucite microchamber consisting of a central loading well surrounded by slightly depressed sidewells that was conceived from the observation that motile sperm migrate to the periphery of microdrops. This device concentrated motile sperm up to 13-fold in the sidewells, yielding a sufficient number for use in ICSI. A microfluidic device has been explored for sperm diagnostic purposes. Kricka et al (1993) designed and fabricated silicon and glass devices for sperm motility evaluation. They evaluated sperm progression along the length of a microchannel (80 μm wide by 20 μm deep) and navigation through a network of branching channels. In initial studies, they demonstrated feasibility and hypothesized that this device could replace conventional methods of motility assessment and semen analysis. Subsequently, they demonstrated that sperm movement within microchannels, judged by the time needed to reach the end of the channel, correlated with forward progression scores (Kricka et al, 1997). However, the design of the device did not give reliable information regarding sperm concentration or percent motility and therefore could only serve as an adjunctive test of motility and forward progression rather than a comprehensive semen analysis tool. Schuster et al (2003) developed a microfluidic device taking advantage of parallel laminar flow streams present at the microscale. In this device, a flowing stream of semen was placed in parallel with a flowing stream of media within a microchannel. Flow within microchannels was maintained by a novel gravity-driven, horizontally oriented pumping system developed specifically for the device (Cho et al, 2003). As discussed, these 2 parallel laminar flow streams mix only by diffusion. Motile sperm demonstrated the ability to actively propel themselves across contacting surface areas and deviated from the initial streamline into the media stream for collection, whereas nonmotile sperm and cellular debris remained in the initial stream and exited the device (Figure 2). . (A) Picture of microfluidic sperm sorter. (B) Three-dimensional view of microfluidic sperm sorter. (C) Theoretical vision for microfluidic sperm sorter. Media flows from left to right. (C, D) Semen sample is loaded into the upper stream inlet, and fresh media is placed in the lower stream inlet. (C, E) Motile sperm are able to deviate from the initial streamline and cross the interface of the laminar flow streams, exiting into the lower stream outlet for recovery. Debris, nonmotile sperm, and some motile sperm are collected in the upper stream outlet. Testing of this laminar flow sorting system was performed with 40 μL of unprocessed human semen, followed by semen samples artificially filled with debris from a stock solution of round immature germ and white blood cells to simulate poor-quality samples. For unprocessed semen, the device consistently produced a sorted fraction with increased motility (mean 98% motile) and improved Kruger strict sperm morphology (mean 22% normal forms) compared with the initial specimen (mean 44% and 10%, respectively). For debris-filled samples, the device not only concentrated motile sperm (mean 98% motile) within the collected fraction, but was also able to produce a round cell:sperm ratio of 1:33 compared with a 10:1 ratio in the starting specimen (Schuster et al, 2003). Microfluidics might be particularly suitable for IVF for a number of reasons (Suh et al, 2003). The microenvironment of a microchannel more closely resembles in vivo fertilization conditions than a culture dish or microdrop. Microfluidic channels allow for nonturbulent bathing of gametes with fresh media throughout insemination and coincubation. Sperm-oocyte interactions occur in an active environment, rather than the static conditions present in a culture dish or droplet. In addition, sperm can be predictably delivered via laminar flow to each oocyte within the microchannel, eliminating the randomness of sperm-oocyte interaction. In a culture dish, sperm can travel randomly in any direction, thereby relying on random sperm movement toward the oocytes; however, in a microchannel environment, sperm movement is limited by the direction of flow, allowing for active transport to the oocytes. Finally, microchannel environments use extremely small volumes of media, theoretically requiring fewer sperm to achieve insemination concentrations equal to standard IVF with larger volumes. Previous investigators have attempted, with some success, to reduce the volume of insemination medium with various low-volume vessels, although none have gained widespread acceptance. Van der Ven et al (1989) tested the use of sterile, nonheparinized hematocrit capillary tubes (75 mm length, 0.9 mm inner diameter) for IVF in humans. Normospermic samples were used with standard culture tubes as controls. Volumes of 5–10 μL containing a range of 500–4000 sperm per oocyte were used in these capillary tubes. Overall fertilization rates between controls and capillary tubes was similar (78% and 66%, respectively), although slightly lower for sperm totals of 500–1000 (56%) compared with 2000–4000 total sperm (79%). Ranoux and Seibel (1990) used embryo cryopreservation straws in volumes up to 200 μL (Ranoux et al, 1988) with 2000–4000 motile sperm. Results compared favorably with controls, with 167 of 322 oocytes (51.8%) fertilized by the straw technique. We have recently demonstrated that mouse IVF can be conducted successfully within microfluidic channels (unpublished data). Not only are lower total numbers of sperm required because of the use of media we have also demonstrated fertilization within microchannels with lower insemination sperm We to develop design that will result in increased and of use. Such microfluidic devices be in clinical not only for but as a for standard This microfluidic sperm sorting device a simple, atraumatic method of motile sperm of normal morphology from both unprocessed normal semen and poor-quality containing significant A of the device the flow at In its it is not of processing an semen however, it provide a means of quickly and easily a small sample of motile sperm of normal morphology for ICSI, insemination in or in an integrated microfluidic device et al, data). In addition, and in the design are in progress that might allow for processing in parallel and increased of flow and sorting (Schuster et al, 2003). As with any new technology, design an important in loading which can allow for of the outcomes. of oocytes and sperm to the device a which time outside of the environment, has significant deleterious effects on gamete and survival. of this time currently a significant Last, the of more but for fluid flow Current studies are focused on a of the gravity-driven, horizontally oriented pumping system et al, from the microfluidic sperm developed by et al (2003) and its application to microfluidic Although of the with microfluidics in IVF has been performed in a the of process and microfluidic technology is Use of microfluidic technology for sperm processing results in a small volume and fraction of motile sperm. Such volumes are to use and translate into a However, laminar sperm have been used for fertilization within a microenvironment (unpublished data). of a microfluidic channel for the oocyte and the stream of sorted sperm would result in of the oocyte with these motile sperm. the oocyte can be to a for for and embryo culture review in Beebe et al, media can be for embryo step the with no cell other than flow along a variety of channels. Miniaturization allows the system to be small and by laboratory not only decreases gamete and embryo but also for greater of multiple in integration of these The development of reliable methods for flow through a network of channels is numerous active and et al, or (McDonald et al, have been designed for microfluidic but the to gamete and embryo must be delivery of fluid at precise rates is important for sperm sorting, culture media and embryo manipulation. A fluid has been for microfluidic sperm sorting (Cho et al, and active regulation of fluid flow with has been used for insemination and embryo et al, 2000). we developed a new microfluidic system with up to of and by on a (Figure et al, these are used as a for the The can a to of small individual on each of the cells cells for a total of and A of these cells would typically a of by a We advantage of the rate and size of the by the microchannels, them at The system advantage of the yet of PDMS microchannels fabricated with and the movement of to fluid through channels. of a can be used to a forward or flow of through the microchannel when to various patterns. The volume of flow per can be by the volume of by the This method of is and are can be and have in devices the size of a With this new we have demonstrated 3 functions necessary for future application of microfluidics for and embryo culture and analysis and and Finally, methods of fabrication and of microfluidic devices must be widespread of this technology for human Current devices have been for purposes. However, development of an IVF laboratory-on-a-chip is a and exciting . (A) A that can be used in with microfluidic (B) for and of (C) that have been in a microfluidic with media of various a microchannel of media is is the is depressed up the PDMS which into the channel to the media. This can be used to or or when into microchannels in this media pumping within the microchannel.

PART I: The Clinical Laboratory The Clinical Laboratory: Organization, Purpose and Practice Physician Office Laboratories (POLS) Principles of Instrumentation Clinical Laboratory Automation Interpreting Laboratory Results Informatics, Imaging and Interoperability Laboratory Statistics Clinical Laboratory Quality Assurance PART II: Clinical Chemistry Evaluation of Renal Function, Wather, Electrolytes, Acid-Base Balance and Blood Gases Metabolic Intermediates, Inorganic Ions and Biochemical Markers of Bone Metabolism Carbohydrates Lipids and Dyslipoproteinemia Specific Proteins Evaluation of Liver Function and Injury Clinical Enzymology Evaluation of Endocrine Function Toxicology and Therapeutic Drug Monitoring PART III: Urine and Other Body Fluids Basic Examination of Urine Cerebrospinal, Synovial, and Serious Body Fluids Andrology Laboratory and Fertility Assessment Laboratory Management of Assisted Reproductive Technology Laboratory Aspects of Gestation Management Laboratory Diagnosis of Gastrointestinal Tract and Pancreatic Disorders PART IV: Hematology, Coagulation and Transfusion Medicine Basic Examination of Blood Hematopoiesis Erythrocytic Disorders Leukocytic Disorders Blood Platelets Coagulation, Fibrinolysis and Hypercoagulation Immunohematology Transfusion Medicine Hemapheresis Tissue Banking and Progenitor Cell PART V: Immunology and Immunopathy Overview of the Immune System and Immunologic Disorders Immunoassays and Immunochemistry Laboratory Evaluation of the Cellular Immune System Laboratory Evaluation of Immunoglobin Function and Humoral Immunity Complement and Kinins: Mediators of Inflamation Cytokines and Adhesion Molecules HLA: The Major Histocompatability Complex of Man The Major Histocompatibility Complex and Disease Immunodeficiency Disorders Clinical and Laboratory Evaluation of Systemic Rheumatic Diseases Vasculitis Organ Specific Autoimmune Diseases Allergic Diseases Diagnosis and Management of Cancer Using Serologic Tumor Markers PART VI: Medical Microbiology Viral Infections Chlamydial, Rickettsial, and Mycoplasmal Infections Medical Microbiology In Vitro Testing of Antimicrobial Agents Spirochete Infections Mycobacteria Mycotic Diseases Medical Parasitology Molecular Pathology of Infectious Diseases Specimen Collection and Handling for Diagnosis of Infectious Diseases PART VII.: Molecular Pathology An Introduction to Molecular Pathology Molecular Diagnostics: Basic Principles and Techniques Polymerase Chain Reaction (PCR) and Other Amplification Technology Hybridization Array Technology Applications for Cytogenetics in Modern Pathology Establishing a Molecular Diagnostics Laboratory Oncoproteins and Early Tumor Detection Molecular Techniques in the Hematopoietic Neoplasms Molecular Diagnosis of Genetic Diseases Parentage Testing: Use of DNA Polymorphism and Other Genetic Markers Forensic Identity Testing by DNA Analysis

STUDY QUESTION: What updates of the International Glossary on Infertility and Fertility Care are required, to reflect contemporary scientific knowledge, social needs, and inclusive definitions, while harmonizing international communication across clinical, research, policy, and public domains? SUMMARY ANSWER: This 4th edition presents 348 consensus-based terms and definitions, including numerous revisions from the previous edition and 79 newly introduced definitions reflecting advances in reproductive science, technology, and evolving social contexts. WHAT IS KNOWN ALREADY: Previous glossary editions (2006, 2009, 2017) established internationally recognized definitions related to clinical practice, research, and policy. The 2017 edition comprised 283 terms and, among many others, expanded the concept of infertility to include not only its recognition as a disease, but also as an impairment of function generating disability. The glossary has been extensively used worldwide and has contributed to international standardization of data collection, appropriate comparison of outcome measures, and provided a reference for all stakeholders including policy makers. STUDY DESIGN, SIZE, DURATION: Under guidance of the organizing committee, 21 professionals from across the world, and representing expertise in different sub-specialties, formed five working groups: clinical definitions; outcome measures; embryology laboratory; clinical and laboratory andrology; and epidemiology, public health and gender related definitions. The definitions from the previous glossary were evaluated and new terms identified. All definitions were then reviewed by an international advisory panel of nine experts that evaluated the glossary from scientific, ethical, cultural, and policy perspectives. PARTICIPANTS/MATERIALS, SETTING, METHODS: Between November 2024 and October 2025, periodical virtual meetings were held within and between working groups and the organizing committee. Following circulation of the first consensually agreed draft, a one-day in-person meeting with representatives of all working groups and members of the international advisory panel was held at ESHRE, June 2025. Most terms and definitions were discussed and agreed. In the absence of agreement, further discussions were held between the organizing committee, working group chairs and members of the advisory panel. It had been determined at the outset that final disagreement would be resolved via a two-third majority vote. All terms and definitions were, however, reached by consensus and adopted following a final round of review and approval by all authors. MAIN RESULTS AND THE ROLE OF CHANCE: The glossary now includes 348 terms. Compared to the previous edition, 14 terms were deleted, numerous terms modified and 79 new terms were added. Modifications reflect current scientific knowledge, technological advancements, and inclusivity related to gender and family structures. Chance does not play a role, as all definitions are consensus-based. LIMITATIONS, REASONS FOR CAUTION: Some terms may require future refinement as scientific knowledge evolves and societal contexts change. The glossary reflects consensus rather than empirical testing of all definitions. WIDER IMPLICATIONS OF THE FINDINGS: This glossary provides a global reference for standardized terminology, supporting clinical care, research, international comparisons, policy making, patient communication, and reproductive health literacy. STUDY FUNDING/COMPETING INTEREST(S): Neither ICMART, responsible for conducting this project, nor any of the participants received specific financial support for their activities in this project. Ferring provided ICMART with a fixed amount to cover venue costs and a one-day hotel accommodation for participants attending the in-person meeting held prior to the ESHRE Congress in June 2025. Disclosures were provided by all authors, and none reported any conflict of interest related to this manuscript. TRIAL REGISTRATION NUMBER: N/A.

Infertility affects nearly 186 million people worldwide and the male partner is the cause in about half of the cases. Meta-regression data indicate an unexplained decline in sperm concentration and total sperm count over the last four decades, with an increasing prevalence of male infertility. This suggests an urgent need to implement further basic and clinical research in Andrology. Andrology developed as a branch of urology, gynecology, endocrinology, and, dermatology. The first scientific journal devoted to andrological sciences was founded in 1969. Since then, despite great advancements, andrology has encountered several obstacles in its growth. In fact, for cultural reasons, the male partner has often been neglected in the diagnostic and therapeutic workup of the infertile couple. Furthermore, the development of assisted reproductive techniques (ART) has driven a strong impression that this biotechnology can overcome all forms of infertility, with a common belief that having a spermatozoon from a male partner (a sort of sperm donor) is all that is needed to achieve pregnancy. However, clinical practice has shown that the quality of the male gamete is important for a successful ART outcome. Furthermore, the safety of ART has been questioned because of the high prevalence of comorbidities in the offspring of ART conceptions compared to spontaneous conceptions. These issues have paved the way for more research and a greater understanding of the mechanisms of spermatogenesis and male infertility. Consequently, numerous discoveries have been made in the field of andrology, ranging from genetics to several "omics" technologies, oxidative stress and sperm DNA fragmentation, the sixth edition of the WHO manual, artificial intelligence, management of azoospermia, fertility in cancers survivors, artificial testis, 3D printing, gene engineering, stem cells therapy for spermatogenesis, and reconstructive microsurgery and seminal microbiome. Nevertheless, as many cases of male infertility remain idiopathic, further studies are required to improve the clinical management of infertile males. A multidisciplinary strategy involving both clinicians and scientists in basic, translational, and clinical research is the core principle that will allow andrology to overcome its limits and reach further goals. This state-of-the-art article aims to present a historical review of andrology, and, particularly, male infertility, from its "Middle Ages" to its "Renaissance", a golden age of andrology.

BACKGROUND: Infertility affects 7%-12% of men, and its etiology is unknown in half of cases. To fill this gap, use of the male genital tract color-Doppler ultrasound (MGT-CDUS) has progressively expanded. However, MGT-CDUS still suffers from lack of standardization. Hence, the European Academy of Andrology (EAA) has promoted a multicenter study ("EAA ultrasound study") to assess MGT-CDUS characteristics of healthy, fertile men to obtain normative parameters. OBJECTIVES: To report (a) the development and methodology of the "EAA ultrasound study," (b) the clinical characteristics of the cohort of healthy, fertile men, and (c) the correlations of both fertility history and seminal features with clinical parameters. METHODS: A cohort of 248 healthy, fertile men (35.3 ± 5.9 years) was studied. All subjects were asked to undergo, within the same day, clinical, biochemical, and seminal evaluation and MGT-CDUS before and after ejaculation. RESULTS: The clinical, seminal, and biochemical characteristics of the cohort have been reported here. The seminal characteristics were consistent with those reported by the WHO (2010) for the 50th and 5th centiles for fertile men. Normozoospermia was observed in 79.6% of men, while normal sperm vitality was present in almost the entire sample. Time to pregnancy (TTP) was 3.0[1.0-6.0] months. TTP was negatively correlated with sperm vitality (Adj.r =-.310, P = .011), but not with other seminal, clinical, or biochemical parameters. Sperm vitality and normal morphology were positively associated with fT3 and fT4 levels, respectively (Adj.r = .244, P < .05 and Adj.r = .232, P = .002). Sperm concentration and total count were negatively associated with FSH levels and positively, along with progressive motility, with mean testis volume (TV). Mean TV was 20.4 ± 4.0 mL, and the lower reference values for right and left testes were 15.0 and 14.0 mL. Mean TV was negatively associated with gonadotropin levels and pulse pressure. Varicocoele was found in 33% of men. CONCLUSIONS: The cohort studied confirms the WHO data for all semen parameters and represents a reference with which to assess MGT-CDUS normative parameters.

BACKGROUND: Infection and inflammation of the reproductive tract are significant causes of male factor infertility. Ascending infections caused by sexually transmitted bacteria or urinary tract pathogens represent the most frequent aetiology of epididymo-orchitis, but viral, haematogenous dissemination is also a contributory factor. Limitations in adequate diagnosis and therapy reflect an obvious need for further understanding of human epididymal and testicular immunopathologies and their contribution to infertility. A major obstacle for advancing our knowledge is the limited access to suitable tissue samples. Similarly, the key events in the inflammatory or autoimmune pathologies affecting human male fertility are poorly amenable to close examination. Moreover, the disease processes generally have occurred long before the patient attends the clinic for fertility assessment. In this regard, data obtained from experimental animal models and respective comparative analyses have shown promise to overcome these restrictions in humans. OBJECTIVE AND RATIONALE: This narrative review will focus on male fertility disturbances caused by infection and inflammation, and the usefulness of the most frequently applied animal models to study these conditions. SEARCH METHODS: An extensive search in Medline database was performed without restrictions until January 2018 using the following search terms: 'infection' and/or 'inflammation' and 'testis' and/or 'epididymis', 'infection' and/or 'inflammation' and 'male genital tract', 'male infertility', 'orchitis', 'epididymitis', 'experimental autoimmune' and 'orchitis' or 'epididymitis' or 'epididymo-orchitis', antisperm antibodies', 'vasectomy'. In addition to that, reference lists of primary and review articles were reviewed for additional publications independently by each author. Selected articles were verified by each two separate authors and discrepancies discussed within the team. OUTCOMES: There is clear evidence that models mimicking testicular and/or epididymal inflammation and infection have been instructive in a better understanding of the mechanisms of disease initiation and progression. In this regard, rodent models of acute bacterial epididymitis best reflect the clinical situation in terms of mimicking the infection pathway, pathogens selected and the damage, such as fibrotic transformation, observed. Similarly, animal models of acute testicular and epididymal inflammation using lipopolysaccharides show impairment of reproduction, endocrine function and histological tissue architecture, also seen in men. Autoimmune responses can be studied in models of experimental autoimmune orchitis (EAO) and vasectomy. In particular, the early stages of EAO development showing inflammatory responses in the form of peritubular lymphocytic infiltrates, thickening of the lamina propria of affected tubules, production of autoantibodies against testicular antigens or secretion of pro-inflammatory mediators, replicate observations in testicular sperm extraction samples of patients with 'mixed atrophy' of spermatogenesis. Vasectomy, in the form of sperm antibodies and chronic inflammation, can also be studied in animal models, providing valuable insights into the human response. WIDER IMPLICATIONS: This is the first comprehensive review of rodent models of both infectious and autoimmune disease of testis/epididymis, and their clinical implications, i.e. their importance in understanding male infertility related to infectious and non-infectious/autoimmune disease of the reproductive organs.

Artificial intelligence (AI) in medicine has gained a lot of momentum in the last decades and has been applied to various fields of medicine. Advances in computer science, medical informatics, robotics, and the need for personalized medicine have facilitated the role of AI in modern healthcare. Similarly, as in other fields, AI applications, such as machine learning, artificial neural networks, and deep learning, have shown great potential in andrology and reproductive medicine. AI-based tools are poised to become valuable assets with abilities to support and aid in diagnosing and treating male infertility, and in improving the accuracy of patient care. These automated, AI-based predictions may offer consistency and efficiency in terms of time and cost in infertility research and clinical management. In andrology and reproductive medicine, AI has been used for objective sperm, oocyte, and embryo selection, prediction of surgical outcomes, cost-effective assessment, development of robotic surgery, and clinical decision-making systems. In the future, better integration and implementation of AI into medicine will undoubtedly lead to pioneering evidence-based breakthroughs and the reshaping of andrology and reproductive medicine.

BACKGROUNDS: Despite a wide spectrum of contraceptive methods for women, the unintended pregnancy rate remains high (45% in the US), with 50% resulting in abortion. Currently, 20% of global contraceptive use is male-directed, with a wide variation among countries due to limited availability and lack of efficacy. Worldwide studies indicate that >50% of men would opt to use a reversible method, and 90% of women would rely on their partner to use a contraceptive. Additional reasons for novel male contraceptive methods to be available include the increased life expectancy, sharing the reproductive risks among partners, social issues, the lack of pharma industry involvement and the lack of opinion makers advocating for male contraception. AIM: The present guidelines aim to review the status regarding male contraception, the current state of the art to support the clinical practice, recommend minimal requirements for new male contraceptive development and provide and grade updated, evidence-based recommendations from the European Society of Andrology (EAA) and the American Society of Andrology (ASA). METHODS: An expert panel of academicians appointed by the EAA and the ASA generated a consensus guideline according to the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) system. RESULTS: Sixty evidence-based and graded recommendations were produced on couple-centered communication, behaviors, barrier methods, semen analysis and contraceptive efficacy, physical agents, surgical methods, actions before initiating male contraception, hormonal methods, non-hormonal methods, vaccines, and social and ethical considerations. CONCLUSION: As gender roles transform and gender equity is established in relationships, the male contribution to family planning must be facilitated. Efficient and safe male-directed methods must be evaluated and introduced into clinical practice, preferably reversible, either hormonal or non-hormonal. From a future perspective, identifying new hormonal combinations, suitable testicular targets, and emerging vas occlusion methods will produce novel molecules and products for male contraception.

This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working group's understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

BACKGROUND: Hyaluronic acid is a glycosaminoglycan widely used in the fields of orthopedics, ophthalmology, and aesthetic medicine due to its significant ability to reduce the synthesis of pro-inflammatory proteins and its activity against oxidative stress, a feature of many degenerative illnesses. OBJECTIVES: The objective of the present review is to provide a comprehensive narrative review of the most recent literature on the use of hyaluronic acid in andrology in order to facilitate the use of this therapeutic device in the common clinical practice of many physicians. Specific conditions covered in the review are Peyronie's disease, premature ejaculation, and penile enlargement. MATERIALS AND METHODS: A broad and comprehensive literature search included Medline, EMBASE, and the Cochrane Libraries, with no time restriction up to December 2020 and restricted to English language publications. Unpublished studies were not included. The study was registered as "The role of hyaluronic acid in andrology: A systematic review and meta-analysis" in PROSPERO with the ID CRD42021223416. DISCUSSION AND CONCLUSION: Hyaluronic acid is a valid choice for the treatment of Peyronie's disease in terms of the resolution of the acute phase of the disease and of contributing to stabilizing the disease as a bridge to potential surgery. Data, furthermore, suggest that hyaluronic acid is frequently associated with an overall clinical improvement, allowing the patient to resume normal sexual activity. With regard to premature ejaculation, data suggests hyaluronic acid-based treatments were effective in prolonging intra-vaginal ejaculation time. Furthermore, hyaluronic acid was found to be safe and well-tolerated, with main adverse events limited to local discomfort, ecchymosis, papule formation, and glans numbness, all of which were reported to resolve spontaneously. Last, with regard to penile enlargement, the overall perception of experts is that hyaluronic acid may be an extremely well-tolerated compound with potential for application in specific areas of male sexual health that are often neglected as compared to more common, and relatively simpler to treat, conditions.

As the specialty of Andrology expands it is important to establish the most important studies that have shaped, and continue to shape, current research and clinical practice. Bibliometric analysis involving a citation rank list is an established means by which to identify the published material within a given field that has greatest intellectual influence. This bibliometric analysis sought to identify the 100 most influential manuscripts in Andrology, as well as the key research themes that have shaped contemporary understanding and management of andrological conditions. The Thompson Reuters Web of Science citation indexing database was interrogated using a number of search terms chosen to reflect the full spectrum of andrological practice. Results were ranked according to citation number and further analysed according to subject, first and senior author, journal, year of publication, institution and country of origin. The Web of Science search returned a total of 24,128 manuscripts. Citation number of the top 100 articles ranged from 2819 to 218 (median 320). The most cited manuscript (by Feldman et al., The Journal of Urology 1994; 2819 citations) reported the prevalence and risk factors for erectile dysfunction (ED) in the Massachusetts Male Ageing Study. The Journal of Urology published the highest number of manuscripts ( n = 11), followed by the New England Journal of Medicine ( n = 10). The most common theme represented within the top 100 manuscripts was erectile dysfunction ( n = 46), followed jointly by hypogonadism and male factor infertility ( n = 24 respectively). Erectile dysfunction should be considered the most widely researched, published and cited field within andrological practice. This study provides a list of the most influential manuscripts in andrology and serves as a reference of what comprises a ‘highly citable’ paper for both researchers and clinicians.

Objective: To update the "Endocrine Treatment of Transsexual Persons: An Endocrine Society Clinical Practice Guideline," published by the Endocrine Society in 2009. Participants: The participants include an Endocrine Society-appointed task force of nine experts, a methodologist, and a medical writer. Evidence: This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation approach to describe the strength of recommendations and the quality of evidence. The task force commissioned two systematic reviews and used the best available evidence from other published systematic reviews and individual studies. Consensus Process: Group meetings, conference calls, and e-mail communications enabled consensus. Endocrine Society committees, members and cosponsoring organizations reviewed and commented on preliminary drafts of the guidelines. Conclusion: Gender affirmation is multidisciplinary treatment in which endocrinologists play an important role. Gender-dysphoric/gender-incongruent persons seek and/or are referred to endocrinologists to develop the physical characteristics of the affirmed gender. They require a safe and effective hormone regimen that will (1) suppress endogenous sex hormone secretion determined by the person's genetic/gonadal sex and (2) maintain sex hormone levels within the normal range for the person's affirmed gender. Hormone treatment is not recommended for prepubertal gender-dysphoric/gender-incongruent persons. Those clinicians who recommend gender-affirming endocrine treatments-appropriately trained diagnosing clinicians (required), a mental health provider for adolescents (required) and mental health professional for adults (recommended)-should be knowledgeable about the diagnostic criteria and criteria for gender-affirming treatment, have sufficient training and experience in assessing psychopathology, and be willing to participate in the ongoing care throughout the endocrine transition. We recommend treating gender-dysphoric/gender-incongruent adolescents who have entered puberty at Tanner Stage G2/B2 by suppression with gonadotropin-releasing hormone agonists. Clinicians may add gender-affirming hormones after a multidisciplinary team has confirmed the persistence of gender dysphoria/gender incongruence and sufficient mental capacity to give informed consent to this partially irreversible treatment. Most adolescents have this capacity by age 16 years old. We recognize that there may be compelling reasons to initiate sex hormone treatment prior to age 16 years, although there is minimal published experience treating prior to 13.5 to 14 years of age. For the care of peripubertal youths and older adolescents, we recommend that an expert multidisciplinary team comprised of medical professionals and mental health professionals manage this treatment. The treating physician must confirm the criteria for treatment used by the referring mental health practitioner and collaborate with them in decisions about gender-affirming surgery in older adolescents. For adult gender-dysphoric/gender-incongruent persons, the treating clinicians (collectively) should have expertise in transgender-specific diagnostic criteria, mental health, primary care, hormone treatment, and surgery, as needed by the patient. We suggest maintaining physiologic levels of gender-appropriate hormones and monitoring for known risks and complications. When high doses of sex steroids are required to suppress endogenous sex steroids and/or in advanced age, clinicians may consider surgically removing natal gonads along with reducing sex steroid treatment. Clinicians should monitor both transgender males (female to male) and transgender females (male to female) for reproductive organ cancer risk when surgical removal is incomplete. Additionally, clinicians should persistently monitor adverse effects of sex steroids. For gender-affirming surgeries in adults, the treating physician must collaborate with and confirm the criteria for treatment used by the referring physician. Clinicians should avoid harming individuals (via hormone treatment) who have conditions other than gender dysphoria/gender incongruence and who may not benefit from the physical changes associated with this treatment.

BACKGROUND: In attempting to formulate potential WHO guidelines for the diagnosis of male infertility, the Evidence Synthesis Group noted a paucity of high-quality data on which to base key recommendations. As a result, a number of authors suggested that key areas of research/evidence gaps should be identified, so that appropriate funding and policy actions could be undertaken to help address key questions. OBJECTIVES: The overall objective of this Consensus workshop was to clarify current knowledge and deficits in clinical laboratory andrology, so that clear paths for future development could be navigated. MATERIALS AND METHODS: Following a detailed literature review, each author, prior to the face-to-face meeting, prepared a summary of their topic and submitted a PowerPoint presentation. The topics covered were (a) Diagnostic testing in male fertility and infertility, (b) Male fertility/infertility in the modern world, (c) Clinical management of male infertility, and (d) The overuse of ICSI. At the meeting in Cairo on February 18, 2019, the evidence was presented and discussed and a series of consensus points agreed. RESULTS: The paper presents a background and summary of the evidence relating to these four topics and addresses key points of significance. Following discussion of the evidence, a total of 36 consensus points were agreed. DISCUSSION: The Discussion section presents areas where there was further debate and key areas that were highlighted during the day. CONCLUSION: The consensus points provide clear statements of evidence gaps and/or potential future research areas/topics. Appropriate funding streams addressing these can be prioritized and consequently, in the short and medium term, answers provided. By using this strategic approach, andrology can make the rapid progress necessary to address key scientific, clinical, and societal challenges that face our discipline now and in the near future.

STUDY QUESTION: Can a tool be developed for authors, reviewers and editors of the ESHRE Journals to improve the quality of published studies which rely on semen analysis data? SUMMARY ANSWER: A basic checklist for authors, reviewers and editors has been developed and is presented. WHAT IS KNOWN ALREADY: Laboratory work which includes semen analysis is burdened by a lack of standardization. This has significant negative effects on the quality of scientific and epidemiological studies, potential misclassification of patients and the potential to impair clinical treatments/diagnoses that rely on accurate semen quality information. Robust methods are available to reduce laboratory error in semen analysis, inducing adherence to World Health Organization techniques, participation in an external quality control scheme and appropriate training of laboratory personnel. However, journals have not had appropriate systems to assess if these methods have been used. STUDY DESIGN, SIZE, DURATION: After discussion at a series of Associate Editor Meetings of the ESHRE Journals the authors of the present text were asked to propose a tool for authors, reviewers and editors of the ESHRE Journals to ensure a high quality assessment of submitted manuscripts which rely on semen analysis data, including a detailed verification of the relevance and the quality of the methods used. PARTICIPANTS/MATERIALS, SETTING, METHODS: N/A. MAIN RESULTS AND THE ROLE OF CHANCE: A basic checklist for authors, reviewers and editors is presented. The checklist contains key points which should be considered by authors when designing studies and which provides essential information for when the submitted manuscript is evaluated. For published articles the answers in the checklist are suitable to be available as supplementary data, which will also reduce the space necessary for technical details in the printed article. LIMITATIONS, REASONS FOR CAUTION: Guidelines such as these should not be used uncritically. It is therefore important that submitting authors, in situations where their study does not comply with the basic requirements for semen analysis, not only explain all methodological deviations but also declare the level of uncertainty in their analyses and how it complies with, or might confound, the aims of the study. WIDER IMPLICATIONS OF THE FINDINGS: The fundamental importance of appropriate and robust methodology to facilitate advances in scientific understanding and patient management and treatment, is now accepted as being paramount. Use of the semen analysis checklist should be part of this process, and when completed and signed by the corresponding author at the time of submitting a manuscript should result in greater transparency, and ultimately uniformity. It is hoped that this initiative will pave the way for wider adoption of the methodology/reporting by other biomedical, epidemiological and scientific journals, and ultimately become the standard of practice for papers reporting semen analysis results obtained in laboratory and clinical andrology. Systems to assist referees, authors and editors to present high quality findings should have a significant impact on the field of reproductive medicine. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained for this work. The authors have no competing interests in relation to the present publication and checklist. TRIAL REGISTRATION NUMBER: N/A.

Basic andrological diagnosis consists of taking the patient's medical history and the couple's history as well as performing a physical examination including genital ultrasound, spermiogram, and hormonal analysis. If needed, a testicular biopsy and genetic testing may also be performed. Recent studies have shown the effect of lifestyle factors on male fertility. Thus, the patient history and clinical/andrological examinations have been broadened to include information on metabolic disorders like obesity and diabetes mellitus. The biggest changes occurred with the publication of the fifth edition of the WHO laboratory manual in 2010 and the introduction of a section on semen analysis in the German Medical Association guidelines (RiliBÄK). The reference values for almost all spermiogram parameters were adapted in an evidence-based approach using worldwide prospective population studies. For central parameters such as sperm motility and morphology, the assessment criteria were changed. New independent markers such as sperm DNA fragmentation rate are now routinely used in clinical diagnosis. For German andrological laboratories, there are now mandatory quality assurance measures for semen analysis (in the German "Rili-BÄK" guidelines). These include duplicate testing of all standard semen parameters and inter-laboratory comparison at regular intervals.

This practical, extensively illustrated handbook covers the procedures that are undertaken in andrology and ART laboratories to analyse and assess male-factor infertility, and to prepare spermatozoa for use in assisted conception therapy. The content is presented as brief, authoritative overviews of the relevant biological background for each area, plus detailed, step-by-step descriptions of the relevant analytical procedures. Each technical section includes pertinent quality control considerations, as well as the optimum presentation of results. In addition to the comprehensive 'basic' semen analysis, incorporating careful analysis of sperm morphology, the handbook provides established techniques for the use of computer-aided sperm analysis and sperm functional assessment. Throughout the handbook the interpretation of laboratory results in the clinical context is highlighted, and safe laboratory practice is emphasized. It is an invaluable resource to all scientists and technicians who perform diagnostic testing for male-factor infertility.